Method for increasing the growth rate, biomass, seed yield, photosynthetic capacity and/or abiotic s

专利摘要:
METHOD TO INCREASE NITROGEN USE EFFICIENCY, PRODUCTION, GROWTH RATE, BIOMASS, VIGOR, OIL CONTENT, SEED PRODUCTION, FIBER PRODUCTION, FIBER QUALITY, FIBER LENGTH, PHOTOSYNTHETIC CAPACITY AND/OR ABIOTIC STRESS TOLERANCE A PLANT, METHOD OF PRODUCTION OF A CULTURE, METHOD FOR SELECTING A TRANSFORMED PLANT, NUCLEIC ACID STRUCTURE, METHOD OF GROWING A CULTURE, PLANT CELL, AND TRANSGENIC PLANT, provides isolated polypeptides that are at least 80% homologous to SEQ ID Nos. 496-794, 28983645 and 3647-4855, isolated polynucleotides that are at least 80% identical to SEQ IDs. No. 1-495 and 795-2897, nucleic acid structures comprising the same, transgenic cells expressing the same, transgenic plants expressing the same, and a method of using the same to increase fertilizer use efficiency, nitrogen use efficiency , yield, growth rate, biomass, vigor, oil content, photosynthetic capacity, seed yield, fiber yield, fiber quality, fiber length and/or a plant's abiotic stress tolerance.
公开号:BR112015015577B1
申请号:R112015015577-4
申请日:2013-12-19
公开日:2022-01-25
发明作者:Adi ETZIONI；Hagai Karchi
申请人:Evogene Ltd；
IPC主号:

专利说明:

FIELD OF APPLICATION AND HISTORY
[001] The present patent application, in some respective applications, relates to isolated polynucleotides and polypeptides, nucleic acid structures comprising the same, transgenic cells comprising the same, transgenic plants exogenously expressing the same, and more particularly , but not exclusively, to methods of using them to increase fertilizer use efficiency (e.g. nitrogen use efficiency), production (e.g. seed production, oil production), biomass, rate of growth, vigor, oil content, fiber production, fiber quality, fiber length, fiber length, photosynthetic capacity and/or abiotic stress tolerance of a plant.
[002] A common approach to promoting plant growth has been and continues to be the use of natural as well as synthetic nutrients (fertilizers). Thus, fertilizers are the fuel behind the “green revolution”, directly responsible for the exceptional increase in crop production during the last 40 years and are considered the number one general expense in agriculture. For example, inorganic nitrogen fertilizers such as ammonium nitrate, potassium nitrate or urea typically account for 40% of costs related to crops such as corn and wheat. Of the three macronutrients provided as main fertilizers [nitrogen (N), phosphate (P) and potassium (K)], nitrogen is often the limiting element in plant growth and all crops have a fundamental dependence on inorganic nitrogen fertilizers. Nitrogen is responsible for the biosynthesis of amino acids and nucleic acids, prosthetic groups, plant hormones, chemical defenses of plants, etc. and it usually needs to be replenished each year, particularly in cereals, which make up more than half of the world's cultivated areas. Thus, nitrogen is translocated to the shoot, where it is stored in the leaves and stem during the rapid stage of plant development and until flowering. In corn, for example, plants accumulate most of their organic nitrogen during the period of grain germination and until flowering. Once the fertilization of the plant occurs, the grains begin to form and become the main nitrogen collectors of the plant. The stored nitrogen can then be redistributed from the leaves and stem that served as storage compartments until grain formation.
[003] Since fertilizer quickly depletes most soil types, it should be provided for growing crops two or three times during the growing season. In addition, the low nitrogen use efficiency (NUE | nitrogen use efficiency) of major crops (e.g. in the range of only 3070%) negatively affects farmer input expenses due to excess fertilizer applied. In addition, inefficient use or overuse of fertilizers are the main factors responsible for environmental problems, such as eutrophication of groundwater, lakes, rivers and seas, nitrate pollution in drinking water, which can cause methemoglobinemia, phosphate pollution, air pollution and the like. However, despite the negative impact of fertilizers on the environment and the limits on fertilizer use, which have been legislated in several countries, fertilizer use is expected to increase in order to support food and fiber production for rapid population growth over limited land resources. For example, it is estimated that by 2050, more than 150 million tons of nitrogen fertilizers will be used worldwide annually.
[004] Increasing nitrogen use efficiency by plants should allow crops to be grown with low input of fertilizers or alternatively be grown in poorer quality soils and therefore have a significant economic impact on developed farming systems and on development.
[005] The genetic improvement of fertilizer use efficiency (FUE|fertilizer use efficiency) in plants can be generated through traditional cultivation or through genetic engineering.
[006] Attempts to generate plants with improved FUE have been described in US Patent Application Publication No. 20020046419 (US Patent Application No. 7,262,055, to Choo, et al.); US Patent Application No. 20050108791, to Edgerton et al.; US Patent Application No. 20060179511, to Chomet et al.; Good, A, et al. 2007 (Engineering nitrogen use efficiency with alanine aminotransferase. Canadian Journal of Botany 85: 252-262 ); and Good AG et al. 2004 (Trends Plant Sci. 9:597-605).
[007] Yanagisawa et al. (Proc. Natl. Acad. Sci.
[008] U.S.A. 2004 101:7833-8) describes transgenic Dof1 plants that show improved growth under low nitrogen conditions.
[009] US Patent No. 6,084,153 to Good et al. discloses the use of a stress-sensitive promoter to control the expression of Alanine Aminotransferase (AlaAT| Alanine Amine Transferase) and transgenic canola plants with improved drought resistance and nitrogen deficiency when compared to control plants.
[0010] Yield is affected by several factors, such as the number and size of plant organs, plant architecture (eg, the number of branches), defined grain length, number of full grains, vigor (p. .eg, the seedling), growth rate, root development, utilization of water, nutrients (eg, nitrogen) and fertilizers, and stress tolerance.
[0011] Crops such as corn, rice, wheat, canola and soybeans account for more than half of total human caloric intake, either through direct consumption of the seeds themselves or through consumption of meat products from seed-raised animals. processed or forage. The seeds are also a source of sugars, proteins and oils and metabolites used in industrial processes. The ability to increase plant production, either by increasing the rate of dry matter accumulation, modifying cellulose or lignin composition, increasing stem strength, increasing meristem size, changing plant branching pattern, leaf firmness, increase in fertilization efficiency, increase in the rate of dry matter accumulation, modification of seed development, improvement of seed filling, or increasing the oil, starch or protein content of seeds would have many applications in agricultural and non-agricultural activities such as in the biotechnological production of pharmaceuticals, antibodies or vaccines.
[0012] Seed or vegetable oils are the main source of energy and nutrition in the human and animal diet. They are also used for the production of industrial products such as paints, inks and lubricants. In addition, plant oils represent renewable sources of long-chain hydrocarbons, which can be used as fuel. As currently used fossil fuels are limited sources and are gradually being depleted, fast-growing biomass crops can be used as alternative fuels or for energy raw materials and can reduce dependence on fossil energy supplies. However, the main obstacle to increasing consumption of plant oils as biofuel is the price of oil, which is still higher than fossil fuel. In addition, the plant's oil production rate is limited through the availability of agricultural land and water. In this way, the increase in plant oil yields from the same growing area can effectively overcome the scarcity in the production space and can lower vegetable oil prices at the same time.
[0013] Studies aimed at increasing plant oil production focus on the identification of genes involved in oil metabolism, as well as genes capable of increasing seed and plant production in transgenic plants. Genes known to be involved in increasing plant oil yields include those that participate in fatty acid synthesis or capture, such as desaturase [e.g., DELTA6, DELTA12, or acyl-ACP (Ssi2; Arabidopsis Information Source ( TAIR; arabidopsis (dot) org/), TAIR No. AT2G43710)], OleosinA (TAIR No. AT3G01570) or FAD3 (TAIR No. AT2G29980) and various transcription factors and activators, such as Lec1 [TAIR No. AT1G21970, Lotan et al. 1998. Cell. 26;93(7):1195-205], Lec2 [TAIR No. AT1G28300, Santos Mendoza et al. 2005, FEBS Lett. 579(21):4666-70], Fus3 (TAIR No. AT3G26790), ABI3 [TAIR No. AT3G24650, Lara et al. 2003. J Biol Chem. 278(23): 21003-11] and Wri1 [TAIR No. AT3G54320, Cernac and Benning, 2004. Plant J. 40(4): 575-85 ].
[0013] Genetic engineering efforts aimed at increasing oil content in plants (eg, in seeds) include upregulation of endoplasmic reticulum (FAD3) and plasitidal (FAD7), fatty acid desaturase in potato (Zabrouskov V ., et al., 2002; Physiol Plant. 116:172-185); overexpressing the transcription factors GmDof4 and GmDof11 ( Wang HW et al., 2007; Plant J. 52:716-29 ); overexpressing glycerol-3-phosphate dehydrogenase in yeast under the control of a plant-specific promoter ( Vigeolas H, et al. 2007, Plant Biotechnol J. 5:431-41 ; US Patent Application No. 20060168684 ); using FAE1 genes from Arabidopsis and SLC1-1 from yeast for improvements in erucic acid and oil content in rapeseed (Katavic V, et al., 2000, Biochem Soc Trans. 28:935-7).
[0014] Several patent applications reveal genes and proteins that can increase the oil content in plants. These include, for example, US Patent Application No. 20080076179 (lipid metabolism protein); US Patent Application No. 20060206961 (Ypr140w polypeptide); US Patent Application No. 20060174373 [synthesis of protein-enhancing triacylglycerols (TEP)]; US Patent Applications N° 20070169219, 20070006345, 20070006346 and 20060195943 (which disclose transgenic plants with increased nitrogen use efficiency that can be used for conversion into fuel or chemical raw materials) and WO2008/122980 (polynucleotides to increase the oil content, growth rate, biomass, production and/or vigor of a plant).
[0015] Abiotic stress conditions (ABS| Abiotic stress; also known as “environmental stress”) such as salinity, drought, flooding, underused temperature and toxic chemical pollution, cause substantial damage to agricultural plants. Most plants have developed strategies to protect themselves against these conditions. However, if the severity and duration of the stress conditions are too great, the effects on plant development, growth and production of most plant species are profound. Furthermore, most plant species are highly susceptible to abiotic stress and therefore require optimal growing conditions for commercial crop crops. Continuous exposure to stress causes important changes in plant metabolism that ultimately leads to cell death and, consequently, generates losses. Drought is a gradual phenomenon, involving abnormally dry periods of time that last long enough to produce serious hydrological imbalances, such as crop damage, lack of water supply, and increased susceptibility to various diseases. In severe cases, drought can last for many years and results in devastating effects on agriculture and water supplies. In addition, drought is associated with increased susceptibility to various diseases. [0017] For most crop plants, the growing regions of the world are very arid. In addition, excessive use of available water results in greater loss of usable agricultural land (desertification), and increased salt accumulation in soils adds to the loss of available water in soils.
[0016] Salinity, high levels of salt, affects one in five hectares of irrigated land. None of the top five food crops, i.e. wheat, corn, rice, potatoes and soybeans, can tolerate excess salt. The harmful effects of salt on plants result both from water deficiency, which leads to osmotic stress (similar to stress caused by drought), and from the effect of excess sodium ions on important biochemical processes. Like freezing and drought, high amounts of salt cause a water deficit; and the presence of high levels of salt makes it difficult for plant roots to extract water from their environment. Soil salinity, therefore, is one of the most important variables that determine whether a plant can thrive. In many parts of the world, considerable areas of land are uncultivated due to the naturally high salinity of the soil. Thus, salinization of soils that are used for agricultural production is a significant and growing problem in regions that rely heavily on agriculture and is being exacerbated by overuse, over-fertilization and water scarcity, generally caused by climate change. and the demands of population growth. Salt tolerance is of particular importance early in the plant's life cycle, as evaporation from the soil surface causes the upward movement of water and salt to accumulate in the topsoil, where the seeds are placed. On the other hand, germination normally occurs at a salt concentration that is higher than the average salt level across the entire soil profile.
[0017] Salt and drought stress signal transduction consists of ionic and osmotic homeostasis signaling pathways. The ionic aspect of salt stress is signaled via the SOS pathway, where a calcium-responsive SOS3-SOS2 protein kinase complex controls the expression and activity of ion transporters such as SOS1. The osmotic component of salt stress involves complex plant reactions that overlap with drought and/or cold stress responses.
[0018] Suboptimal temperatures affect plant growth and development throughout its life cycle. Thus, low temperatures reduce the germination rate and high temperatures result in leaf necrosis. In addition, mature plants exposed to excess heat can experience heat shock, which can arise in various organs, including leaves and especially fruits, when transpiration is insufficient to overcome heat stress. Heat also damages cellular structures, including organelles and the cytoskeleton, and impairs membrane function. Heat shock can produce a decrease in overall protein synthesis, accompanied by the expression of heat shock proteins, eg, chaperones, which are involved in the rearrangement of heat-denatured proteins. High temperature damage to pollen almost always occurs in conjunction with drought stress and rarely occurs under good irrigation conditions. The combined stress can alter the plant's metabolism in new ways. Excessive cold conditions, eg low temperatures but above freezing, affect crops of tropical origin such as soybeans, rice, corn and cotton. Typical cold damage includes wilting, necrosis, chlorosis, or loss of ions from cell membranes. The underlying mechanisms of cold sensitivity are not fully understood yet, but likely involve the level of membrane saturation and other physiological deficiencies. Excessive light conditions, which occur under bright atmospheric conditions subsequent to the cool nights of late summer/autumn, can lead to photoinhibition of photosynthesis (disruption of photosynthesis). In addition, the cold can lead to yield losses and lower product quality through late maturation of maize.
[0019] Common Aspects of Drought, Cold and Salt Stress Response [Reviewed in Xiong and Zhu (2002) Plant Cell Environ. 25:131-139] include: (a) transient changes in cytoplasmic calcium levels prior to the signaling event; (b) signal transduction through calcium dependent protein kinases (CDPKs | calcium dependent protein kinases) and/or activated by mitogen and protein phosphatases; (c) increases in abscisic acid levels in response to stress, triggering a subset of the responses; (d) inositol phosphates as signal molecules (at least for a subset of transient stress-responsive changes; (e) activation of phospholipases, which in turn generate a diverse array of second messenger molecules, some of which can regulate the activity of stress-responsive kinases; (f) the induction of late embryogenesis abundant (LEA | late embryogenesis abundant) genes, including the CR/RD CRT/DRE responsive genes; (g) increased levels of antioxidants and osmolytes such as proline and soluble sugars; and (h) the accumulation of reactive oxygen species, such as superoxide, hydrogen peroxide, and hydroxyl radicals. Abscisic acid biosynthesis is regulated by osmotic stress in several steps. Osmotic stress signaling, both ABA-dependent and independent, they first modify the constitutively expressed transcription factors, leading to the expression of transcriptional activators of previous response, which then activate downstream stress tolerance effector genes.
[0020] Several genes that increase tolerance to cold or salt stress may also improve protection from drought stress, including, for example, the transcription factor AtCBF/DREB1, OsCDPK7 (Saijo et al. 2000, Plant J. 23 : 319-327) or AVP1 (a vacuolar pyrophosphatase-proton pump, Gaxiola et al. 2001, Proc. Natl. Acad. Sci. USA 98: 11444-11449).
[0021] Studies have shown that plant adaptations to adverse environmental conditions are complex genetic traits of a polygenic nature. Conventional growing media and horticultural improvements use selective breeding techniques to identify plants that display desirable traits. However, selective breeding is tedious, time consuming and has unpredictable results. In addition, limited germplasm resources to improve yields and incompatibility in crosses between distantly related plant species represent significant problems encountered in conventional breeding. Advances in genetic engineering allowed man to modify the germplasm of plants by expressing genes of interest in plants. This technology has the ability to generate crops or plants with improved economic, agronomic or horticultural traits.
[0022] Genetic engineering efforts focused on imparting abiotic stress tolerance to transgenic crops have been described in several publications [Apse and Blumwald (Curr Opin Biotechnol. 13:146-150, 2002), Quesada et al. (Plant Physiol. 130:951-963, 2002), Holmstrom et al. (Nature 379: 683-684, 1996), Xu et al. (Plant Physiol 110: 249-257, 1996), Pilon-Smits and Ebskamp (Plant Physiol 107: 125-130, 1995) and Tarczynski et al. (Science 259: 508-510, 1993)].
[0023] Several patents and patent applications disclose the genes and proteins that can be used to increase the tolerance of plants to abiotic stress. These include, for example, US Patent Nos. 5,296,462 and 5,356,816 (to increase tolerance to cold stress); U.S. Patent No. 6,670,528 (to increase ABST); US Patent No. 6,720,477 (to increase ABST); US Patent Application Serial Nos. 09/938842 and 10/342224 (to increase ABST); US Patent Application Serial No. 10/231035 (to increase ABST); WO2004/104162 (to increase ABST and biomass); WO2007/020638 (to increase ABST, biomass, vigor/or production); WO2007/049275 (to increase ABST, biomass, vigor/or production); WO2010/076756 (to increase ABST, biomass and/or production); WO2009/083958(to increase water use efficiency, fertilizer use efficiency, biotic-abiotic stress tolerance, production and/or biomass); WO2010/020941 (to increase nitrogen use efficiency, tolerance to abiotic stress, production and/or biomass); WO2009/141824 (to increase the usefulness of the plant); WO2010/049897 (to increase plant production).
[0024] Nutrient deficiency causes adaptations of root architecture, particularly notable, for example, is root proliferation within nutrient-rich areas to enhance nutrient uptake. Nutrient deficiency also causes the activation of plant metabolic pathways that maximize absorption, assimilation and distribution processes as well as by activating architectural changes. Engineering the expression of triggered genes can make the plant show changes in its architecture and improved metabolism, too, under other conditions.
[0025] Furthermore, it is widely known that plants generally respond to water deficiency by creating a system of deeper roots that allow access to moisture located in deeper layers of the soil. Triggering this effect will allow plants to access nutrients and water located in deeper soil horizons, particularly those readily dissolved in water, such as nitrates.
[0026] Cotton and cotton by-products provide raw materials that are used to produce a wide variety of consumer-based products in addition to textiles, including cotton foodstuffs, animal feed, fertilizer and paper. The production, marketing, consumption and trade of cotton-based products generate an excess of $100 billion annually in the US alone, making cotton the number one crop in terms of added value.
[0027] Although 90% of the value of cotton as a crop resides in the fiber (lint), production and fiber quality has declined due to general erosion in the genetic diversity of cotton varieties and an increased vulnerability of the crop to environmental conditions.
[0028] There are many varieties of cotton plants, from which cotton fibers with a range of characteristics can be obtained and used for various applications. Cotton fibers can be characterized according to a variety of properties, some of which are considered highly desirable within the textile industry for the production of increasingly higher quality products and optimal exploitation of modern spinning technologies. Commercially desirable properties include length, length uniformity, fineness, maturity ratio, reduced fiber yield, micron, bundle strength, and single fiber strength. Much effort has been put into improving the characteristics of cotton fibers, mainly focusing on fiber length and fiber fineness. In particular, there is a great demand for cotton fibers of specific lengths.
[0029] A cotton fiber is composed of a single cell that has differentiated from an epidermal cell covering the seed, developing through four stages, namely initiation, elongation, stages of thickening and secondary cell wall maturation. More specifically, the elongation of a cotton fiber begins in the epidermal cell of the ovule immediately after flowering, after which the cotton fiber rapidly elongates for about 21 days. The elongation of the fiber is then completed, and a secondary cell wall is formed and grown through maturation, becoming a mature cotton fiber.
[0030] Several candidate genes that are associated with the elongation, formation, quality and production of cotton fibers have been disclosed in various patent applications, such as US Patent No. 5,880,100 and Serial No. US Patent Applications 08/580,545, 08/867,484 and 09/262,653 (which describe genes involved in the elongation phase of cotton fiber); WO0245485 (which improve fiber quality by modulating sucrose synthase); U.S. Patent Nos. 6,472,588 and WO0117333 (which enhance fiber quality by transformation with DNA sucrose phosphate-encoding synthase); WO9508914 (which uses a fiber-specific promoter and a coding sequence encoding cotton peroxidase); WO9626639 (which uses an ovary-specific promotion sequence to express plant growth, modifying hormones in cotton ovum tissue, altering fiber quality characteristics such as fiber dimension and strength); U.S. Patent No. 5,981,834, U.S. Patent No. 5,597,718, U.S. Patent No. 5,620,882, U.S. Patent No. 5,521,708 and U.S. Patent No. 5,495,070 ( coding sequences to alter the characteristics of plants that produce transgenic fibers); US Patent Application No. 2002049999 and US Patent No. 2003074697 (which express a genetic code for endoxyloglucan transferase, catalase or peroxidase to improve cotton fiber characteristics); WO 01/40250 (which improves cotton fiber quality by modulating transcription factor gene expression); WO 96/40924 (an associated cotton fiber transcriptional initiation regulatory region, which is expressed in cotton fiber); EP0834566 (a gene which controls the fiber formation mechanism in the cotton plant); WO2005/121364 (which improves cotton fiber quality by modulating gene expression); WO2008/075364 (which improves quality tolerance, production/biomass/vigor and/or abiotic stress tolerance of plant fiber).
[0031] WO Publication No. 2004/104162 discloses methods for increasing tolerance to abiotic stress and/or biomass in plants generated in this way.
[0032] WO Publication No. 2004/111183 discloses nucleotide sequences for regulating gene expression in trichomes and plant structures and methods using the same.
[0033] WO Publication No. 2004/081173 discloses novel plant-derived regulatory sequences and structures and methods of using such sequences to direct the expression of exogenous polynucleotide sequences in plants.
[0034] WO Publication No. 2005/121364 discloses polynucleotides and polypeptides involved in the development of plant fibers and method of use thereof, in order to increase fiber quality, production and/or biomass of a fiber-producing plant.
[0035] WO Publication No. 2007/049275 discloses isolated polypeptides, polynucleotides that encode the same, transgenic plants that express the same and method of using the same to increase the efficiency in the use of fertilizers, the tolerance to abiotic stress of the plant and biomass.
[0036] WO Publication No. 2007/020638 discloses methods for increasing tolerance to abiotic stress and/or biomass in plants and plants thus generated.
[0037] WO Publication No. 2008/122980 discloses gene structures and methods for increasing the oil content, growth rate and biomass of plants.
[0038] WO Publication No. 2008/075364 discloses polynucleotides involved in plant fiber development and methods of using them.
[0039] WO Publication No. 2009/083958 discloses methods for increasing water use efficiency, fertilizer use efficiency, tolerance to biotic/abiotic stress, yield and biomass in plants and in plants generated thereby.
[0040] WO Publication No. 2009/141824 discloses isolated polynucleotides and methods for using the same to increase plant utility.
[0041] WO Publication No. 2009/013750 discloses genes, structures and methods for increasing tolerance to abiotic stress, biomass and/or production in plants thus generated.
[0042] WO Publication No. 2010/020941 discloses methods for increasing nitrogen use efficiency, abiotic stress tolerance, production and biomass in plants and in plants generated therefrom.
[0043] WO Publication No. 2010/076756 discloses isolated polynucleotides to increase abiotic stress tolerance, production, biomass, growth rate, vigor, oil content, fiber production, fiber quality and/or efficiency in the use of nitrogen from a plant.
[0044] Publication WO2010/100595 discloses isolated polynucleotides and polypeptides and methods of using the same to increase plant production and/or agricultural traits.
[0045] WO Publication No. 2010/049897 discloses isolated polynucleotides and polypeptides and methods of using them to increase plant production, biomass, growth rate, vigor, oil content, plant abiotic stress tolerance and plant efficiency. nitrogen use.
[0046] Publication WO2010/143138 discloses isolated polynucleotides and polypeptides and methods of using them to increase nitrogen use efficiency, fertilizer use efficiency, production, growth rate, vigor, biomass, oil content, tolerance to abiotic stress and/or water use efficiency.
[0047] WO Publication No. 2011/080674 discloses isolated polynucleotides and polypeptides and methods of using them to increase plant production, biomass, growth rate, vigor, oil content, plant abiotic stress tolerance and plant efficiency. nitrogen use.
[0048] Publication WO2011/015985 discloses polynucleotides and polypeptides for enhancing desirable plant qualities.
[0049] Publication WO2011/135527 discloses isolated polynucleotides and polypeptides for increasing plant production and/or agricultural traits.
[0050] Publication WO2012/028993 discloses isolated polynucleotides and polypeptides and methods of using them to increase nitrogen use efficiency, production, growth rate, vigor, biomass, oil content and/or abiotic stress tolerance.
[0051] Publication WO2012/085862 discloses isolated polynucleotides and polypeptides and methods of using the same to improve plant properties.
[0052] Publication WO2012/150598 discloses isolated polynucleotides and polypeptides and methods of using them to increase plant production, biomass, growth rate, vigor, oil content, plant abiotic stress tolerance and nitrogen use efficiency .
[0053] Publication WO2013/027223 discloses isolated polynucleotides and polypeptides and methods of using the same to increase plant production and/or agricultural traits.
[0054] Publication WO2013/080203 discloses isolated polynucleotides and polypeptides and methods of using them to increase nitrogen use efficiency, production, growth rate, vigor, biomass, oil content and/or abiotic stress tolerance.
[0055] Publication WO2013/098819 discloses isolated polynucleotides and polypeptides and methods of using the same to increase plant production.
[0056] Publication WO2013/128448 discloses isolated polynucleotides and polypeptides and methods of using them to increase plant production, biomass, growth rate, vigor, oil content, plant abiotic stress tolerance and nitrogen use efficiency . SUMMARY OF THE INVENTION
[0057] According to one aspect of some applications of the present invention, there is provided a method for increasing nitrogen use efficiency, production, growth rate, biomass, vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide at least 80% identical to the SEQ ID. No. [Sequence Identification No.]: 496-794, 2898-3645, 3647-4854 or 4855, thus increasing nitrogen use efficiency, production, growth rate, biomass, vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of the plant.
[0058] According to an aspect of some applications of the present invention, there is provided a method for increasing nitrogen use efficiency, production, growth rate, biomass, vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide selected from the group consisting of SEQ IDs. N° 496-794, 2898-4854 and 4855, thus increasing the efficiency in the use of nitrogen, production, growth rate, biomass, vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of the plant.
[0059] In accordance with one aspect of some applications of the present invention, there is provided a method of producing a crop, comprising culturing a crop plant transformed with an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide of at least , 80% homologous to the amino acid sequence selected from a group consisting of SEQ IDs. N° 496-794, 2898-3645, 3647-4854 and 4855, characterized by the harvest plant being derived from plants selected to increase nitrogen use efficiency, increase production, increase growth rate, increase biomass, increase vigor, increase in oil content, increase in seed production, increase in fiber production, increase in fiber quality, increase in fiber length, increase in photosynthetic capacity and/or increase in tolerance to abiotic stress compared to a wild-type plant of the same species that was grown under the same growing conditions, and the crop plant having increased nitrogen use efficiency, increased yield, increased growth rate, increased biomass, increased vigor, increased of oil content, increase in seed production, increase in fiber production, increase in fiber quality, increase in fiber length, increase in photosynthetic capacity and/or increase in stress tolerance to biotic, thus producing the harvest.
[0060] According to an aspect of some applications of the present invention, there is provided a method for increasing nitrogen use efficiency, production, growth rate, biomass, vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence at least 80% identical to the SEQ ID. No. 1-495, 795-2896 or 2897, thereby increasing nitrogen use efficiency, production, growth rate, biomass, vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of the plant.
[0061] According to an aspect of some applications of the present invention, there is provided a method for increasing nitrogen use efficiency, production, growth rate, biomass, vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ IDs. N° 1-495, 795-2896 and 2897, thus increasing nitrogen use efficiency, production, growth rate, biomass, vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of the plant.
[0062] In accordance with one aspect of some applications of the present invention, there is provided a method of producing a crop, comprising culturing a crop plant transformed with an exogenous polynucleotide that comprises a nucleic acid sequence which is, at least, least 80% identical to the nucleic acid sequence selected from the group consisting of SEQ IDs. N° 1-495, 795-2896 and 2897, characterized in that the crop plant is derived from plants (parent plants) selected to increase nitrogen use efficiency, increase production, increase growth rate, increase biomass, increase vigor, increase in oil content, increase in seed production, increase in fiber production, increase in fiber quality, increase in fiber length, increase in photosynthetic capacity and/or increase in tolerance to abiotic stress compared to a wild-type plant of the same species that was grown under the same growing conditions, and the crop plant having increased nitrogen use efficiency, increased yield, increased growth rate, increased biomass, increased vigor, increased of oil content, increase in seed production, increase in fiber production, increase in fiber quality, increase in fiber length, increase in photosynthetic capacity and/or increase in tolerance to and abiotic stress, thus producing the crop.
[0063] In accordance with one aspect of some applications of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide, which comprises an amino acid sequence at least 80% homologous to the established amino acid sequence. in the SEQ IDs. N° 496-794, 2898-3645, 3647-4854 or 4855, characterized by the amino acid sequence being able to increase nitrogen use efficiency, production, growth rate, biomass, vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of a plant.
[0064] In accordance with one aspect of some applications of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence encoding a polypeptide, which comprises the amino acid sequence selected from the group consisting of SEQ IDs. Nos. 496-794, 2898-4854 and 4855.
[0065] In accordance with one aspect of some applications of the present invention, there is provided an isolated polynucleotide comprising a nucleic acid sequence at least 80% identical to the SEQ ID. No. 1-495, 795-2896 or 2897, characterized by the nucleic acid sequence being able to increase nitrogen use efficiency, production, growth rate, biomass, vigor, oil content, seed production, fiber production , fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of a plant.
[0066] In accordance with one aspect of some applications of the present invention, there is provided an isolated polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ IDs. No. 1495, 795-2896 and 2897.
[0067] In accordance with one aspect of some applications of the present invention, there is provided a nucleic acid backbone comprising the isolated polynucleotide of some applications of the invention and a promoter for directing transcription of the nucleic acid sequence in a host cell.
[0068] In accordance with one aspect of some applications of the present invention, there is provided an isolated polypeptide comprising an amino acid sequence at least 80% homologous to the SEQ ID. N° 496-794, 2898-3645, 3647-4854 or 4855, characterized by the amino acid sequence being able to increase nitrogen use efficiency, production, growth rate, biomass, vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of a plant.
[0069] In accordance with one aspect of some applications of the present invention, there is provided an isolated polypeptide comprising the amino acid sequence selected from the group consisting of SEQ IDs. Nos. 496-794, 2898-4854 and 4855.
[0070] In accordance with one aspect of some applications of the present invention, there is provided a plant cell that exogenously expresses the polynucleotide of some applications of the invention or the nucleic acid structure of some applications of the invention.
[0071] In accordance with one aspect of some applications of the present invention, there is provided a plant cell that exogenously expresses the polypeptide of some applications of the invention.
[0072] According to one aspect of some applications of the present invention, there is provided a transgenic plant, comprising the nucleic acid structure of some applications of the invention, or the plant cell of some applications of the invention.
[0073] According to one aspect of some applications of the present invention, there is provided a method of growing a crop, the method comprising sowing seeds and/or planting seedlings of a transformed plant with the polynucleotide isolated from some applications of the invention or the nucleic acid structure of some applications of the invention, characterized by the plant being derived from selected plants with at least one trait selected from the group consisting of increased nitrogen use efficiency, increased tolerance to abiotic stress, increased biomass, increased rate growth rate, vigor increase, production increase and fiber production increase, fiber quality increase, fiber length increase, photosynthetic capacity increase and oil content increase compared to an unprocessed plant, thereby increasing the harvest.
[0074] In accordance with an aspect of some applications of the present invention, there is provided a method of selecting a transformed plant having increased nitrogen use efficiency, production, growth rate, biomass, vigor, oil content, production of seed, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance as compared to a wild type plant of the same species grown under the same growing conditions, the method comprising: (a) providing plants transformed with an exogenous polynucleotide encoding a polypeptide comprising an amino acid sequence at least 80% homologous to the amino acid sequence selected from the group consisting of SEQ IDs. N° 496-794, 2898-3645, 3647-4854 and 4855,(b) select, from the plants, a plant having increased efficiency in the use of nitrogen, production, growth rate, biomass, vigor, oil content , seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance, thus selecting the plant with increased nitrogen use efficiency, production, growth rate, biomass , vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance compared to a wild type plant of the same species that is grown under the same conditions of cultivation.
[0075] According to an aspect of some applications of the present invention, a method of selecting a transformed plant is provided, having increased efficiency in nitrogen use, production, growth rate, biomass, vigor, oil content, production seed yield, fiber yield, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance as compared to a wild-type plant of the same species grown under the same growing conditions, the method comprising: (a) provide plants transformed with an exogenous polynucleotide encoding a polypeptide comprising an amino acid sequence at least 80% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NOS: 1-495, 795-2896 and 2897,(b) select, from the plants, a plant having increased efficiency in the use of nitrogen, production, growth rate, biomass, vigor, oil content, seed production, fiber production, quality of fiber, fiber length, photosynthetic capacity and/or abiotic stress tolerance, thus selecting the plant with increased nitrogen use efficiency, production, growth rate, biomass, vigor, oil content, seed production , fiber yield, fiber quality, fiber length, photosynthetic capacity, and/or abiotic stress tolerance compared to a wild-type plant of the same species that is grown under the same growing conditions.
[0076] In accordance with some applications of the invention, the nucleic acid sequence encodes an amino acid sequence from a group consisting of SEQ IDs. Nos. 496-794, 2898-4854 and 4855.
[0077] In accordance with some applications of the invention, the nucleic acid sequence is selected from the group consisting of SEQ IDs. No. 1495, 795-2896 and 2897.
[0078] According to some applications of the invention, the polynucleotide consists of the nucleic acid sequence selected from the group consisting of SEQ IDs. No. 1-495, 795-2896 and 2897.
[0079] In accordance with some applications of the invention, the nucleic acid sequence encodes the amino acid sequence selected from a group consisting of SEQ IDs. Nos. 496-794, 2898-4854 and 4855.
[0080] According to some applications of the invention, the host cell is a plant cell.
[0081] According to some applications of the invention, the plant cell forms part of a plant.
[0082] According to some applications of the invention, the method further comprises the cultivation of the plant that expresses the exogenous polynucleotide under abiotic stress.
[0083] According to some applications of the invention, abiotic stress is selected from a group consisting of desalinity, drought, osmotic stress, water deprivation, flooding, etiolation, low temperature, high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency, nitrogen deficiency, nutrient excess, air pollution and UV irradiation.
[0084] According to some applications of the invention, production comprises seed production or oil production.
[0085] According to some applications of the invention, the method further comprises the cultivation of the plant that expresses the exogenous polynucleotide under nitrogen-limiting conditions.
[0086] According to some applications of the invention, the promoter is heterologous to the isolated polynucleotide and/or to the host cell.
[0087] According to some applications of the invention, the isolated polynucleotide is heterologous to the plant cell.
[0088] According to some applications of the invention, the untransformed plant is a wild-type plant of identical genetic base.
[0089] According to some applications of the invention, the untransformed plant is a wild-type plant of the same species.
[0090] According to some applications of the invention, the untransformed plant is grown under identical growing conditions.
[0091] According to some applications of the invention, the method further comprises selecting a plant having increased efficiency in the use of nitrogen, production, growth rate, biomass, vigor, oil content, seed production, fiber production , fiber quality, fiber length, photosynthetic capacity, and/or abiotic stress tolerance compared to a wild-type plant of the same species that is grown under the same growing conditions.
[0092] Unless defined otherwise, all technical and/or scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which the invention belongs. While similar or equivalent methods and materials to those described herein may be used in the practice or testing of applications of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent's claim framework, including definitions, will prevail. Furthermore, the materials, methods and examples are illustrative only and are not intended to be necessarily limiting. BRIEF DESCRIPTION OF THE DRAWINGS
[0093] Some applications of the invention are described here, by way of example only, with reference to the accompanying drawings. Now, with specific reference to the drawings in detail, it is emphasized that the features shown are exemplary and for the purposes of illustrative discussion of applications of the invention. In this regard, the description accompanying the drawings makes it apparent to those skilled in the art how applications of the invention can be practiced. In the drawings: Figure 1 is a schematic illustration of a modified pGI binary plasmid containing the novel At6669 promoter (SEQ ID NO: 4880) and the GUSintron (pQYN_6669) used to express the isolated polynucleotide sequences of the invention. RB - right edge of T-DNA; LB - left edge of T-DNA; MCS - Multiple Cloning Site; RE - any restriction enzyme; NOS pro = nopaline synthase promoter; NPT-II = neomycin phosphotransferase gene; NOS ter = nopaline synthase terminator; Poly-A signal (polyadenylation signal); GUSintron - the GUS reporter gene (coding sequence and intron). The isolated polynucleotide sequences of the invention were cloned into the vector while replacing the GUSintron reporter gene.
[0094] Figure 2 is a schematic illustration of the modified binary plasmid pGI containing the novel At6669 promoter (SEQ ID NO: 4880) (pQFN or pQFNc) used to express the isolated polynucleotide sequences of the invention. RB [right border] - right border of the T-DNA; LB [left border] - left border of the T-DNA; MCS [multiple cloning site]- Multiple cloning site; RE [restriction enzyme] - any restriction enzyme; NOS pro [nopaline synthase promoter] = nopaline synthase promoter; NPT-II [neomycin phosphotransferase] = neomycin phosphotransferase gene; NOS ter [nopalinha synthase terminator] = nopaline synthase terminator; Poly-A signal (polyadenylation signal). The isolated polynucleotide sequences of the invention were cloned into the MCS of the vector.
[0095] Figures 3A-F are images that describe the visualization of the root development of transgenic plants exogenously expressing the polynucleotide of some applications of the invention when grown on clear agar plates under normal conditions (Figures 3A-B), stress osmotic (15% PEG. Figs. 3C-D) or nitrogen limitation (Figures 3E-F). The different transgenes were grown on clear agar plates for 17 days (7 days in the nursery and 10 days after transplantation). Plates were photographed every 3-4 days starting on day 1 after transplantation Fig. 3A - An image of a photograph of plants taken 10 days after transplanting onto agar plates when grown under normal (standard) conditions. Fig. 3B - An image of the root analysis of the plants shown in Fig. 3A in which the measured root lengths are represented by arrows. Fig. 3C - An image of a photograph of plants taken 10 days after transplantation on Agar plates, grown under highly osmotic conditions (15% PEG). Fig. 3D - A root analysis image of the plants shown in Fig.3C in which the measured root lengths are represented by arrows. Fig. 3E - An image of a photograph of plants taken 10 days after transplantation on agar plates, grown under low nitrogen conditions. Fig. 3F - An image of the root analysis of the plants shown in Fig. 3E in which the measured root lengths are represented by arrows.
[0096] Figure 4 is a schematic illustration of the modified pGI binary plasmid containing the Root Promoter (pQNa RP) used to express the isolated polynucleotide sequences of the invention. RB - right edge of T-DNA; LB - left edge of T-DNA; NOS pro = nopaline synthase promoter; NPT-II = neomycin phosphotransferase gene; NOS ter = nopaline synthase terminator; Poly-A signal (polyadenylation signal; sequences of the isolated polynucleotide, in accordance with some applications of the present invention, were cloned into the MCS [Multiple Cloning Site] of the vector.
[0097] Figure 5 is a schematic illustration of plasmid pQYN.
[0098] Figure 6 is a schematic illustration of plasmid pQFN.
[0099] Figure 7 is a schematic illustration of plasmid pQFYN.
[00100] Figure 8 is a schematic illustration of the modified binary plasmid pGI (pQXNc) used to express the isolated polynucleotide sequences of some applications of the invention. RB - T-DNA right edge; LB - T-DNA left edge; NOS pro = nopaline synthase promoter; NPT-II = neomycin phosphotransferase gene; NOS ter = nopaline synthase terminator; RE = any restriction enzyme; Poly-A signal (polyadenylation signal); 35S
[00101] - the 35S promoter (pqfnc; SEQ ID NO: 4876). Polynucleotide sequences isolated from some applications of the invention were cloned into the MCS (multiple cloning site) of the vector.
[00102] Figures 9A-B are schematic illustrations of the plasmids pEBbVNi tDNA (Figure 9A) and pEBbNi tDNA (Figure 9B), used in the experiments with Brachypodium. The pEBbVNi tDNA (Figure 9A) was used for expression of polynucleotide sequences isolated from some applications of the invention in Brachypodium. pEBbNi tDNA (Figure 9B) was used for transformation into Brachypodium as a negative control. “RB [right border]” = right border; “2LBregion” = 2 repetitions of left edge; "35S" = 35S promoter (SEQ ID NO: 4892); “NOS ter” = nopaline synthase terminator; "Bar ORF" - BAR opening reading frame (GenBank Accession No. JQ293091.1; SEQ ID No.: 5436); The polynucleotide sequence isolated from the same applications of the invention was cloned into the Multiple Cloning site of the vector using one or more of the indicated restriction enzyme site(s). DESCRIPTION OF SPECIFIC APPLICATIONS OF THE INVENTION
[00103] The present inventors have identified new polypeptides and polynucleotides that can be used to generate nucleic acid structures, transgenic plants and increase nitrogen use efficiency, fertilizer use efficiency, production, growth rate, vigor, biomass, of oil, fiber production, fiber quality, fiber length, photosynthetic capacity, abiotic stress tolerance and/or a plant's water use efficiency.
[00104] Thus, as shown in the Examples section below, the present inventors use bioinformatics tools to identify polynucleotides that improve/increase fertilizer use efficiency (eg, nitrogen use efficiency), production (p. .eg seed production, oil production, oil content), growth rate, biomass, vigor, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of a plant. Genes that affect the trait of interest were identified (SEQ ID NO: 496-794 for polypeptides; and SEQ ID NO: 1-495 for polynucleotides), based on the expression profiles of gens from various ecotypes of Arabidopsis, Barley , Sorghum, Corn, Tomato and Millet, and accessions in various tissues and culture conditions, homology with genes known to affect the trait of interest and use the digital expression profile in specific tissues and conditions (Tables 1 and 3-99 , Examples 1 and 3-11 of the Examples section below). Homologous (eg, orthologs) polypeptides and polynucleotides with the same function have also been identified [SEQ ID. No. 2898-4855 (for polypeptides) and SEQ ID. No. 795-2897 (for polynucleotides); Table 2, Example 2 of the Examples section that follows]. Polynucleotides from some applications of the invention were cloned into binary vectors (Example 12, Table 100) and were further transformed into Arabidopsis and Brachypodium plants (Examples 13-15). Transgenic plants that overexpress the identified polynucleotides were found to exhibit increased biomass, growth rate, vigor, and yield under normal growing conditions or under nitrogen-limiting growing conditions (Tables 101-128; Examples 16- 20) and increased tolerance under abiotic stress conditions (eg, nutrient deficiency) compared to controlled plant growth under the same growing conditions. Taken together, these results suggest the use of the novel polynucleotides and polypeptides of the present invention (e.g., SEQ ID NO: 496-794 and 2898-4855 and SEQ ID NO: 1-495 and 795-2897) to increase nitrogen use efficiency, fertilizer use efficiency, yield (eg oil yield, seed yield and oil content), growth rate, biomass, vigor, fiber yield, fiber quality, length fiber, photosynthetic capacity, water use efficiency and/or abiotic stress tolerance of a plant.
[00105] Thereby, in accordance with an aspect of some applications of the present invention, there is provided a method for increasing fertilizer use efficiency (e.g. nitrogen use efficiency), oil content, production, rate of growth, biomass, vigor, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of a plant, comprising expressing within the plant an exogenous polynucleotide, comprising a nucleic acid sequence encoding a polypeptide, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86% , at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or, say, 100% homologous to the amino acid sequence selected from the group consisting of SEQ IDs. N° 496-794, 2898-3645, 3647-4854 and 4855, thereby increasing fertilizer use efficiency (eg nitrogen use efficiency), oil content, production, growth rate, biomass, vigor, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of the plant.
[00106] As used herein, the term "plant yield" refers to the amount (eg, as determined by weight or size) or quantity (numbers) of tissues or organs produced by plants or by growing season. Therefore, increasing seed production per plant can affect the economic benefit that one can derive from the plant in a given growing area and/or growing season.
[00107] It is important to note that plant production can be affected by several parameters, including but not limited to plant biomass; plant vigor; growth rate; seed production; quantity of seeds or grains; seed or grain quality; oil production; oil, starch and/or protein content in harvested organs (eg, seeds or plant parts of the plant); number of flowers (flores) per panicle (expressed as a proportion of the number of filled seeds over the number of primary panicles); harvest index; number of plants cultivated per area; number and size of organs harvested per plant and per area; number of plants per growing area (density); number of organs harvested in the field; total area of the sheet; carbon assimilation and carbon partitioning (the distribution/allocation of carbon within the plant); shade resistance; number of harvestable organs (eg, seeds), seeds per pod, weight per seed; and modified architecture [such as increased stem diameter, thickness, or improved physical properties (eg, elasticity)].
[00108] As used herein, the term "seed production" refers to the number or weight of seeds per plant, seeds per pod or area of cultivation, or the weight of a single seed, or oil extracted per seed. Therefore, seed production can be affected by seed dimensions (eg, length, width, perimeter, area and/or volume), number of seeds (full) and seed filling rate and seed content. seed oil. Therefore, the increase in seed production per plant can affect the economic benefit one can derive from the plant in a given growing area and/or growing season; and increasing seed production per crop area can be achieved by increasing seed production per plant, and/or increasing the number of plants grown in a given area.
[00109] The term “seed” (also referred to as “grain” or “core”), as used herein, refers to a small embryonic plant confined in a covering called a seed coat (usually with some stored food), the product of the matured ovum of gymnosperm and angiosperm plants that occurs after fertilization and some growth within the mother plant.
[00110] The term “oil content”, as used herein, refers to the amount of lipids in a particular plant organ, whether the seeds (seed oil content) or the vegetable portion of the plant (vegetable oil content). ) and is expressed as a percentage of dry weight (10% seed moisture) or wet weight (for the vegetable portion).
[00111] It should be noted that the oil content is affected by the intrinsic oil production of a tissue (e.g. seed, plant part) as well as the mass or size of the oil producing tissue per plant or per plant. growth period.
[00112] In one application, increasing the oil content of the plant can be achieved by increasing the size/mass of tissue(s) of a plant, which comprises oil per growing season. In this way, increasing the oil content of a plant can be achieved by increasing production, growth rate, biomass and plant vigor.
[00113] As used herein, the term “plant biomass” refers to the amount (e.g., measured in grams of air-dried tissue) of tissue produced from the plant in a period of cultivation, which also may determine or affect plant yield or yield per growing area. An increase in plant biomass can occur in the whole plant or in parts of it such as above-ground (harvestable) parts, plant biomass, roots and seeds.
[00114] As used herein, the term "growth rate" refers to the increase in plant organ/tissue size per period (can be measured in cm2 per day or cm/day).
[00115] As used here, the term “photosynthetic capacity” (also known as “Amax”) is a measure of the maximum rate at which leaves are able to fix carbon during photosynthesis. It is typically measured as the amount of carbon dioxide that is fixed per square meter per second, for example, as μmol m-2 sec-1. Plants are able to increase their photosynthetic capacity by various modes of action, such as through increase in total leaf area (eg, by increasing leaf area, increasing number of leaves, and increasing plant vigor, e.g., ability of the plant to produce new leaves over the course of time) , as well as by increasing the plant's ability to efficiently carry out carbon fixation in the leaves. Thus, the increase in total leaf area can be used as a reliable measurement parameter for increasing photosynthetic capacity.
[00116] As used herein, the term “plant vigor” refers to the amount (measured by weight) or tissue produced by the plant in a given period. Therefore, the increase in vigor can determine or affect plant production or production per growing season or growing area. In addition, early vigor (seed and/or seedling) results in better position in the field.
[00117] Improving early vigor is an important objective of modern rice breeding programs in temperate and tropical rice cultivars. Long roots are important for proper soil fixation in pre-sprouted rice. When rice is sown directly in flooded fields and where plants must quickly emerge through the water, longer shoots are associated with vigor. Where drill seeding is practised, longer mesocotyls and coleoptiles are important for good seedling emergence. The ability to project early vigor into plants would be of great importance in agriculture. For example, low early vigor has been a limitation in the introduction of maize (Zea mays L.) hybrid based on the Corn Belt germplasm in the European Atlantic.
[00118] It should be noted that a plant yield can be determined under stress (eg, abiotic stress, nitrogen limiting conditions) and/or non-stress (normal) conditions.
[00119] As used here, the term “non-stress conditions” refers to growing conditions (e.g. water, temperature, light-dark cycles, humidity, salt concentration, soil fertilizer concentration, supply of nutrient such as nitrogen, phosphorus and/or potassium), which do not significantly go beyond the daily climatic and other abiotic conditions that plants may encounter and which allow for optimal growth, metabolism, reproduction and/or viability of a plant at any stage in its life cycle (eg, in a crop plant from seed to mature plant and back to seed again). Those skilled in the art are aware of normal soil and climatic conditions for a given plant in a given geographic location. It should be noted that while non-stress conditions may include some mild variations from ideal conditions (which vary from one plant type/species to another), such variations do not cause the plant to cease growth without the ability to resume growth. growth.
[00120] The term “abiotic stress”, as used herein, refers to any adverse effect on the metabolism, growth, reproduction and/or viability of the plant. Consequently, abiotic stress can be induced by suboptimal environmental growing conditions such as salinity, osmotic stress, water deprivation, drought, flooding, freezing, low or high temperature, heavy metal toxicity, anaerobiosis, nutrients (eg, nitrogen deficiency or limited nitrogen), air pollution or UV irradiation. The implications of abiotic stress are discussed in the History section.
[00121] The term “abiotic stress tolerance”, as used herein, refers to the ability of a plant to withstand an abiotic stress without undergoing a substantial change in metabolism, growth, productivity and/or viability.
[00122] Plants are subjected to a range of environmental challenges. Several of these challenges, including salt stress, general osmotic stress, aridity stress and freeze stress, have the ability to impact whole plant and cellular water availability. Not surprisingly, then, the plant's responses to this collection of stresses are related. Zhu (2002) Ann. Rev. Biol plant. 53: 247-273 et al. Note that "most studies on water stress signaling have focused on saline stress primarily because plant responses to salt and drought are closely related and the mechanisms overlap." Many examples of similar responses and pathways to this set of stresses have been documented. For example, CBF transcription factors have demonstrated salt, freeze, and drought resistance conditioning (Kasuga et al. (1999) Nature Biotech. 17: 287-291). The Arabidopsis rd29B gene is induced in response to salt stress and dehydration, a process that is mediated largely through an ABA signal transduction process (Uno et al. (2000) Proc. Natl. Acad. Sci. USA 97: 11632 -11637), resulting in altered activity of transcription factors that bind to an upstream element within the rd29B promoter. In Mesembryanthemum crystallinum (ice plant), Patharker and Cushman showed that a calcium-dependent protein kinase (McCDPK1) is induced by exposure to drought and salt stresses (Patharker and Cushman (2000) Plant J. 24: 679-691). Stress-induced kinase has also been shown to phosphorylate a transcription factor, likely altering its activity, although transcriptional levels of the target transcription factor are not altered in response to salt or drought stress. Similarly, Saijo et al. demonstrated that a rice drought/salt-induced calmodulin-dependent protein kinase (OsCDPK7) conferred high salt and drought tolerance for rice when overexpressed (Saijo et al. (2000) Planta J. 23: 319-327).
[00123] Exposure to dehydration evokes similar survival strategies in plants, as does frost stress (see, e.g., Yelenosky (1989) Planta Physiol 89: 444-451) and drought stress induces frost tolerance (see, e.g., Siminovich et al. (1982) Planta Physiol 69: 250-255; and Guy et al. (1992) Plant 188: 265-270 ). In addition to inducing cold acclimatization proteins, strategies that allow plants to survive in low water conditions may include, eg, reduced surface area, or production of surface oil or wax. In another example, the plant's high solute content prevents evaporation and water loss due to heat, drought, salinity, osmotic and the like, thus providing a better tolerance to the above stresses.
[00124] It will be understood that some pathways involved in resistance to one stress (as described above) will also be involved in resistance to other stresses, regulated by the same genes or homologues. Of course, resistance pathways are related, not identical, and therefore not all genes that control resistance to one stress will control resistance to other stresses. Nevertheless, if a gene conditions resistance to one of these stresses, it would be evident to the person skilled in the art to test for resistance to these related stresses. Stress resistance assessment methods are also provided in the Examples section below.
[00125] As used here, the expression “WUE|water use efficiency” refers to the level of organic matter produced per unit of water consumed by the plant, that is, the dry weight of a plant in in relation to plant water use, eg biomass produced by unit transpiration.
[00126] As used here, the term “fertilizer use efficiency” refers to the metabolic process(es) that lead to an increase in plant production, biomass, vigor and rate of growth per unit of fertilizer applied. The metabolic process can be the uptake, propagation, absorption, accumulation, reallocation (within the plant) and utilization of one or more minerals and organic portions absorbed by the plant, such as nitrogen, phosphates and/or potassium.
[00127] As used herein, the term "fertilizer limiting conditions" refers to growing conditions that include a level (eg, concentration) of an applied fertilizer that is below the level required for normal plant metabolism. , growth, reproduction and/or viability.
[00128] As used here, the term “nitrogen use efficiency (NUE)” refers to the metabolic process(es) that lead to an increase in plant production, biomass, vigor and growth rate per unit of nitrogen applied. The metabolic process can be the uptake, propagation, absorption, accumulation, reallocation (within the plant) and utilization of nitrogen taken up by the plant.
[00129] As used herein, the term “nitrogen limiting conditions” refers to growing conditions that include a level (eg, concentration) of applied nitrogen (ie, ammonia or nitrate) that is below the level necessary for normal plant metabolism, growth, reproduction and/or viability.
[00130] Improved plant NUE and FUE are translated in the field into similar amounts of crop yield while implementing less fertilizer, or improved yields gained by implementing the same fertilizer levels. Thus, improved NUE or FUE has a direct effect on plant production in the field. Thus, the polynucleotides and polypeptides of some applications of the invention positively affect plant production, seed production and plant biomass. Furthermore, the benefit of improved plant NUE will certainly improve crop quality and seed biochemical constituents such as protein production and oil production.
[00131] It should be noted that an improved ABST will give plants improved vigor also under non-stress conditions, resulting in crops that have improved biomass and/or yield, eg stretched fibers for the cotton industry, higher oil.
[00132] The term "fiber" is normally inclusive of thick-walled conduction cells, such as vessels and tracheids, and for fibrillar aggregates of many single fiber cells. Hence, the term “fiber” refers to (a) xylem thick-walled conduction and non-conduction cells; (b) fibers of extraaxillary origin, including those from phloem, bark, soil tissue and epidermis; and (c) fibers from the stems, leaves, roots, seeds and flowers or inflorescences (such as those of Sorghum vulgare used in the manufacture of brushes and brooms).
[00133] Examples of plant that produce fiber include but are not limited to agricultural crops such as cotton, cotton silk tree (Paina, Ceiba pentandra), desert willow, creosote bush, winterfat, balsa, kenaf, rosala, jute , abaca sisal, flax, corn, sugar cane, hemp, ramie, kapok, coconut fiber, bamboo, Spanish moss and Agave spp. (eg sisal).
[00134] As used herein, the term "fiber quality" refers to at least one fiber parameter that is agriculturally desired or required in the fiber industry (also described below). Examples of such parameters include, but are not limited to, fiber length, fiber strength, fiber suitability, fiber weight per unit length, maturity ratio, and uniformity (also described below).
[00135] The quality of cotton fiber (gauze) is typically measured according to the length, strength and fineness of the fiber. Consequently, the quality of gauze is considered higher when the fiber is longer, stronger and thinner.
[00136] As used herein, the term "fiber production" refers to the quantity or quality of fibers produced from the fiber-producing plant.
[00137] As used herein, the term "increase" refers to at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10 %, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70% , at least about 80% increase in fertilizer use efficiency, nitrogen use efficiency, yield, seed yield, biomass, growth rate, vigor, oil content, fiber yield, fiber quality, seed length fiber, photosynthetic capacity, and/or abiotic stress tolerance of a plant compared to a native plant or wild-type plant [i.e., a plant not modified with the biomolecules (polynucleotide or polypeptides) of the invention, e.g., a plant non-transformed species of the same species that is grown under the same (eg, identical) growth conditions].
[00138] The expression "expressing within the plant an exogenous polynucleotide", as used herein, refers to the upregulation of the expression level of an exogenous polynucleotide within the plant, introducing the exogenous polynucleotide into a plant or plant cell and expressing it by recombinant means, as also described below.
[00139] As used herein, "express" refers to expression at the mRNA and, optionally, at the polypeptide level.
[00140] As used herein, the term "exogenous polynucleotide" refers to a heterologous nucleic acid sequence that may not be naturally expressed within the plant (e.g., a nucleic acid sequence from different species) or in which overexpression in the plant is desired. The exogenous polynucleotide can be introduced into the plant in a stable or transient manner, so as to produce a ribonucleic acid (RNA | ibonucleic acid) molecule and/or a polypeptide molecule. It should be noted that the exogenous polynucleotide may comprise a nucleic acid sequence that is identical or partially homologous to an endogenous plant nucleic acid sequence.
[00141] The term "endogenous", as used herein, refers to any polynucleotide or polypeptide that is present and/or naturally expressed within a plant or a cell thereof.
[00142] In accordance with some applications of the invention, the exogenous polynucleotide of the invention comprises a nucleic acid sequence that encodes a polypeptide having an amino acid sequence of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89 %, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% , at least about 97%, at least about 98%, at least about 99%, or, say, 100% homologous to the amino acid sequence selected from the group consisting of SEQ IDs. No. 496-794, 2898-3645, 36474854 and 4855.
[00143] Homologous sequences include both orthologous and paralogous sequences. The term "paralog" refers to gene duplications within the genome of a species, leading to paralogous genes. The term “orthologous” refers to homologous genes in different organisms due to ancestral relationship. Thus, orthologs are evolutionary counterparts derived from unique ancestral genes in the last common ancestor of two given species (Koonin EV and Galperin MY (Sequence - Evolution - Function: Computational Approaches in Comparative Genomics. Boston: Kluwer Academic; 2003. Chapter 2, Evolutionary Concept). in Genetics and Genomics. Available at: ncbi (dot) nlm (dot) nih (dot) gov/books/NBK20255) and, therefore, are highly likely to have the same function.
[00144] One option to identify orthologs in monocotyledonous plant species is to perform a reciprocal explosion search. This can be done through a first explosion, involving blasting the sequence of interest against any database of the sequence, such as the publicly available NCBI database which can be found at: ncbi (dot) nlm (dot) nih ( point) gov. If rice orthologs are sought, the sequence of interest would be detonated again, eg, the 28,469 full-length cDNA clones of Oryza Sativa Nipponbare available from NCBI. The explosion results can be filtered. The full length sequences from the filtered or unfiltered results are then blasted back (second burst) against the organism sequences from which the sequence of interest is derived. The results of the first and second bursts are then compared. An ortholog is identified when the sequence resulting in the highest score (best hit) in the first burst identifies the query string (the original sequence of interest) as the best hit in the second burst. Using the same rationale, a paralog (homologous to a gene in the same organism) is found. In the case of large sequence families, the ClustalW program can be used [ebi (dot) ac (dot) uk/Tools/clustalw2/index (dot) html], followed by a neighboring union tree (wikipedia (dot) org/ wiki/Neighbor-joining) which helps in visualizing the grouping.
[00145] Homology (eg, percent homology, sequence identity + sequence similarity) can be determined using any homology comparison software that calculates pairwise sequence alignment.
[00146] As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to residues in the two sequences which are the same when aligned. When percent sequence identity is used in reference to proteins, it is recognized that the positions of residues that are not identical often differ by conservative amino acid substitutions, in which amino acid residues are replaced by other amino acid residues with chemical properties. similar (eg, charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ by conservative substitutions, the percentage of sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are referred to as having "sequence similarity" or "similarity". Means for making such an adjustment are well known to those skilled in the art. Typically, this involves achieving a conservative substitution such as a partial rather than a total mismatch, thereby increasing the percentage of sequence identity. Thus, for example, when a score of 1 is assigned to an identical amino acid and when a score of zero is assigned to a non-conservative substitution, a score of between zero and 1 will be assigned to a conservative substitution. The conservative substitution score is calculated, eg, according to the algorithm of Henikoff S and Henikoff JG. [Amino acid substitution matrices from protein blocks. process natl. academy Sci. U.S.A. 1992, 89(22): 10915-9].
[00147] Identity (eg, percentage homology) can be determined using any homology comparison software, including, for example, the National Center of Biotechnology Information's BlastN software [NCBI] as well as using default parameters.
[00148] According to some applications of the invention, the identity is an overall identity, that is, an identity over the entire amino acid or nucleic acid sequence of the invention and not just over parts thereof.
[00149] In accordance with some applications of the invention, the term "homology" or "homologue" refers to the identity of two or more nucleic acid sequences; or identity of two or more amino acid sequences; or amino acid sequence identity to one or more nucleic acid sequences.
[00150] According to some applications of the invention, homology is global homology, that is, homology over the entire amino acid or nucleic acid sequence of the invention and not just over parts thereof.
[00151] The degree of homology or identity between two or more sequences can be determined using various known sequence comparison tools. Following is a non-limiting description of such tools, which can be used in conjunction with some applications of the invention.
[00152] Global pairwise alignment was defined by SB Needleman and CD Wunsch, “A general method applicable to the search of similarities in the amino acid sequence of two proteins” Journal of Molecular Biology, 1970, pages 443-53, volume 48 ).
[00153] For example, when starting with a polypeptide sequence and comparing with other polypeptide sequences, Needleman-Wunsch's EMBOSS-6.0.1 algorithm (available at emboss(dot)sourceforge(dot)net/apps/cvs/emboss/apps/ needle(dot)html) can be used to find the optimal alignment (including gaps) of two strings along with their total lengths - a "Global Alignment". Default parameters for the Needleman-Wunsch algorithm (EMBOSS-6.0.1) include: gapopen=10;gapextend=0.5; datafile= EBLOSUM62; brief=YES.
[00154] According to some applications of the invention, the parameters used with the EMBOSS-6.0.1 tool (for protein-protein comparison) include: gapopen=8; gapextend=2;datafile=EBLOSUM62; brief=YES.
[00155] According to some applications of the invention, the threshold used to determine homology using the Needleman-Wunsch EMBOSS-6.0.1 algorithm is 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
[00156] When starting with a polypeptide sequence and comparing to polynucleotide sequences, the OneModel FramePlus algorithm (Halperin, E., Faigler, S. and Gill-More, R. (1999) - FramePlus: aligning DNA to protein sequences. Bioinformatics, 15, 867-873) (available from biocceleration(dot)com/ Products(dot)html) can be used with the following default parameters: model=frame+_p2n.model mode=local.
[00157] According to some applications of the invention, the parameters used with the OneModel FramePlus algorithm are model=frame+_p2n.model, mode=qglobal.
[00158] According to some applications of the invention, the threshold used to determine homology using the OneModel FramePlus algorithm is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
[00159] When starting with a polynucleotide sequence and comparing with other polynucleotide sequences, Needleman-Wunsch's EMBOSS-6.0.1 algorithm (available at emboss(dot)sourceforge(dot)net/apps/cvs/emboss/apps/need le (dot)html) can be used with the following default parameters. (EMBOSS-6.0.1) gapopen=10; gapextend=0.5; datafile= EDNAFULL; brief=YES.
[00160] According to some applications of the invention, the parameters used with the Needleman-Wunsch EMBOSS-6.0.1 algorithm are gapopen=10; gapextend=0.2; datafile= EDNAFULL; brief=YES.
[00161] According to some applications of the invention, the threshold used to determine homology using the Needleman-Wunsch EMBOSS-6.0.1 algorithm for comparing polynucleotides with polynucleotides is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% .
[00162] According to some applications, the determinations of the degree of homology additionally require the use of the Smith-Waterman algorithm (for protein-protein comparison or nucleotide-nucleotide comparison).
[00163] Default parameters for the GenCore 6.0 SmithWaterman algorithm include: model =sw.model.
[00164] According to some applications of the invention, the threshold used to determine homology using the SmithWaterman algorithm is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
[00165] In accordance with some applications of the invention, global homology is performed on sequences preselected by local homology to the polypeptide or polynucleotide of interest (e.g., 60% identity over 60% sequence length) prior to performing overall homology to the polypeptide or polynucleotide of interest (e.g., 80% overall homology over the entire sequence). For example, homologous sequences are selected using BLAST software with the Blastp and tBlastn algorithms acting as filters for the first stage and the needle (EMBOSS package) or Structure+algorithmic alignment for the second stage. The local identity (Blast alignments) is defined with a very permissive cutoff - 60% Identity in a range of 60% of the sequence lengths, because it is only used as a filter for the global alignment phase. In this specific application (when the local identity is used) the standard Blast packet filtering is not used (by setting the parameter “-F F”).
[00166] In the second stage, homologs are defined based on the overall identity of at least 80% to the main polypeptide sequence.
[00167] According to some applications of the invention, two distinct ways to discover the ideal global alignment for protein or nucleotide sequences are used: 1. Between two proteins (following the blastp filter): EMBOSS-6.0.1 algorithm of Needleman-Wunsch with the following parameters modified: gapopen=8 gapextend=2.The rest of the parameters remain unchanged from the default options listed here:Default qualifiers (Required):[-asequence] sequence Sequence file name and optional format, or reference (US entry).[-bsequence] seqall Sequence(s) filename and format optional, or reference (US entry)-floating gappen [10,0 for any sequence]. The open gap penalty is a rating taken when a gap is created. The best value depends on the choice of comparison arrangement. The default value assumes you are using the EBLOSUM62 array for the protein sequences and the EDNAFULL array for the nucleotide sequences (floating-point number from 1.0 to 100.0). Floating-gapextend [0.5 for any sequence] . The gap extension penalty is added to the standard gap penalty for each base or residue in the gap. This is the time for which the gap is penalized. Normally, few large gaps and not many small gaps are expected, so the penalty on gap length should be less than the gap penalty. An exception is when one or more sequences are read alone with possible sequence errors in which case you would expect many single-base gaps. This result can be obtained by setting the open gap penalty to zero (or very low) and using the gap extension penalty to control the gap classification, (floating point number from 0.0 to 10.0).[ -outfile] alignment [*.needle] Output alignment file nameAdditional Qualifiers (Optional):-datafile matrixf [EBLOSUM62 for protein, EDNAFULL for DNA] . This is a file of the sorting array used when comparing sequences. By default this is the 'EBLOSUM62* file (for proteins) or the 'EDNAFULL' file (for nucleic sequences) These files are found in the 'data' directory of the EMBOSS installation. Advanced Qualifiers (Spontaneous):-[no]brief boolean [Y] Brief identity and similarity.Associated Qualifiers:"-asequence" Associated Qualifiers.-sbeginl integer Start sequence to be used.-sendl integer End sequence to be used.-sreversel boolean Reverse (if DNA) . -sask.1 boolean Asks to start/stop/reverse.-nucleotide1 boolean Sequence is nucleotide.-sproteinal boolean Sequence is protein.-slowerl boolean Lowercase.-supperl boolean Uppercase.-sformat1 string Input sequence format .-sdbnamel string Database name.-sidl string Input name.-uf ol string UFO Resources.-fformat1 string Resource Format.-fopenfilei string Resource file name."-bsequence" Associated Qualifiers.-sbegin2 integer Start each sequence to be used.-send2 integer End each sequence to be used.-sreverse2 boolean Reverse (if DNA).-sask∑ boolean Ask to start/stop/reverse.— snucleotide2 boolean Sequence is nucleotide.-sproteina∑ boolean String is proteina.-slower2 boolean Lowercase.-supper2 boolean Uppercase.-aformat2 string Input string format.~sdbname2 string Database name.-sid2 string Input name.-uf o2 string UFO resources. -f for mat2 string Resource Format.-fopenfile2 string Resource filename.—outfile" Associated Qualifiers.-aformat3 string Alignments Format.-aextension3 string Filename Extension.-adirectory3 string Output Directory.-aname3 string Filename of base.-awidth3 integer Width of the alignment.-aaccshow3 boolean Show the accession number in the header.-adesshow3 boolean Show the description in the header.-ausashow3 boolean Show the complete USA in the alignment.-aglobal3 boolean Show the complete string in the alignment. General Qualifiers: -auto boolean Off: Turn on requests.-stdout boolean Write first file to standard output.-boolean filter Read first file from standard input. writes first file to standard output.—Options boolean Prompts for default and additional values. Writes program debug output .dbg Logs some/all command-line options. Logs command-line options. More information about associated and general qualifiers can be found with -help -verbose. Log warnings. Log errors. Log fatal errors. Log program expiration messages.2. Between a protein sequence and a nucleotide sequence (following the rblastn filter) Application of Gencore 6.0 Onemodel using the structure+algorithm with the following parameters: model=frame+_p2n.model mode=qglobal - q=protein.sequence -db= nucleotide .sequence. The rest of the parameters remain unchanged from the default options: Usage:om —model—<model_fname> [—q=]query [-db=]database [options].—model=<model_fname> Specifies the model you want to run. All models supplied by Compugen are located in the directory $CGNROOT/models/.Valid command line parameters:—dev=<dev name> Selects the device to be used by the application.Valid devices are:bic — Bioccelerator (valid for SW ,XSW, FRAME N2P, and FRAME_P2N models) .Xlg - BioXL/G (valid for all models except XSW).xlp - BioXL/P (valid for SW, FRAME+_N2P, and FRAME_P2N models).xlh - BioXL/H (valid for SW, FRAME+_N2P, and FRAME_P2N models). for SW,FRAME+—N2P, and FRAME_P2N models).soft — Software device (for all models).-q=<query> Defines the query set. Queries can be a sequence file or a database reference. You can specify a query by name or by accession number. The format is automatically detected. However, you can specify a format using the -qfmt parameter. If a query is not specified, the program prompts you for one. If the query set is a database reference, an output file is produced for each string in the query.—db=<database Choose the database set. The name>database set can be a string file or a database reference. The database format is automatically detected. However, you can specify a format using the parameter —dfmt.-qacc Add this parameter to the command line if a query is specified using accession numbers.—dacc Add this parameter to the command line if a database is specified using accession numbers.-dfmt/- Choose database/query format type.qfmt=< format_type> Possible formats are: fasta - fasta with autodetected sequence type.fastap — fastan — protein sequence fasta, nucleic sequence fasta.gcg - gcg format, type is autodetected. gcg9 format, the type is autodetected.gcg9 format protein sequence.gcg9 format nucleic sequence.nbrf sequence, the type is autodetected.nbrf protein sequence. nbrf nucleic sequence. embl and swissprot format. genbank (nucleic) format. blast format.nbrf_gcg sequence, type is autodetected.nbrf protein sequence—gcg. nucleic sequence nbrf—gcg.raw ascii sequence, the type is autodetected.raw ascii protein sequence, raw ascii nucleic sequence.pir codata format, the type is autodetected.gcg profile (only valid for-qfmt in SW, XSW, FRAME P2N and FRAME+_P2N) .The name of the output file.The suffix of the name of the output file.Penalty on gap opening. This parameter is not valid for FRAME+. For FrameSearch the default is 12.0. For other surveys the default is 10.0. Penalty for gap extension. This parameter is not valid for FRAME+. For FrameSearch the default is 4.0. For other models: the default for protein searches is 0.05 and the default for nucleic searches is 1.0. The penalty for opening a gap in the query string. The default is 10.0. Valid for XSW.A penalty for extending a gap in the sequence of questions. The default is 0.05. Valid for XSW. The position in the query string to start the search. The position in the query string to stop the search. Performs a translated search, relevant to a nucleic query against a protein database. The nucleic query is translated into six reading frames and a result is provided for each frame.for SW and XSW.-dtrans Performs a translated search, relevant to a protein query against a DNA database. Each database entry is translated into six reading frames and a result is given for each cruadro. Specifies the comparison arrangement to be used in the search. The arrangement must be in BLAST format. If the array file is not found in $CGNROOT/tables/matrix, specify the full path as the value of the -matrix parameter.Translation table. The default location for the table is $CGNROOT/tables/trans. Restricts the search to only the top strand of the nucleic sequence query/database. The maximum size of the output hitlist. The default is 50. The number of documentation lines preceding each alignment. The default is 10. The rating that places a limit on the display of results. Ranks less than the minimum value of -thr min or greater than the value of —thr_max are not shown. Valid options are: quality.Score.Score.The highest threshold of the rating. Results greater than the —thr max value are not shown. The lowest sort threshold. Results less than the —thr min value are not shown.The number of alignments recorded in the output file.Does not display the alignment.Note: the "-align" and "-noalign" parameters are mutually exclusive.-outfmt=<format_name> Specifies the output format type. The default format is PFS. Possible values are: PFS - Text format PFSFASTA - FASTA text format BLAST - BLAST text format -nonorm Does not perform classification normalization.-norm=<norm_name> Specify the normalization method. Valid options are:log - normalization of algorithm.std - normalization of standard.stat - Statistical method of PeacockNote: parameters "—nonorm" and "—norm" cannot be used together.Note: Parameters -xgapop, -xgapext, - fgapop, -fgapext, —ygapop, -ygapext, -delop, and -delext only apply to FRAME+.- xgapop=<n> The penalty for opening a gap by inserting a (triple) codon. The default is 12.0.- xgapext=<n> The penalty for extending a gap when inserting a (triple) codon. The default is 4.0.- ygapop=<n> The penalty for opening a gap when deleting an amino acid. The default is 12.0. The penalty for gap extension when excluding an amino acid. The default is 4.0. The penalty for opening a gap when inserting a DNA base. The default is €.0. The penalty for extending a gap when entering a DNA base. The default is 7.0. The penalty for opening a gap when deleting a DNA base. The default is €.0. The penalty for extending a gap when excluding a DNA base. The default is 7.0. No output to the screen is produced. The name of the host under which the server works. By default, the application uses a specific organizer in the $CGNROOT/cgnhosts file. Do not access the background when the device is busy. This option is not relevant for Parseq pseudo-device or Soft.-batch Run the job in the background. When this option is specified, the file "SCGNROOT/defaults/batch.defaults" is used to choose the batch command. If the file does not exist, the command "at now" is used to run the job.Note: The parameters "—batch" and "—wait" are mutually exclusive, -version Print the version number of the software.-help Display this help message. For more specific help type: "om -model=<model fname> -help".
[00168] According to some a local homology or a local identity.
[00169] The other tools are not limited to the BlastP, BlastN, BlastXor TBLASTN software from the National Center for Biotechnology Information (NCBI), FASTA and the Smith-Waterman algorithm.
[00170] A tblastn search allows the comparison between a protein sequence and translations of six structures from a nucleotide database. It can be a very productive way of finding homologous protein-encoded regions in unannotated nucleotide sequences, such as expressed sequence tags [ESTs expressed sequence tags] and draft genome records [HTG draft genome records], located in the databases of BLAST est and htgs data, respectively.
[00171] Default parameters for blastp include: Maximum target sequences: 100; Expected limit: e- 5; Word Size: 3: Maximum matches in a query range: 0; Classification parameters: Array - BLOSUM62; filters and camouflage: Filter - regions of low complexity.
[00172] Local alignment tools that can be used include, but are not limited to, the tBLASTX algorithm, which compares the conceptual translation products of six structures from a sequence of nucleotide queries (both strands) against a sequence database of protein. Default parameters include: Maximum Target Sequences: 100; Expected threshold: 10; Word Size: 3: Maximum matches in a query range: 0; Classification parameters: Array - BLOSUM62; filters and camouflage: Filter - regions of low complexity.
[00173] According to some applications of the invention, the exogenous polynucleotide of the invention encodes a polypeptide having an amino acid sequence of at least 80%, at least 81%, at least 82%, at least 83%, at least 84 %, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94 %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or, say, 100% identical to the amino acid sequence selected from the consistent group of SEQ ID. No. 496-794, 2898-3645, 3647-4854 and 4855.
[00174] In accordance with some applications of the invention, the exogenous polynucleotide of the invention encodes a polypeptide having an amino acid sequence selected from the group consisting of IDs. SEQ Nos. 496-794, 2898-4854 and 4855.
[00175] According to some applications of the invention, the method to increase fertilizer use efficiency, nitrogen use efficiency, production, biomass, growth rate, vigor, oil content, fiber production, fiber quality, fiber length, photosynthetic capacity, and/or abiotic stress tolerance of a plant is effected by expressing within the plant an exogenous polynucleotide, comprising a nucleic acid sequence that encodes a polypeptide, at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88% , at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or say mos, 100% identical to the amino acid sequence selected from the group consisting of SEQ IDs. N° 496-794, 2898-3645, 3647-4854 and 4855, thus increasing fertilizer use efficiency, nitrogen use efficiency, production, biomass, growth rate, vigor, oil content, fiber production , fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of a plant.
[00176] In accordance with some applications of the invention, the exogenous polynucleotide encodes a polypeptide consisting of an amino acid sequence established by SEQ IDs. No. 496-794, 2898-4854 or 4855.
[00177] According to an aspect of some applications of the invention, the method for increasing fertilizer use efficiency, nitrogen use efficiency, production, biomass, growth rate, vigor, oil content, fiber production, quality fiber length, fiber length, photosynthetic capacity and/or abiotic stress tolerance of a plant is effected by expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence selected from a group consisting of the following: SEQ ID N° 496-794, 2898-4854 and 4855, thus increasing the efficiency in the use of fertilizer, efficiency in the use of nitrogen, production, biomass, growth rate, vigor, oil content, fiber production, quality of fiber, fiber length, photosynthetic capacity and/or abiotic stress tolerance of the plant.
[00178] According to an aspect of some applications of the invention, there is provided a method for increasing fertilizer use efficiency, nitrogen use efficiency, yield, biomass, growth rate, vigor, oil content, fiber production , fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding a polypeptide selected from the group consisting of SEQ IDs. N° 496-794, 2898-4854 and 4855, thus increasing the efficiency in the use of fertilizer, efficiency in the use of nitrogen, production, biomass, growth rate, vigor, oil content, fiber production, quality of fiber, fiber length, photosynthetic capacity and/or abiotic stress tolerance of the plant.
[00179] In accordance with some applications of the invention, the exogenous polynucleotide encodes a polypeptide consisting of the amino acid sequence established by SEQ IDs. No. 496-794, 2898-4854 or 4855.
[00180] In accordance with some applications of the invention, the exogenous polynucleotide comprises a nucleic acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83% , at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 96% at least about 97%, at least about 98%, at least about 99%, e.g., 100% identical to the nucleic acid sequence selected from the group consisting of SEQ IDs. No. 1-495, 795-2896 and 2897.
[00181] According to an aspect of some applications of the invention, there is provided a method for increasing fertilizer use efficiency, nitrogen use efficiency, yield, biomass, growth rate, vigor, oil content, fiber production , fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence, at least about 80%, at least about 81% , at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g., 100% identical to the nucleic acid sequence selected from the group consisting of SEQ IDs. N° 1-495, 795-2896 and 2897, thus increasing the efficiency in the use of fertilizer, efficiency in the use of nitrogen, production, biomass, growth rate, vigor, oil content, fiber production, quality of fiber, fiber length, photosynthetic capacity and/or abiotic stress tolerance of the plant.
[00182] According to some applications of the invention, the exogenous polynucleotide is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84% , at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 97% at least about 98%, at least about 99%, e.g., 100% identical to the polynucleotide selected from the group consisting of SEQ IDs. No. 1-495, 795-2896 and 2897.
[00183] In accordance with some applications of the invention, the exogenous polynucleotide is established by SEQ IDs. No. 1-495, 795-2896 or 2897.
[00184] According to some applications of the present invention, the method for increasing fertilizer use efficiency, nitrogen use efficiency, production, growth rate, biomass, vigor, oil content, seed production, fiber production , fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of a plant, also comprises selecting a plant with an increase in fertilizer use efficiency, nitrogen use efficiency, production, growth rate , biomass, vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance compared to a wild type plant of the same species that is grown in the same cultivation conditions.
[00185] It should be noted that the selection of a transformed plant, having an increase in the tract compared to a native plant (eg, untransformed) grown under the same cultivation conditions, is carried out by selecting for the tract, p .eg, validating the ability of the transformed plant to exhibit tract enhancement, well known assays (eg, seedling analyses, greenhouse assays), as will be described later.
[00186] According to an aspect of some applications of the present invention, there is provided a method of selecting a transformed plant, having an increase in fertilizer use efficiency, nitrogen use efficiency, production, growth rate, biomass, vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance compared to a wild type plant of the same species grown under the same growing conditions, the method comprising: (a) providing plants transformed with an exogenous polynucleotide encoding a polypeptide comprising an amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 82% 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90 %, at least about 91 %, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97% , at least about 98%, at least about 99%, e.g., 100% homologous (e.g., having a sequence similarity or sequence identity) to the amino acid sequence selected from the group consisting of SEQ IDs . N° 496-794, 2898-3645, 3647-4854 and 4855,(b) select, from the plants, a plant with increased fertilizer use efficiency, nitrogen use efficiency, production, growth rate, biomass , vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or tolerance to abiotic stress, thus selected, the plant having increased efficiency in the use of fertilizers, efficiency in nitrogen use, production, growth rate, biomass, vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or tolerance to abiotic stress compared to a plant in the wild type of the same species that is grown under the same growing conditions.
[00187] According to an aspect of some applications of the present invention, there is provided a method of selecting a transformed plant, having an increase in fertilizer use efficiency, nitrogen use efficiency, production, growth rate, biomass, vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance compared to a wild type plant of the same species grown under the same growing conditions, the method comprising: (a) providing plants transformed with an exogenous polynucleotide encoding a polypeptide comprising an amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83% , at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 97% at least about 98%, at least about 99%, e.g., 100% identical to the nucleic acid sequence selected from the group consisting of SEQ IDs. 1495, 795-2896 and 2897,(b) select, from said plants, a plant having increased fertilizer use efficiency, nitrogen use efficiency, production, growth rate, biomass, vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance, thereby selecting the plant to have increased fertilizer use efficiency, nitrogen use efficiency, production, growth rate, biomass, vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance compared to a wild type plant of the same species that is grown under the same growing conditions.
[00188] As used herein, the term "polynucleotide" refers to a single-stranded or double-stranded nucleic acid sequence that is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a sequence polynucleotide genomics and/or a composite polynucleotide sequence (eg, a combination of the above sequences).
[00189] The term “isolated” refers to at least partially separated from the natural environment, eg from a plant cell.
[00190] As used herein, the term "complementary polynucleotide sequence" refers to a sequence that results from reverse transcription of messenger RNA using reverse transcriptase or any other RNA-dependent DNA polymerase. Such a sequence may subsequently be amplified in vivo or in vitro using a DNA-dependent DNA polymerase.
[00191] As used herein, the term "polynucleotide genomic sequence" refers to a sequence derived (isolated) from a chromosome and, as such, represents a contiguous portion of a chromosome.
[00192] As used herein, the term "composite polynucleotide sequence" refers to a sequence that is at least partially complementary and at least partially genomic. The composite sequence may include some exonal sequences necessary to encode the polypeptide of the present invention, as well as some intervening intronic sequences. Intronic sequences can be from any source, including other genes, and will typically include preserved interlaced signal sequences. These intronic sequences may also include cis-acting expression regulatory elements.
[00193] Nucleic acid sequences encoding the polypeptides of the present invention can be optimized for expression. Examples of these sequence modifications include, but are not limited to, an altered G/C content from a more careful approach than that commonly found in the plant species of interest and the removal of codons atypically found in the plant species commonly called optimization. of codons.
[00194] The term "codon optimization" refers to the selection of appropriate DNA nucleotides for use within a structural gene or fragment thereof that addresses codon utilization within the plant of interest. Therefore, an optimized gene or nucleic acid sequence refers to a gene in which the nucleotide sequence of a native or naturally occurring gene has been modified in order to use statistically preferred or statistically favored codons within the plant. Typically, the nucleotide sequence is examined at the DNA level and in the coding region optimized for expression in the determined plant species using any suitable procedure, e.g., as described in Sardana et al. (1996, Plant Cell Reports 15:677-681). In this method, the standard deviation of codon utilization, a measure of codon utilization tendency, can be calculated by first finding the square of the proportional deviation of utilization of each codon of the native gene relative to that of highly expressed plant genes, followed by a calculation of the square of the mean deviation. The formula used is: 1 SDCU = n = 1 N [(Xn - Yn) /Yn] 2/N, where Xn refers to the frequency of use of the codon n in highly expressed plant genes, where Yn refers to the frequency of use of codon n in the gene of interest and N refers to the total number of codons in the gene of interest. A Codon Usage Table of highly expressed dicot genes is compiled using data from Murray et al. (1989, Nuc AcIDs Res. 17:477-498).
[00195] One method of optimizing the nucleic acid sequence according to the preferred codon usage for a particular plant cell type is based on the direct use, without performing any extra statistical calculations, of Codon Optimization Tables such as those available online in the Codon Usage Database through the DNA bank of the NIAS (National Institute of Agrobiological Sciences|National Institute of Agrobiological Sciences) in Japan (kazusa (dot) or (dot) jp/codon/). The Codon Usage Database contains Codon Usage Tables for several different species, with each Codon Usage Table having been statistically determined based on data present in Genbank.
[00196] Using the Tables above to determine the most preferred or most favored codons for each amino acid in a particular species (e.g. rice), a naturally occurring nucleotide sequence encoding a protein of interest may have the codon optimized for that particular plant species. This is accomplished by replacing codons that may have a statistically low incidence in the genome of the particular species with corresponding codons, relative to an amino acid, that are statistically more favored. However, one or more less favored codons can be selected to exclude existing restriction sites, to create new ones at potentially useful junctions (5' and 3' ends for adding signal peptide or termination cassettes, internal sites that can be used to cut and reassemble segments to produce a correct full-length sequence), or to eliminate nucleotide sequences that could negatively affect the stability or expression of the mRNA.
[00197] The naturally occurring coding nucleotide sequence may, prior to any modification, already contain a codon number that corresponds to a statistically favored codon in a particular plant species. Therefore, codon optimization of the native nucleotide sequence may comprise determining which codons, within the native nucleotide sequence, are not statistically favored with respect to a particular plant, and modifying those codons according to a plant codon usage table. particular to produce a codon-optimized derivative. A modified nucleotide sequence may be fully or partially optimized for plant codon utilization provided that the protein encoded by the modified nucleotide sequence is produced at a higher level than the protein encoded by the corresponding naturally occurring or native gene. The structure of synthetic genes by altering codon usage is described, e.g., in PCT (Patent Cooperation Treaty) Patent Application 93/07278.
[00198] According to some applications of the invention, the exogenous polynucleotide is a non-coding RNA.
[00199] As used herein, the term "non-coding RNA" refers to an RNA molecule that does not encode an amino acid sequence (a polypeptide). Examples of such non-coding RNA molecules include, but are not limited to, an antisense RNA, a pre-miRNA (a precursor of a microRNA), or a precursor of an RNA that interacts with Piwi (piRNA).
[00200] Non-limiting examples of non-encoded RNA polynucleotides are provided in SEQ IDs. No. 217, 218, 219, 287, 288, 495, 997, 1003, 1543 and 1703.
[00201] Thus, the invention encompasses the nucleic acid sequences described above, fragments thereof, hybridizing sequences found therein, sequences homologous thereto, sequences encoding similar polypeptides with different codon usages, altered sequences characterized by mutations such as deletion, introduction or substitution of one or more nucleotides, naturally occurring or induced by man, either randomly or in an objectified manner.
[00202] According to some applications of the invention, the exogenous polynucleotide encodes a polypeptide, comprising an amino acid sequence, at least 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 90% at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g., 100% identical to the amino acid sequence of a plant whose naturally occurring ortholog of the polypeptide selected from the group consisting of SEQ IDs. Nos. 496-794 and 2898-4855.
[00203] According to some applications of the invention, the polypeptide comprises an amino acid sequence, at least 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84 %, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91% , at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g., 100% identical to the amino acid sequence of a plant whose naturally occurring ortholog of the polypeptide selected from the group consisting of SEQ IDs. Nos. 496-794 and 2898-4855.
[00204] The invention provides an isolated polynucleotide comprising a nucleic acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84 %, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91% , at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g., 100% identical to the polynucleotide selected from the group consisting of SEQ IDs. No. 1-495, 795-2896 and 2897.
[00205] According to some applications of the invention, the nucleic acid sequence is capable of increasing nitrogen use efficiency, fertilizer use efficiency, oil content, production, growth rate, vigor, biomass, fiber production , fiber quality, fiber length, photosynthetic capacity, abiotic stress tolerance and/or water use efficiency of a plant.
[00206] In accordance with some applications of the invention, the isolated polynucleotide comprising a nucleic acid sequence is selected from the group consisting of SEQ IDs. No. 1-495, 795-2896 and 2897.
[00207] In accordance with some applications of the invention, the isolated polynucleotide is set forth by SEQ IDs. No. 1-495, 795-2896 or 2897.
[00208] The invention provides an isolated polynucleotide, comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 89% at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 95% 96%, at least about 97%, at least about 98%, at least about 99%, or, say, 100% homologous to the amino acid sequence selected from the group consisting of SEQ IDs. No. 496-794, 2898-3645, 3647-4854 and 4855.
[00209] According to some applications of the invention, the amino acid sequence is able to increase nitrogen use efficiency, fertilizer use efficiency, oil content, production, growth rate, vigor, biomass, fiber production, fiber quality, fiber length, photosynthetic capacity, abiotic stress tolerance and/or a plant's water use efficiency.
[00210] The invention provides an isolated polynucleotide, comprising a nucleic acid sequence encoding a polypeptide, comprising the amino acid sequence selected from the group consisting of SEQ IDs. Nos. 496-794, 2898-4854 and 4855.
[00211] In accordance with one aspect of some applications of the invention, there is provided a nucleic acid backbone, comprising the isolated polynucleotide of the invention and a promoter for directing transcription of the nucleic acid sequence in a host cell.
[00212] The invention provides an isolated polypeptide comprising an amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84% , at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 97% at least about 98%, at least about 99%, or, say, 100% homologous to an amino acid sequence selected from the group consisting of SEQ IDs. No. 496-794, 2898-3645, 3647-4854 and 4855.
[00213] According to some applications of the invention, the polypeptide comprising an amino acid sequence is selected from the group consisting of SEQ IDs. Nos. 496-794, 2898-4854 and 4855.
[00214] In accordance with some applications of the invention, the polypeptide is set forth by SEQ IDs. No. 496-794, 2898-4854 or 4855.
[00215] The invention also encompasses fragments of the polypeptides described above and of polypeptides having mutations, such as deletions, introductions or substitutions of one or more amino acids, naturally occurring or induced by man, either randomly or in an objectified manner.
[00216] The term “plant”, as used herein, encompasses whole plants, grafted plants, ancestors and progenies of plants and plant parts, including seeds, shoots, stems, roots (including the tubers), rhizomes, grafts and cells, plant tissues and organs. The plant can be in any form, including suspension cultures, embryos, meristematic regions, callous tissue, leaves, gametophytes, sporophytes, pollen and microspores. Plants which are particularly useful in the methods of the invention include all plants belonging to the superfamily Viridiplantae, in particular monocots and dicots, including a forage or forage legume, ornamental plant, food crop, tree or shrub selected from the list comprising Acacia spp. ., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Buchá frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina , Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia ob longa, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalypfus spp., Euclea schimperi, Eulalia vi/losa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp. , Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesli, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus spp., Oryza spp., Peltophorum a fricanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopis cineraria, Pseupontosuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficiillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp., Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrots, cauliflower , celery, kale, flax, kale, lentil, rapeseed, okra, onion, potato, rice, soybean, straw, beet, sugar cane, sunflower, tomato, pumpkin tea, corn, wheat, barley, rye, oats, peanuts, peas, lentils and alfalfa, cotton, rapeseed, canola, pepper, sunflower, tobacco, eggplant, eucalyptus, a tree, an ornamental plant, a perennial grass and a forage crop. Alternatively, algae and other non-Viridiplantae can be used for the methods of the present invention.
[00217] According to some applications of the invention, the plant used by the method of the invention is a crop plant such as rice, corn, wheat, barley, peanut, potato, sesame, olive, palm oil, banana, soybean, sunflower , canola, sugar cane, alfalfa, millet, pulses (beans, peas), flax, lupinus, rapeseed, tobacco, poplar and cotton.
[00218] According to some applications of the invention, the plant is a dicotyledonous plant.
[00219] According to some applications of the invention, the plant is a monocotyledonous plant.
[00220] According to some applications of the invention, there is shown a plant cell exogenously expressing the polynucleotide of some applications of the invention, the nucleic acid structure of some applications of the invention and/or the polypeptide of some applications of the invention.
[00221] According to some applications of the invention, expression of the exogenous polynucleotide of the invention within the plant is affected by the transformation of one or more plant cells with the exogenous polynucleotide, followed by the generation of a mature plant from the transformed cells and growing the mature plant under conditions suitable for expressing the exogenous polynucleotide within the mature plant.
[00222] In accordance with some applications of the invention, transformation is effected by introducing a nucleic acid backbone into the plant cell that includes the exogenous polynucleotide of some applications of the invention and at least one promoter to direct transcription of the exogenous polynucleotide into a host cell (a plant cell). Additional details of suitable transformation approaches are presented below.
[00223] As mentioned, the nucleic acid backbone, according to some applications of the invention, comprises a promoter sequence and the isolated polynucleotide, according to some applications of the invention.
[00224] In accordance with some applications of the invention, the isolated polynucleotide is operably linked to the promoter sequence.
[00225] A coding nucleic acid sequence is "operably linked" to a regulatory sequence (e.g., the promoter) if the regulatory sequence is capable of exerting a regulatory effect on the coding sequence linked thereto.
[00226] As used herein, the term "promoter" refers to a region of DNA that is upstream of the transcriptional start site of a gene to which RNA polymerase binds to initiate RNA transcription. The promoter controls where (eg, what portion of a plant) and/or when (eg, at what stage or condition in an organism's lifetime) the gene is expressed.
[00227] In accordance with some applications of the invention, the promoter is heterologous to the isolated polynucleotide and/or the host cell. As used herein, the term "heterologous promoter" refers to a promoter from a different species or from the same species, but from a different gene locus, from the isolated plinucleotide sequence.
[00228] According to some applications of the invention, the isolated polynucleotide is heterologous to the plant cell.
[00229] Any suitable promoter sequence can be used by the nucleic acid structure of the present invention. Preferably, the promoter is a constitutive, tissue-specific or abiotic stress-inducible promoter.
[00230] According to some applications of the invention, the promoter is a plant promoter that is suitable for expression of the exogenous polynucleotide in a plant cell.
[00231] Promoters suitable for expression in wheat include, but are not limited to, the wheat SPA promoter (SEQ ID NO: 4856; Albanietal, Plant Cell, 9: 171-184, 1997, which is fully incorporated herein by reference), LMW wheat [SEQ ID. No. 4857 (longer LMW promoter) and SEQ ID. No. 4858 (LMW promoter)] and glutenin-1 HMW [SEQ ID. No. 4859 (longer glutenin-1 wheat LMW promoter) and SEQ ID. No. 4860 (HMW glutenin-1 wheat promoter); Thomas and Flavell, The Plant Cell 2:1171-1180; Furtado et al., 2009 Plant Biotechnology Journal 7:240-253 , each is fully incorporated herein by reference] alpha, beta, and gamma gliadin wheat [e.g., SEQ ID NO: 4861 (alpha gliadin wheat, genome B, district Attorney); SEQ ID No. 4862 (gamma gliadin wheat promoter); EMBO 3:1490-15, 1984, which is fully incorporated herein by reference] wheat TdPR60 [SEQ ID. No. 4863 (longer promoter TdPR60 wheat) or SEQ ID. No. 4864 (TdPR60 wheat promoter); Kovalchuk et al., Plant Mol Biol 71:81-98, 2009, which is fully incorporated herein by reference], maize Ub1 promoter [Nongda cultivars 105 (SEQ ID NO: 4865); GenBank: DQ141598.1; Taylor et al., Plant Cell Rep 1993 12: 491-495, which is fully incorporated herein by reference), and cultivars B73 (SEQ ID NO: 4866); Christensen, AH, et al. Plant Mol. Biol. 18 (4), 675-689 (1992), which is fully incorporated herein by reference), rice actin 1 (SEQ ID NO: 4867; Mc Elroy et al. 1990, The Plant Cell, Vol. 2, 163171, which is fully incorporated herein by reference), and rice GOS2 [SEQ ID. No. 4868 (longest promoter of rice GOS2) and SEQ ID. No. 4869 (GOS2 rice promoter); De Pater et al. Plant J. 1992; 2: 837-44, which is fully incorporated herein by reference), Arabidopsis Phol [SEQ ID. No. 4870 (Arabidopsis Pho1 Promoter); Hamburger et al., Plant Cell. 2002 ; 14:889-902, which is fully incorporated herein by reference), ExpansinB promoters, e.g., rice ExpB5 [SEQ ID. No. 4871 (longest promoter of rice ExpB5) and SEQ ID NO: 4872 (promoter of rice ExpB5) and Barley ExpBl [SEQ ID. No. 4873 (ExpBl barley promoter), Won et al. Mol Cells. 2010; 30:369-76, which is fully incorporated herein by reference), barley SS2 (sucrose synthase 2) [(SEQ ID NO: 4874), Guerin and Carbonero, Plant Physiology May 1997 vol. 114 no. 155-62, which is fully incorporated herein by reference], and rice PG5a [SEQ ID. No. 4875, US 7,700,835, Nakase et al., Plant Mol Biol. 32:621-30, 1996, which is fully incorporated herein by reference].
[00232] Suitable constitutive promoters include, for example, the CaMV 35S promoter [SEQ ID. No. 4876 (CaMV 35S Promoter (QFNC)); SEQ ID No. 4877 (PJJ 35S from Brachypodium); SEQ ID No. 4878 (CaMV 35S Promoter (OLD)) (Odell et al., Nature 313:810-812, 1985)], At6669 Arabidopsis promoter (SEQ ID No. 4879 (Arabidopsis At6669 Promoter (OLD)); see PCT Publication No. WO04081173A2 or the Novel At6669 promoter (SEQ ID No. 4880 (At6669 Arabidopsis Promoter (NEW)); Maize Ub1 promoter [Nongda 105 cultivars (SEQ ID No. 4865); GenBank: DQ141598.1; Taylor et al. al., Plant Cell Rep 1993 12: 491-495, which is fully incorporated herein by reference), and cultivars B73 (SEQ ID NO: 4866); Christensen, AH, et al. Plant Mol. Biol. 18 (4) , 675-689 (1992), which is fully incorporated herein by reference), rice actin 1 (SEQ ID NO: 4867, McElroy et al., Plant Cell 2:163-171, 1990); pEMU (Last et al., Theor. Appl. Genet. 81:581-588, 1991); CaMV 19S (Nilsson et al., Physiol. Plant 100:456-462, 1997); rice GOS2 [SEQ ID. No. 4868 (longest promoter of rice GOS2) and SEQ ID. No. 4869 (GOS2 rice promoter); De Pater et al., Plant J Nov;2(6):837-44, 1992]; RBCS promoter (SEQ ID NO: 4881); Rice cyclophilin (Bucholz et al, Plant Mol Biol. 25(5):837-43, 1994); corn histone H3 (Lepetit et al, Mol. Gen. Genet. 231: 276-285, 1992); Actin 2 (An et al., Plant J. 10(1);107-121, 1996) and Synthetic Super MAS (Ni et al., The Plant Journal 7: 661-76, 1995). Other constitutive promoters include those in U.S. Patent Nos. 5,659,026, 5,608,149, 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463 and 5,608,142.
[00233] Suitable tissue-specific promoters include, but are not limited to, sheet-specific promoters [e.g., AT5G06690 (Thioredoxin) (high expression, SEQ ID NO: 4882), AT5G61520 (AtSTP3) (low expression, ID SEQ. No. 4883) described in Buttner et al 2000 Plant, Cell and Environment 23, 175-184 , or the promoters described in Yamamoto et al., Plant J. 12:255-265, 1997 ; Kwon et al., Plant Physiol. 105:357-67, 1994; Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994; Gotor et al., Plant J. 3:509-18, 1993; Orozco et al., Plant Mol. Biol. 23:11291138, 1993; and Matsuoka et al., Proc. natl. academy Sci. USA 90:9586-9590, 1993; as well as the STP3 Arabidopsis promoter (AT5G61520) ( Buttner et al., Plant, Cell and Environment 23:175-184, 2000 )], seed-preferred promoters [e.g., Napin (originating from Brassica napus, being characterized by a seed-specific promoter activity; Stuitje AR et al. Plant Biotechnology Journal 1 (4): 301-309; SEQ ID No: 4884 (Brassica napus NAPIN Promoter) from the seed-specific genes (Simon, et al. ., Plant Mol. Biol. 5, 191, 1985; Scofield, et al., J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al., Plant Mol. Biol. 14: 633, 1990), rice PG5a (SEQ ID NO: 4875; US 7,700,835), early development of Arabidopsis BAN (AT1G61720) seeds (SEQ ID NO: 4885, US 2009/0031450 A1), late development of Arabidopsis ABI3 seeds (AT3G24650) (ID SEQ NO: 4886 (Arabidopsis ABI3 (AT3G24650) Longest Promoter) or 4887 (Arabidopsis ABI3 (AT3G24650) Promoter)) (Ng et al., Plant Molecular Biology 54: 25-38, 2004), Brazil nut albumin ( Pearson' et al ., Plant Mol. Biol. 18: 235-245, 1992), legumes (Ellis, et al. Plant Mol. Biol. 10: 203-214, 1988), Glutelin (rice) (Takaiwa, et al., Mol. Gen. Genet. 208: 15 -22, 1986; Takaiwa, et al., FEBS Letts. 221: 43-47, 1987), Zein (Matzke et al, Plant Mol Biol, 143).323-32 1990), napA (Stalberg, et al, Planta 199 : 515-519, 1996), wheat SPA (SEQ ID No. 4856; Albanietal, Plant Cell, 9: 171-184, 1997), sunflower oil (Cummins, et al., Plant Mol. Biol. 19: 873-876, 1992)], endosperm-specific promoters [e.g., LMW wheat (SEQ ID NO: 4857 (LMW Wheat Longest Promoter), and SEQ ID NO: 4858 (Wheat LMW promoter)] and glutenin -1 HMW [SEQ ID NO: 4859 (longer glutenin-1 LMW promoter from wheat) and SEQ ID NO: 4860 (glutenin-1 promoter HMW from wheat); Thomas and Flavell, The Plant Cell 2:1171-1180 , 1990; Mol Gen Genet 216:81-90, 1989; NAR 17:461-2), wheat alpha, beta and gamma gliadins (SEQ ID NO: 4861 (wheat alpha gliadin promoter (genome B)); SEQ ID No. 4862 (wheat gamma gliadin promoter); EMBO 3 :1409-15, 1984), barley ltrl promoter, barley B1, C, D hordein (Theor Appl Gen 98:1253-62, 1999; Plant J 4:343-55, 1993; Mol Gen Genet 250:750-60, 1996), barley DOF (Mena et al, The Plant Journal, 116(1): 53-62, 1998), Biz2 (EP99106056.7), Barley SS2 (SEQ ID No. 4874 (Barley SS2 Promoter); Guerin and Carbonero Plant Physiology 114: 155-62, 1997), wheat Tarp60 Kovalchuk et al., Plant Mol Biol 71:81-98, 2009, barley D-hordein (D-Hor) and B-hordein (B-Hor) (Agnelo Furtado, Robert J. Henry and Alessandro Pellegrineschi (2009)], synthetic promoter (Vicente-Carbajosa et al., Plant J. 13: 629-640, 1998), rice prolamin NRP33 , rice globulin Glb-1 (Wu et al, Plant Cell Physiology 39(8) 885-889, 1998), rice alpha globulin REB/OHP-1 (Nakase et al. Plant Mol. Biol. 33: 513-S22 , 1997), rice ADP glucose PP (Trans Res 6:157-68, 1997), corn ESR gene family (Plant J 12:235-46, 1997), sorghum gamma kaphyrin (PMB 32:1029 -35, 1996)], embryonic specific promoters [e.g., rice OSH1 (Sato et al, Proc. Natl. Acad. Sci. USA, 93: 8117-8122), KNOX (Postma-Haarsma et al., Plant Mol Biol 39:257-71, 1999 ), rice oleosin (Wu et at, J. Biochem., 123:386, 1998)], and specific flower promoters [e.g., AtPRP4, chalene synthase (chsA) (Van der Meer, et al., Plant Mol. Biol. 15, 95-109, 1990), LAT52 (Twell et al. Mol. Gen. Genet. 217:240-245; 1989), Arabidopsis apetala-3 (Tilly et al., Development. 125:1647-57, 1998 ), Arabidopsis APETALA 1 (AT1G69120, API) (SEQ ID NO: 4888 (Arabidopsis (AT1G69120) APETALA 1)) (Hempel et al., Development 124:3845-3853, 1997)], and root promoter [e.g. ., the ROOTP promoter [SEQ ID. No.: 4889]; rice ExpB5 (SEQ ID NO: 4872 (rice promoter ExpB5); or SEQ ID NO: 4871 (longer rice promoter ExpB5)) and barley promoter ExpBl (SEQ ID NO: 4873) (Won et al. Mol. Cells 30: 369-376, 2010); Arabidopsis ATTPS-CIN promoter (AT3G25820) (SEQ ID No. 4890; Chen et al., Plant Phys 135:1956-66, 2004); Arabidopsis Pho1 promoter (SEQ ID No: 4870, Hamburger et al., Plant Cell. 14: 889-902, 2002), which is also mildly stress-induced].
[00234] Abiotic stress-inducible promoters include, but are not limited to, salt-inducible promoters such as RD29A (Yamaguchi-Shinozalei et al., Mol. Gen. Genet. 236:331-340, 1993); drought-inducible promoters such as the rab17 promoter gene (Pla et al., Plant Mol. Biol. 21:259-266, 1993), the maize rab28 promoter gene (Busk et al., Plant J. 11:1285 -1295, 1997 and the corn Ivr2 promoter gene (Pelleschi et al., Plant Mol. Biol. 39:373-380, 1999); heat-inducible promoters such as the tomato hsp80 promoter (U.S. Patent No. 5,187 .267).
[00235] The nucleic acid backbone of some applications of the invention may further include an appropriate selectable marker and/or origin of replication. According to some applications of the invention, the nucleic acid structure used is a bridging vector, which can propagate either in E. coli (where the structure comprises an appropriate selectable marker and the origin of replication) or can be compatible with propagation. in the cells. The structure according to the present invention can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.
[00236] The nucleic acid backbone of some applications of the invention can be used to stably or transiently transform plant cells. In stable transformation, the exogenous polynucleotide is integrated into the plant genome and, as such, represents a stable, inherited trait. In transient transformation, the exogenous polynucleotide is expressed by the transformed cell but is not integrated into the genome and, as such, represents a transient trait.
[00237] There are several methods for introducing foreign genes into both monocots and dicots ( Potrykus, I., Annu. Rev. Plant. Physiol, Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al. , Nature (1989) 338:274-276).
[00238] Cause principle methods of stable integration of exogenous DNA into plant genomic DNA include two main approaches: (i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J. and Vasil, L.K., Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S. and Arntzen, C.J., Butterworth Publishers, Boston, Mass. (1989) p. 93-112.
[00239] (ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J. and Vasil, L.K., Academic Publishers, San Diego, Calif. (1989) p. 52-68; including methods for direct uptake of DNA into protoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. Brief electrical shock-induced DNA uptake of plant cells: Zhang et al. Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986) 319:791-793. Injection of DNA into plant cells or tissues by particle bombardment, Klein et al. Bio/Technology (1988) 6:559563; McCabe et al. Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990) 79:206-209; by the use of micropipette systems: Neuhaus et al., Theor. App. Gene (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant. (1990) 79:213-217; transformation of glass fiber particles or silicon carbide from cell cultures, embryos or callous tissue, US Patent No. 5,464,765 or by direct incubation of DNA with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, G.P. and Mantell, S.H. and Daniels, W. Longman, London, (1985) p. 197-209; and Ohta, Proc. natl academy Sci. USA (1986) 83:715-719.
[00240] The Agrobacterium system includes the use of plasmid vectors that contain defined segments of DNA that integrate into the genomic DNA of the plant. Plant tissue inoculation methods vary depending on the plant species and Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiating whole-plant differentiation. See, e.g., Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplemental approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable for breeding transgenic dicots.
[00241] There are several methods of direct transfer of DNA to plant cells. In electroporation, protoplasts are briefly exposed to a strong electric field. In microinjection, DNA is mechanically injected directly into cells using very small micropipettes. In microparticle bombardment, DNA is absorbed into microprojectiles such as magnesium sulfate crystals or tungsten particles, and the microprojectiles are physically accelerated in plant cells or tissues.
[00242] After the stable transformation, plant propagation is carried out. The most common method of plant propagation is by seed. Regeneration by seed propagation, however, presents the deficiency that due to heterozygosity there is a lack of uniformity in the culture, since the seeds are produced by the plants according to the genetic variances governed by Mendelian norms. Basically, each seed is genetically different and each will grow with its own specific traits. Therefore, it is preferable that the transformed plant be produced in such a way that the regenerated plant has identical traits and characteristics of the transgenic plant arrangement. Therefore, it is preferred that the transformed plant is regenerated by micropropagation which provides for rapid, consistent reproduction of the transformed plants.
[00243] Micropropagation is a process of growing new generation plants from a piece of tissue that has been taken from a selected plant arrangement or cultivar. This process allows for the mass reproduction of plants that have the preferred tissue expressing the fusion protein. The plants of the new generation that are produced are genetically identical to the original plant and present all its characteristics. Micropropagation allows the mass production of quality plant material in a short period of time and offers a rapid multiplication of selected cultivars in order to preserve the characteristics of the original transgenic or transformed plant. The advantages of plant cloning are the speed of plant multiplication and the quality and uniformity of the plants produced.
[00244] Micropropagation is a multi-stage procedure that requires changing the culture medium or culture conditions between stages. Thus, the micropropagation process involves four basic stages: Stage one, initial tissue culture; stage two, tissue culture multiplication; stage three, plant differentiation and formation; and fourth stage, culture and strengthening in a greenhouse. During stage one, the initial tissue culture, the tissue culture is established and certified to be free of contaminants. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production objectives. During stage three, tissue samples grown in stage two are split and grown into individual seedlings. In stage four, the transformed seedlings are transferred to a greenhouse for strengthening, where the plants' light tolerance is gradually increased so that they can be grown in the natural environment.
[00245] According to some applications of the invention, transgenic plants are generated by the transient transformation of leaf cells, meristematic cells or the whole plant.
[00246] Transient transformation can be effected by any of the direct DNA transfer methods described above or by viral infection using modified plant viruses.
[00247] Viruses that have been shown to be useful for transforming plant hosts include CaMV, tobacco mosaic virus (TMV|tobacco mosaic virus), bromine mosaic virus (BMV|brome mosaic virus) and common mosaic virus. common bean (BV or BCMV|beam common mosaic virus). Transformation of plants using plant viruses is described in U.S. Patent No. 4,855,237 (bean golden mosaic virus; BGV), EP-A 67,553 (TMV), Japanese Published Application No. 6314693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudoviral particles for use in expressing foreign DNA in many hosts, including plants, are described in WO 87/06261.
[00248] According to some applications of the invention, the virus used for transient transformation is avirulent and thus is unable to cause severe symptoms such as reduced growth rate, mosaic, ring spots, leaf curl, yellowing, streaking, vesicle formation, tumor formation and corrosion. A suitable avirulent virus may be a naturally occurring avirulent virus or an artificially attenuated virus. Virus attenuation can be accomplished using methods well known in the art, including, but not limited to, sublethal heating, chemical treatment, or by targeted mutagenesis techniques as described, e.g., by Kurihara and Watanabe (Molecular Plant Pathology 4: 259-269, 2003), Galon et al. (1992), Atreya et al. (1992) and Huet et al. (1994).
[00249] Suitable viral strains can be obtained from available sources, eg, from the ATCC|American Type Culture Collection, or by isolating infected plants. Isolation of viruses from infected plant tissues can be accomplished by techniques well known in the art, as described, e.g., by Foster and Tatlor, Eds. “Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)”, Humana Press, 1998. The tissues of an infected plant are believed to quickly contain a high concentration of a virus. suitable, preferably young leaves and flower petals, are crushed in a buffer solution (eg, phosphate buffer solution) to produce a virus-infected sap that can be used in subsequent inoculations.
[00250] The structure of plant RNA viruses for the introduction and expression of non-viral exogenous polynucleotide sequences in plants is demonstrated by the above references, as well as by Dawson, WO et al., Virology (1989) 172:285- 292; Takamatsu et al. EMBO J. (1987) 6:307-311; French et al. Science (1986) 231:1294-1297; Takamatsu et al. FEBS Letters (1990) 269:73-76; and U.S. Patent No. 5,316,931.
[00251] When the virus is a DNA virus, suitable modifications can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid to facilitate the structure of the desired viral vector with the foreign DNA. Then the virus can be extracted from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA which is then replicated by the bacterium. Transcription and translation of this DNA will produce the coat protein that will encapsulate the viral DNA. If the virus is an RNA virus, the virus is usually cloned as a cDNA and introduced into a plasmid. Then the plasmid is used to make all the structures. Then, the RNA virus is produced by transcribing the viral sequence from the plasmid and translating the viral genes to produce the coat protein(s) that encapsulate the viral RNA.
[00252] In one application, a viral polynucleotide from a plant is provided, in which the native coat protein encoding the sequence has been excluded from a viral polynucleotide, a non-native plant viral coat protein encoding the sequence, and a non-native promoter. native, preferably the subgenomic promoter of the non-native coat protein encoding the sequence, capable of expressing itself in the plant host, in the envelope of the plant recombinant viral polynucleotide and ensuring a systemic infection of the host by the plant recombinant viral polynucleotide, was introduced . Alternatively, the coat protein gene can be inactivated by introducing the non-native polynucleotide sequence into it, so that a protein is produced. The recombinant plant viral polynucleotide may contain one or more additional non-native subgenomic promoters. Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or polynucleotide sequences in the plant host and unable to recombine with each other and with native subgenomic promoters. Non-native (foreign) polynucleotide sequences can be introduced adjacent to the plant-native viral subgenomic promoter, or the native viral subgenomic promoter and the plant non-native viral subgenomic promoter, if more than one polynucleotide sequence is included. Non-native polynucleotide sequences are transcribed or expressed in the host plant under the control of the subgenomic promoter to produce the desired products.
[00253] In a second application, a recombinant plant viral polynucleotide is provided, as in the first application, except that the native coat protein encoding the sequence is placed adjacent to one of the genomic promoters of the non-native coat protein rather than a non-native coat protein coding sequence.
[00254] In a third application, a recombinant plant viral polynucleotide is provided, in which the native coat protein gene is adjacent to its subgenomic promoter and one or more non-native subgenomic promoters have been inserted into the polynucleotide viral. The introduced non-native subgenomic promoters are able to transcribe or express adjacent genes in a plant host and are unable to recombine with each other and with native subgenomic promoters. Non-native polynucleotide sequences can be introduced adjacent to the plant's non-native viral subgenomic promoters so that the sequences are transcribed or expressed in the host plant under the control of the subgenomic promoter to produce the desired product.
[00255] In a fourth application, a recombinant plant viral polynucleotide is provided, as in the third application, such that the native coat protein coding sequence is replaced by a non-native coat protein coding sequence.
[00256] Viral vectors are encapsulated by the coat proteins encoded by the recombinant plant viral polynucleotide to produce a recombinant plant virus. Recombinant plant viral polynucleotide or recombinant plant virus is used to infect appropriate host plants. The recombinant plant viral polynucleotide is capable of replication in the host, systemic diffusion in the host, and transcription or expression of foreign gene(s) (exogenous polynucleotide) in the host to produce the desired protein.
[00257] Techniques for inoculating viruses into plants can be found in Foster and Taylor, eds. “Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)”, Humana Press, 1998; Maramorosh and Koprowski, eds. “Methods in Virology” 7 vols, Academic Press, New York 1967-1984; Hill, S.A. "Methods in Plant Virology", Blackwell, Oxford, 1984; Walkey, D.G.A. "Applied Plant Virology", Wiley, New York, 1985; and Kado and Agrawa, eds. “Principles and Techniques in Plant Virology”, Van Nostrand-Reinhold, New York.
[00258] In addition to the above, the polynucleotide of the present invention can also be introduced into a chloroplast genome, thereby allowing chloroplast expression.
[00259] A technique for introducing exogenous polynucleotide sequences into the genome of chloroplasts is known. This technique involves the following procedures. First, plant cells are chemically treated to reduce the number of chloroplasts per cell to approximately one. Then, the exogenous polynucleotide is introduced through particle bombardment into the cells with the aim of introducing at least one molecule of the exogenous polynucleotide into the chloroplasts. Exogenous polynucleotides are selected to be integrable into the chloroplast genome through homologous recombination that is readily effected by chloroplast-inherent enzymes. To that end, the exogenous polynucleotide includes, in addition to a gene of interest, at least an extension of the polynucleotide that is derived from the chloroplast genome. In addition, the exogenous polynucleotide includes a selectable marker that serves, by sequential selection procedures, to verify that all or substantially all copies of the chloroplast genomes, after such selection, will include the exogenous polynucleotide. Additional details relating to this technique are found in U.S. Patent Nos. 4,945,050 and 5,693,507 which are incorporated herein by reference. In this way, a polypeptide can be produced by the chloroplast protein expression system and integrates into the inner membrane of the chloroplast.
[00260] According to some applications, a method is provided to improve nitrogen use efficiency, fertilizer use efficiency, yield, growth rate, biomass, vigor, oil content, seed yield, fiber yield, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of a grafted plant, the method comprising providing a graft that does not transgenically express a polynucleotide encoding a polypeptide at least 80% homologous to the sequence of amino acids selected from the group consisting of SEQ IDs. No 496-794 and 2898-4855 and a plant rhizome transgenically expressing a polynucleotide encoding a polypeptide at least about 80%, at least about 81%, at least about 82%, at least about at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 89% 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96 %, at least about 97%, at least about 98%, at least about 99%, e.g., 100% homologous (or identical) to the amino acid sequence selected from the group consisting of SEQ IDs. N° 496-794, 2898-3645, 3647-4854 and 4855 (eg, in a constitutive or responsive form to abiotic stress), thereby improving nitrogen use efficiency, fertilizer use efficiency, production , growth rate, biomass, vigor, oil content, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance of the grafted plant.
[00261] In some applications, the plant graft is non-GMO.
[00262] Several applications refer to a grafted plant, exhibiting an increase in nitrogen use efficiency, fertilizer use efficiency, production, growth rate, biomass, vigor, oil content, seed production, fiber production , fiber quality, fiber length, photosynthetic capacity and/or abiotic stress tolerance, comprising a graft that does not transgenically express a polynucleotide encoding a polypeptide at least 80% homologous to the selected amino acid sequence from the group consisting of IDs SEQ Nos. 496-794 and 2898-4855 and a plant rhizome transgenically expressing a polynucleotide encoding a polypeptide, at least about 80%, at least about 81%, at least about 82%, at least about at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 89% 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96 %, at least about 97%, at least about 98%, at least about 99%, e.g., 100% homologous (or identical) to the amino acid sequence selected from the group consisting of SEQ IDs. No. 496-794, 2898-3645, 3647-4854 and 4855.
[00263] In some applications, the plant rhizome transgenically expresses a polynucleotide that encodes a polypeptide, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 90% at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g. e.g., 100% homologous (or identical) to the amino acid sequence selected from the group consisting of SEQ IDs. 496-794, 2898-3645, 3647-4854 and 4855 in a stress-responsive manner.
[00264] In accordance with some applications of the invention, the plant rhizome transgenically expresses a polynucleotide encoding a polypeptide selected from the group consisting of SEQ IDs. Nos. 496-794, 2898-4854 and 4855.
[00265] In accordance with some applications of the invention, the plant rhizome transgenically expresses a polynucleotide, comprising a nucleic acid sequence, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 89% at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g., 100% identical to the polynucleotide selected from the group consisting of SEQ IDs. No. 1-495 and 795-2897.
[00266] In accordance with some applications of the invention, the plant rhizome transgenically expresses a polynucleotide selected from the group consisting of SEQ IDs. No. 1-495 and 795-2897.
[00267] Since the processes that increase nitrogen use efficiency, fertilizer use efficiency, oil content, production, seed production, fiber production, fiber quality, fiber length, photosynthetic capacity, rate of A plant's growth, biomass, vigor and/or tolerance to abiotic stress may involve multiple genes acting either additively or synergistically (see, for example, Quesda et al., Plant Physiol. 130:951-063, 2002), the present The invention also provides for expressing a plurality of exogenous polynucleotides in a single host plant to thereby achieve a superior effect on nitrogen use efficiency, fertilizer use efficiency, oil content, yield, seed yield, fiber yield, fiber quality, fiber length, photosynthetic capacity, growth rate, biomass, vigor and/or tolerance to abiotic stress.
[00268] Expression of a plurality of exogenous polynucleotides in a single host plant can be accomplished by co-introducing multiple nucleic acid structures, each including a different exogenous polynucleotide, into a single plant cell. The transformed cell can then be regenerated into a mature plant using the methods described above.
[00269] Alternatively, expression of a plurality of exogenous polynucleotides in a single host plant may be effected by co-introducing, in a single plant cell, a single nucleic acid structure including a plurality of different exogenous polynucleotides. This structure can be designed with a single promoter sequence that can transcribe a polycistronic messenger RNA including all the different exogenous polynucleotide sequences. In order to allow co-translation of the different polypeptides encoded by polycistronic messenger RNA, the polynucleotide sequences can be linked through an internal ribosome entry site (IRES|internal ribosome entry site) sequence that facilitates the translation of the positioned polynucleotide sequences. downstream of the IRES sequence. In this case, a transcribed polycistronic RNA molecule that encodes the different polypeptides described above will be translated both at the 5' end that has the cap and in the two internal sequences of the IRES of the polycistronic RNA molecule to thus produce all the different polypeptides in the cell. . Alternatively, the structure may include several promoter sequences, each linked to a different exogenous polynucleotide sequence.
[00270] The plant cell transformed with the framework, including a plurality of different exogenous polynucleotides, can be regenerated into a mature plant using the methods described above.
[00271] Alternatively, expression of a plurality of exogenous polynucleotides in a single host plant may be effected by introducing different nucleic acid structures, including different exogenous polynucleotides, into a plurality of plants. The regenerated transformed plants can then be crossed and the resulting progeny selected to obtain superior traits of abiotic stress tolerance, water use efficiency, fertilizer use efficiency, growth, biomass, yield and/or vigor, using conventional techniques. of plant breeding.
[00272] According to some applications of the invention, the method further comprises culturing the plant expressing the exogenous polynucleotide under abiotic stress.
[00273] Non-limiting examples of abiotic stress conditions include salinity, osmotic stress, drought, water deprivation, excess water (eg, flooding, waterlogging), etiolation, low temperature (eg, cold stress ), high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency (eg, nitrogen deficiency or nitrogen limitation), nutrient excess, air pollution, and UV irradiation.
[00274] According to some applications of the invention, the method further comprises culturing the plant that expresses the exogenous polynucleotide under fertilizer-limiting conditions (eg, nitrogen-limiting conditions). Non-limiting examples include growing the plant in soils with low nitrogen content (40-50% nitrogen of the present content under normal and ideal conditions), or even in severe nitrogen deficiency (0 to 10% nitrogen of the present content under normal and ideal conditions).
[00275] Thus, the invention encompasses plants that exogenously express the polynucleotide(s), nucleic acid structures and/or polypeptide(s) of the invention.
[00276] Once expressed within the plant cell or the entire plant, the level of the polypeptide encoded by the exogenous polynucleotide can be determined by methods well known in the art, such as activity assays, Western blots using antibodies capable of specifically binding to the polypeptide , Enzyme-linked immunosorbent assay (ELISA | enzyme-linked immunosorbent assay), radioimmunoassays (RIA|radio-immuno-assays), immunohistochemistry, immunocytochemistry, immunofluorescence and other similar methods.
[00277] Methods for determining the level of exogenous polynucleotide RNA transcribed in the plant are well known in the art and include, e.g., Northern blot analysis, reverse transcription polymerase chain reaction (RT-PCR|reverse transcription) analysis polymerase chain reaction) (including quantitative, semi-quantitative or real-time RT-PCR) and in situ hybridization to RNA.
[00278] Sequence information and annotations not covered by the present teachings can be leveraged in favor of classical improvement. Thus, the subsequence data of those polynucleotides described above can be used as markers for marker assisted selection (MAS | marker assisted selection), in which a marker is used for indirect selection of a genetic determinant(s) of a trait. interest (eg, biomass, growth rate, oil content, yield, abiotic stress tolerance, water use efficiency, nitrogen use efficiency, and/or fertilizer use efficiency). The nucleic acid data of the present teachings (DNA or RNA sequence) may contain or be linked to polymorphic sites or genetic markers in the genome such as restriction fragment length polymorphism (RFLP), microsatellites and polymorphism nucleotide polymorphism (SNP|single nucleotide polymorphism), genetic fingerprinting (DFP|DNA fingerprinting), amplified fragment length polymorphism (AFLP|amplified fragment length polymorphism), expression level polymorphism, encoded polypeptide polymorphism and any other polymorphism in the DNA or RNA sequence.
[00279] Examples of marker assisted selections include, but are not limited to, selection for a morphological trait (e.g., a gene that affects shape, coloration, male sterility, or resistance such as the presence or absence of edge, leaf sheath color, height, grain color, rice aroma); selection of a biochemical trait (eg, a gene that encodes a protein that can be extracted and observed; eg, isozymes and storage proteins); selection of a biological trait (eg, (pathogenic races or insect biotypes based on the host pathogen or interaction with the host parasite may be used as a marker since the genetic makeup of an organism may affect its susceptibility to pathogens) or parasites).
[00280] The polynucleotides and polypeptides described above can be used in a wide variety of economical plants, safely and cost-effectively.
[00281] Plant strains that exogenously express the polynucleotide or polypeptide of the invention are screened to identify those that show the greatest increase in the desired plant trait.
[00282] Therefore, in accordance with a further application of the present invention, there is provided a method of evaluating a trait from a plant, the method comprising: (a) expressing in a plant or a part thereof the nucleic acid structure of some applications of the invention; and (b) evaluating a plant trait against a wild-type plant of the same type (e.g., not transformed with the claimed biomolecules); thus evaluating the trait of the plant.
[00283] In accordance with some applications of the invention, there is provided a method of producing a crop, comprising culturing a crop of a plant expressing an exogenous polynucleotide, comprising a nucleic acid sequence encoding a polypeptide of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 86% 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94 %, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or, say, 100% homologous to the selected amino acid sequence from a group consisting of the SEQ IDs. N° 496-794, 2898-3645, 3647-4854 and 4855, characterized in that said plant is derived from a plant selected to increase fertilizer use efficiency, increase nitrogen use efficiency, increase tolerance to abiotic stress, increase in water use efficiency, increase in growth rate, increase in biomass, increase in vigor, increase in oil content, increase in production, increase in seed production, increase in fiber production, increase in fiber quality, increase in fiber length and/or increase in photosynthetic capacity compared to a control plant, thus producing the crop.
[00284] According to one aspect of some applications of the present invention, there is provided a method of producing a culture, comprising cultivating a plant culture transformed with an exogenous polynucleotide that encodes a polypeptide, at least 80% at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 87% at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 94% 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or, say, 100% homologous (e.g., identical) to the amino acid sequence selected from the group consisting of the SEQ IDs. N° 496-794, 2898-3645, 3647-4854 and 4855, characterized by the crop plant being derived from plants selected for an increase in abiotic stress tolerance, increase in water use efficiency, increase in growth rate, increase in vigor, increase in biomass, increase in oil content, increase in production, increase in seed production, increase in fiber production, increase in fiber quality, increase in fiber length, increase in photosynthetic capacity and/or increase in fertilizer use efficiency (eg, increased nitrogen use efficiency) compared to a wild type plant of the same species that is grown under the same growing conditions, and the crop plant having increased stress tolerance abiotic, increase in water use efficiency, increase in growth rate, increase in vigor, increase in biomass, increase in oil content, increase in production, increase in seed production, increase in fiber production, increase in q fiber quality, increased fiber length, increased photosynthetic capacity, and/or increased fertilizer use efficiency (eg, increased nitrogen use efficiency), thereby producing the crop.
[00285] In accordance with some applications of the invention, the polypeptide is selected from the group consisting of SEQ IDs. Nos. 496-794, 2898-4854 and 4855.
[00286] In accordance with one aspect of some applications of the invention, there is provided a method of producing a culture, comprising culturing a culture of a plant expressing an exogenous polynucleotide that comprises a nucleic acid sequence that is at least about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 86% 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93 %, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g. 100 % identical to the nucleic acid sequence selected from the group consisting of SEQ IDs. N° 1-495, 795-2896 and 2897, characterized in that said plant is derived from a plant (main plant) that was transformed to express the exogenous polynucleotide and that was selected to increase tolerance to abiotic stress, increase efficiency in use of water, increase in growth rate, increase in vigor, increase in biomass, increase in oil content, increase in production, increase in seed production, increase in fiber production, increase in fiber quality, increase in fiber length , increased photosynthetic capacity and/or increased fertilizer use efficiency (eg, increased nitrogen use efficiency) compared to a control plant, thereby producing the crop.
[00287] In accordance with one aspect of certain applications of the invention, there is provided a method of producing a culture, comprising cultivating a plant culture transformed with an exogenous polynucleotide, at least 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 88%, at least about 88% at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or say, 100% identical to the nucleic acid sequence selected from the group consisting of SEQ IDs. N° 1- 495, 795-2896 and 2897, characterized by the crop plant being derived from selected plants to increase tolerance to abiotic stress, increase efficiency in water use, increase growth rate, increase vigor, increase biomass , increase in oil content, increase in production, increase in seed production, increase in fiber production, increase in fiber quality, increase in fiber length, increase in photosynthetic capacity and/or increase in fertilizer use efficiency ( e.g., increased nitrogen use efficiency) compared to a wild-type plant of the same species that is grown under the same growing conditions, and the crop plant having an increased tolerance to abiotic stress, increased efficiency in water use, increase in growth rate, increase in vigor, increase in biomass, increase in oil content, increase in production, increase in seed production, increase in fiber production, increase in fiber quality bra, increase in fiber length, increase in photosynthetic capacity, and/or increase in fertilizer use efficiency (eg, increase in nitrogen use efficiency) thereby producing the crop.
[00288] In accordance with some applications of the invention, the exogenous polynucleotide is selected from the group consisting of SEQ IDs. No. 1-495, 795-2896 and 2897.
[00289] According to one aspect of some applications of the invention, there is provided a method for growing a crop, comprising sowing seeds and/or planting seedlings of a plant transformed with the exogenous polynucleotide of the invention, for example, the polynucleotide that encodes the polypeptide of some applications of the invention, characterized in that the plant is derived from selected plants with at least one trait selected from the group consisting of increased tolerance to abiotic stress, increased efficiency in water use, increased rate of growth, vigor increase, biomass increase, oil content increase, production increase, seed production increase, fiber production increase, fiber quality increase, fiber length increase, photosynthetic capacity increase and/or or increased fertilizer use efficiency (increased nitrogen use efficiency) compared to an untransformed plant.
[00290] According to some applications of the invention, the method of growing a crop comprises sowing seeds and/or planting seedlings of a plant transformed with an exogenous polynucleotide, comprising a nucleic acid sequence encoding a polypeptide, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 86% at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g. 100% identical to IDs SEQ N° 496-794, 2898-3645, 3647-4854 or 4855, characterized in that the plant is derived from selected plants with at least one trait selected from the group consisting of increased tolerance to abiotic stress, increased efficiency in water use , increase in growth rate, increase in vigor, increase in biomass, increase in oil content, increase in production, increase in seed production, increase in fiber production, increase in fiber quality, increase in fiber length, increase photosynthetic capacity and/or increased efficiency in the use of fertilizers (increased efficiency in the use of nitrogen) compared to an untransformed plant, thus increasing the harvest.
[00291] In accordance with some applications of the invention, the polypeptide is selected from the group consisting of SEQ IDs. Nos. 496-794, 2898-4854 and 4855.
[00292] In accordance with some applications of the invention, the method of growing a crop comprises sowing seeds and/or planting seedlings of a plant transformed with an exogenous polynucleotide, the nucleic acid sequence comprising at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87 %, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94% , at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g., 100% identical to SEQ IDs. N° 1-495, 795-2896 or 2897, characterized by the plant being derived from selected plants with at least one trait selected from the group consisting of increased tolerance to abiotic stress, increased efficiency in water use, increased rate growth rate, vigor increase, biomass increase, oil content increase, production increase, seed production increase, fiber production increase, fiber quality increase, fiber length increase, photosynthetic capacity increase and /or increased efficiency in the use of fertilizers (increased efficiency in the use of nitrogen) compared to an untransformed plant, thus increasing the harvest.
[00293] In accordance with some applications of the invention, the exogenous polynucleotide is selected from the group consisting of SEQ IDs. No. 1-495, 795-2896 and 2897.
[00294] The effect of transgene (the exogenous polynucleotide encoding the polypeptide) on abiotic stress tolerance can be determined using known methods, as detailed below and in the Examples section below.
[00295] Abiotic Stress Tolerance - Transformed (i.e., expressing the transgene) and untransformed (wild type) plants are exposed to a condition of abiotic stress such as water deprivation, suboptimal temperature (low temperature, high temperature), deficiency of nutrients, nutrient excess, a stress condition caused by salt, osmotic stress, heavy metal toxicity, anaerobiosis, smog, and UV irradiation.
[00296] Salinity Tolerance Assay - Transgenic plants with tolerance to high salt concentrations are expected to show better germination, seedling vigor or growth in conditions with high salt levels. Salt stress can be effected in many ways such as, e.g., irrigating the plants with a hyperosmotic solution, growing the plants hydroponically in a hyperosmotic growing solution (e.g., Hoagland's solution), or subjecting the plants culture in hyperosmotic growth medium [eg, 50% Murashige-Skoog medium (MS medium)]. As different plants vary considerably in their tolerance to salinity, the concentration of salt in irrigation water, growing solution or growing medium can be adjusted according to the specific characteristics of the cultivar or specific plant variety in order to inflict a mild or moderate effect on plant physiology and/or morphology (for guidelines regarding appropriate concentration, see, Bernstein and Kafkafi, Root Growth Under Salinity Stress In: Plant Roots, The Hidden Half 3rd ed. Waisel Y, Eshel A and Kafkafi U. (editors) Marcel Dekker Inc., New York, 2002 and the reference therein).
[00297] For example, the salinity tolerance test can be performed by irrigating plants at different stages of development with increasing concentrations of sodium chloride (eg, 50 mM, 100 mM, 200 mM, 400 mM NaCl) applied from below and above to ensure even dispersion of the salt. After exposure to the stress condition, plants are often monitored until physiological and/or morphological effects appear in wild-type plants. In this way, external phenotypic appearance, degree of wilting and overall success to reach maturity and production progeny are compared between control and transgenic plants.
[00298] Quantitative tolerance parameters measured include, but are not limited to, average wet and dry weight, growth rate, leaf size, leaf cover (general leaf area), weight of seeds generated, average size of seeds and the number of seeds produced per plant. Transformed plants that do not exhibit substantial physiological and/or morphological effects, or that exhibit greater biomass than wild-type plants, are identified as abiotic stress-tolerant plants.
[00299] Osmotic Tolerance Test - Osmotic stress assays (including sodium chloride and mannitol assays) are performed to determine whether an osmotic stress phenotype was specific to sodium chloride or whether it was a phenotype related to general osmotic stress. Osmotic stress-tolerant plants may have more drought and/or freeze tolerance. For germination experiments under saline and osmotic stress, the medium is supplemented, eg, with 50 mM, 100 mM, 200 mM NaCl or with 100 mM, 200 mM NaCl, 400 mM mannitol.
[00300] Drought Tolerance Assay/Osmotic Assay - Drought Tolerance is performed to identify genes that confer better plant survival after acute water deprivation. To analyze whether transgenic plants are more drought tolerant, an osmotic stress produced by the non-ionic sorbitol osmolyte in the medium can be performed. Control and transgenic plants are germinated and grown on plant agar plates for 4 days, after which they are transferred to plates containing 50 mM sorbitol. The treatment causes growth retardation, so both control and transgenic plants are compared, measuring plant weight (wet and dry), yield and growth rates measured as time to flowering.
[00301] Conversely, soil-based dry screens are performed with plants overexpressing the polynucleotides detailed above. Seeds from control Arabdopsis plants, or from other transgenic plants that overexpress the polypeptide of the invention, are germinated and transferred to pots. Drought stress is obtained after irrigation is stopped, accompanied by placing the pots on absorbent paper to improve the rate of soil drying. Transgenic and control plants are compared with each other when most control plants develop severe wilting. Plants are given water again after getting a significant fraction of the control plants requiring severe wilting. Plants are ranked compared to controls with respect to each of two criteria: tolerance to drought conditions and recovery (survival) after re-watering.
[00302] Cold stress tolerance - To analyze cold stress, mature plants (25 days old) are transferred to chambers at 4°C for 1 or 2 weeks, with constitutive light. Afterwards, the plants are returned to the greenhouse. Two weeks later, the damage caused by the cooling period, resulting in growth retardation and other phenotypes, is compared between both control and transgenic plants, measuring plant weight (wet and dry) and comparing measured growth rates. such as time to flowering, plant size, production and similar parameters.
[00303] Heat Stress Tolerance - Heat stress tolerance is achieved by exposing plants to temperatures above 34°C for a certain period. Plant tolerance is examined after transferring plants back to 22°C for recovery and evaluation after 5 days against internal controls (non-transgenic plants) or plants not exposed to either cold stress or heat stress .
[00304] Efficiency in water use - can be determined as the biomass produced by unit transpiration. To analyze WUE, relative leaf water content can be measured in control and transgenic plants. The fresh weight (FW | fresh weight) is recorded immediately; then, the leaves are placed for 8 hours in distilled water at room temperature in the dark and the turgid weight (TW | turgid weight) is recorded. The total dry weight (DW | dry weight) is recorded after drying the sheets at 60°C at constant weight. The relative water content (RWC | relative water content) is calculated according to the following Formula I: Formula IRWC = [(FW - DW) / (TW - DW)] x 100
[00305] Fertilizer Use Efficiency - To analyze whether transgenic plants are more responsive to fertilizers, the plants were grown on agar plates or pots with a limited amount of fertilizer, as described, for example, in Examples 15-17 below and in Yanagisawa et al (Proc Natl Acad Sci USA. 2004; 101:7833-8. The plants are analyzed for their general size, time to flowering, production, bud and/or grain protein content. The parameters verified are the general size of the mature plant, its wet and dry weight, the weight of the seeds generated, the average size of the seed and the number of seeds produced per plant. Other parameters that can be tested are: the chlorophyll content of the leaves (such as the nitrogen status of the plant and the degree of leaf greenness are highly correlated), the amino acid and total protein content of the seeds or other parts of the plant such as leaves or buds, the oil content, etc. Similarly, instead to supply nitrogen in limiting amounts, phosphate or potassium can be added in increasing concentrations. Again the same measured parameters are the same as listed above. In this way, the nitrogen use efficiency (NUE), the phosphate use efficiency (PUE | phosphate use efficiency) and the potassium use efficiency (KUE | potassium use efficiency) are evaluated, verifying the capacity of the transgenic plants to grow under nutrient restriction conditions.
[00306] Nitrogen use efficiency - To analyze whether transgenic plants (eg Arabidopsis plants) are more responsive to nitrogen, plants are grown in 0.75-3 mM (nitrogen deficiency conditions) or 6 -10 mM (adequate nitrogen concentration). Plants are allowed to grow for an additional 25 days or until seed production. Then, the plants are analyzed for their overall size, time to flowering, production, bud and/or grain/seed protein content. The parameters verified can be the general size of the mature plant, its wet and dry weight, the weight of the seeds generated, the average size of the seed and the number of seeds produced per plant. Other parameters that can be tested are: the chlorophyll content of the leaves (as the nitrogen status of the plant and the degree of leaf greenness are highly correlated), the amino acid and total protein content of the seeds or other parts of the plant like the leaves or buds the oil content. Transformed plants that do not exhibit substantial physiological and/or morphological effects, or that exhibit higher levels of the measured parameters than wild-type plants, are identified as nitrogen-efficient plants.
[00307] Nitrogen use efficiency test using seedlings - The test is performed according to Yanagisawa-S. et al. with minor modifications (“Metabolic engineering with Dofl transcription factor in plants: Improved nitrogen assimilation and growth under low-nitrogen conditions” Proc. Natl. Acad. Sci. USA 101, 7833-7838). Briefly, transgenic plants that are grown for 7-10 days in 0.5 x MS [Murashige-Skoog] supplemented with a selection agent are transferred to two nitrogen limiting conditions: MS medium in which the combined nitrogen concentration ( NH4NO3 and KNO3) was 0.75 mM (nitrogen deficiency conditions) or 6-15 mM (adequate nitrogen concentration). The plants were allowed to grow for an additional 30-40 days and were then photographed, individually removed from the agar (the bud without the roots) and immediately weighed (fresh weight) for further statistical analysis. Structures for which only T1 seeds are available are sown on selective medium and at least 20 seedlings (each representing an independent transformation event) are carefully transferred to the nitrogen-limiting medium. For structures in which T2 seeds are available, different transformation events are analyzed. Generally, 20 plants selected at random from each event are transferred to the nitrogen-limiting medium and allowed to grow for an additional 3-4 weeks and weighed individually at the end of that period. The transgenic plants are compared to control plants grown in parallel under the same conditions. Mock transgenic plants expressing the uidA reporter gene (GUS) under the same promoter, or transgenic plants carrying the same promoter but lacking a reporter gene, are used as controls.
[00308] Determination of nitrogen - The procedure for determining the concentration of N (nitrogen) in the structural parts of plants involves the method of digestion of potassium persulfate to convert organic N into NO3-(Purcell and King 1996 Argon. J. 88:111-113), the modified Cd-mediated reduction of NO3- to NO2- (Vodovotz 1996 Biotechniques 20:390-394) and the measurement of nitrite by the Griess assay (Vodovotz 1996, supra). Absorbance values are measured at 550 nm against a standard NaNO2 curve. The procedure is described in detail in Samonte et al. 2006 Agro. J. 98:168-176.
[00309] Germination tests - Germination tests compare the percentage of seeds from transgenic plants that could complete the germination process to the percentage of seeds from control plants that are treated in the same way. Normal conditions are considered, for example, incubations at 22°C under daily cycles of 22 hours light and 2 hours dark. The evaluation of seedling germination and vigor is carried out between 4 and 14 days after planting. The basal medium is 50% MS medium (Murashige and Skoog, 1962 Plant Physiology 15, 473-497).
[00310] Germination is also verified under unfavorable conditions such as cold (incubation at temperatures below 10°C instead of 22°C) or using solutions for seed inhibition that contain high concentrations of an osmolyte such as sorbitol (in of 50 mM, 100 mM, 200 mM, 300 mM, 500 mM and up to 1000 mM) or by applying increasing concentrations of salt (from 50 mM, 100 mM, 200 mM, 300 mM, 500 mM NaCl).
[00311] The effect of the transgene on the vigor, growth rate, biomass, production and/or oil content of the plant can be determined using known methods.
[00312] Plant Vigor - Plant vigor can be calculated by increasing growth parameters such as leaf area, fiber length, rosette diameter, plant fresh weight and similar parameters per period.
[00313] Growth Rate - The growth rate can be measured using digital analysis of cultivated plants. For example, terrain-based images of greenhouse-grown plants can be captured every 3 days and the rosette area can be calculated by digital analysis. Rosette area growth is calculated using the difference in rosette area between sampling days divided by the difference in days between samples.
[00314] The assessment of growth rate can be performed by measuring the produced plant biomass, leaf size or root length per period (can be measured in cm2 per day of leaf area). The Relative Growth Area can be be calculated using Formula II. Formula II: Area of the Relative Growth Rate = Regression coefficient of the area over time.
[00315] Thus, the relative growth rate of area is in units of area units (eg, mm2/day or cm2/day) and the relative growth rate of length is in units of length units ( eg cm/day or mm/day).
[00316] For example, RGR can be determined for plant height (Formula III), SPAD (Formula IV), Number of tillers (Formula V), root length (Formula VI), plant growth (Formula VII), number of leaves (Formula VIII), rosette area (Formula IX), rosette diameter (Formula X), plot coverage (Formula XI), leaf blade area (Formula XII) and leaf area (Formula XIII).Formula III : Plant Height Relative Growth Rate = Plant Height Regression Coefficient Over Time Course (measured in cm/day).Formula IV: SPAD Relative Growth Rate = Regression coefficient of SPAD measurements over time time course.Formula V: Relative growth rate of Number of tillers = Regression coefficient of Number of tillers over time course (measured in units of “number of tillers/day”).Formula VI: Relative growth rate of root length = Regression coefficient of root length over the course of temp o (measured in cm per day).
[00317] Plant growth rate analysis - was calculated according to Formula VII, below.Formula VII: Relative plant growth rate = Regression coefficient of plant weight over time course (measured in grams per day ).Formula VIII: Relative growth rate of the number of leaves = Regression coefficient of the number of leaves over the course of time (measured in number per day).Formula IX: Relative growth rate of rosette areas = Regression coefficient of rosette area over time course (measured in cm2 per day).Formula X: Relative growth rate of rosette diameter = Regression coefficient of rosette diameter over time course (measured in cm per day) .Formula XI: Relative growth rate of plot coverage = Plot regression coefficient over time course (measured in cm2 per day).Formula XII: Relative growth rate of leaf blade area = Area regression coefficient from the leaf to over time course (measured in cm2 per day).Formula XIII: Relative growth rate of leaf area = Regression coefficient of leaf area over time course (measured in cm2 per day).Formula XIV:Weight of 1000 Seeds = number of seeds in the sample/weight of the sample X 1000.
[00318] The Harvest Index can be calculated using Formulas XV, XVI, XVII, XVIII and XXXVII below. Formula XV: Harvest Index (seed) = Average seed production per plant/average dry weight.Formula XVI: Harvest Index (Sorghum) = Average grain dry weight per Head/(average vegetable dry weight per Head + average dry weight per head)Formula XVII: Crop Index (Corn) = Average grain weight per plant/(average plant dry weight per plant plus average grain weight per plant)
[00319] Harvest Index (for barley) - The Harvest Index is calculated using Formula XVIII.Formula XVIII:Harvest Index (for barley and wheat) = Average ear dry weight per plant / (average plant dry weight per plant + average ear weight per plant)
[00320] The following is an unlimited list of additional parameters that can be detected in order to show the effect of transgenics on the desired plant tracts.Formula XIX: Grain circularity = 4 x 3.14 (area of grain / perimeter 2)Formula XX:internode volume = 3.14 x (d/2) 2 x l.Formula XXI:Standardized ear weight per plant + plant dry weight.Formula XXII:Root/Sprouts Ratio = total root weight at harvest / total weight of the plant part above the ground at the time of harvest. (=RBiH/BiH)Formula XXIII:Ratio between the number of pods per node on the main stem together pods = Total number of pods on the main stem / total number of nodes on the main stem.Formula XXIV:Ratio of the total number of seeds in the main stem to number of seeds on side branches = Total number of seeds on main stem in pod set / Total number of seeds on side shoot in pod set.Formula XXV: Relative Petiole Area = (Petiole Area) / Area of rosette (measured in %). Formula XXVI: percentage (%) of reproductive tillers = Number of reproductive tillers / number of tillers) X 100.Formula XXVII: Ear Index = Average ear weight per plant / (average plant dry weight per plant plus Average Ear Weight per plant plant)Formula XXVIII: Relative growth rate of root cover = Regression coefficient of root cover over time course.Formula XXIX: Seed Oil Production = Seed production per plant (g.) * % Oil in the seed.Formula XXX: Shoot/root ratio = total weight of the plant part above the ground at the time of harvest / total weight of the root at harvest. (=RBiH/BiH)Formula XXXI: Spikelet Index = Average spikelet weight per plant / (average plant dry weight per plant plus average spikelet weight per plant)Formula XXXII: % Canopy Coverage = (1-(PAR_ABAIXO/PAR_ABOVE ))x100. Formula XXXIII: Leaf Mass Fraction = Leaf / Shoot Area.Formula XXXIV: Relative Growth Rate Based on Dry Weight = Dry Weight Regression Coefficient Over Time Course.Formula XXXV:Total Dry Matter (for Corn ) = Normalized ear weight per plant + plant dry weight.Formula XXXVI:
Formula XXXVII:Harvest Index (Brachypodium) = Average grain weight/vegetable dry weight (vegetable + spikelet) per plant.Formula XXXVIII:Sorghum Harvest Index* (* when plants are not dry) = FW (fresh weight| fresh weight) of the Heads/(FW of the Heads + FW of the Plants)
[00321] Grain filling rate [mg/day] - Dry matter accumulation rate in the grain. Grain fill rate is calculated using Formula XXXIX.Formula XXXIX:Grain fill rate [mg/day] = [Grain weight*cob-1 x 1000] / [Number of grain*cob-1] x Duration of grain filling].
[00322] Grain protein concentration - The protein content in the grain (g of protein in the grain m-2) is estimated as the product of the mass of the grain N (g of N of the m-2) multiplied by the conversion rate of N/protein of k-5,13 (Mosse 1990, supra). The protein concentration in the grain is estimated as the ratio of the protein content in the grain per unit mass of the grain (g of protein in the grain kg-1 of the grain).
[00323] Fiber Length - Fiber length can be measured using a fibrograph. The fibrograph system was used to compute the length in terms of “Average Upper Half” length. The upper half mean (UHM | upper half mean) is the average length of the longest half of the fiber distribution. The fibrograph measures length in span lengths at a given percentage point (cottoninc (dot) com/ClassificationofCotton/ Pg=4#Length).
[00324] According to some applications of the invention, the increase in corn production can be manifested as one or more of the following: increase in the number of plants per growing area, increase in the number of ears per plant, increase in the number of rows per ear, number of grains per ear row, grain weight, thousand grain weight (weight per 1000), ear length/diameter, increase in oil content per grain and increase in starch content per grain.
[00325] As mentioned, the increase in plant production can be determined by several parameters. For example, an increase in rice production can be manifested by an increase in one or more of the following: number of plants per growing area, number of panicles per plant, number of spikelets per panicle, number of flowers per panicle, increase in seed filling rate, increase in weight per thousand grains (weight per 1000), increase in oil content per seed, increase in starch content per seed, among others. An increase in production can also result in the modified architecture, or it can occur because of the modified architecture.
[00326] Similarly, the increase in soybean production can be manifested by an increase in one or more of the following: number of plants per growing area, number of pods per plant, number of seeds per pod, increase in the rate filling of the seed, increase in the weight of 1000 seeds (weight per 1000), reduction in pod tearing, increase in oil content per seed, increase in protein content per seed, among others. An increase in production can also result in the modified architecture, or it can occur because of the modified architecture.
[00327] Increased canola production can be manifested by an increase in one or more of the following: number of plants per growing area, number of pods per plant, number of seeds per pod, increase in seed filling rate , increase in the weight of 1000 seeds (weight per 1000), reduced pod tearing, increased oil content per seed, among others.
[00328] The increase in cotton production can be manifested by an increase in one or more of the following: number of plants per growing area, number of bolls per plant, number of seeds per boll, increase in seed filling rate , increase in the weight of a thousand seeds (weight per 1000), increase in the oil content per seed, improvement in fiber length, fiber strength, among others. An increase in production can also result in the modified architecture, or it can occur because of the modified architecture.
[00329] Oil Content - The oil content of a plant can be determined by extracting the oil from the seed or vegetable portion of the plant. Briefly, lipids (oil) can be removed from the plant (eg, seed) by grinding the plant tissue in the presence of specific solvents (eg, hexane or petroleum ether) and extracting the oil in a continuous extractor. . Indirect analysis of oil content can be performed using various known methods such as Nuclear Magnetic Resonance Spectroscopy (NMR), which measures the resonance energy absorbed by hydrogen atoms in the liquid state of the sample [See, p. eg, Conway TF. and Earle FR., 1963, Journal of the American Oil Chemists' Society; Springer Berlin / Heidelberg, ISSN: 0003-021X (Print) 1558-9331 (Online)]; Near Infrared Spectroscopy (NI | near infrared), which uses the absorption of near infrared energy (1100-2500 nm) by the sample; and a method described in W0/2001/023884, which is based on extracting oil with solvent, evaporating the solvent in a gas stream that forms oil particles and directing a light into the gas stream and on the oil particles, which forms a detectable reflected light.
[00330] Thus, the present invention is of high agricultural value to promote the production of commercially desired crops (e.g., biomass from a plant organ such as poplar wood, or from a reproductive organ such as the number of seeds or seed biomass).
[00331] Any of the transgenic plants described above or parts thereof may be processed to produce a food, feed, protein or oil preparation, such as for ruminant animals.
[00332] The transgenic plants described above, which exhibit an increased oil content, can be used to produce vegetable oil (by extracting the oil from the plant).
[00333] Vegetable oil (including seed oil and/or vegetable portion oil) produced according to the method of the invention can be combined with a variety of other ingredients. The specific ingredients included in a product are determined according to the intended use. Exemplary products include animal feed, chemical modification feedstock, biodegradable plastic, blended food product, edible oil, biofuel, cooking oil, lubricant, biodiesel, snack foods, cosmetics and fermentation process feedstock. Exemplary products to be incorporated into vegetable oil include animal feeds, human food products such as extruded snacks, breads, as a food binding agent, aquaculture feeds, fermentable blends, food supplements, sports drinks, nutritional food bars, multivitamin supplements, beverages diets and cereals.
[00334] According to some applications of the invention, the oil comprises a seed oil.
[00335] According to some applications of the invention, the oil comprises an oil of the vegetable portion (the oil of the vegetable portion of the plant).
[00336] According to some applications of the invention, the plant cell forms a part of the plant.
[00337] According to another application of the present invention, a food or feed is provided, comprising the plants or a part thereof of the present invention.
[00338] As used herein, the term "about" refers to ±10%.
[00339] The terms “comprises”, “comprising”, “includes”, “including”, “presenting” and their conjugations mean “including, but not limited to”.
[00340] The term “consisting of” means “including and limited to”.
[00341] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the product. claimed composition, method or structure.
[00342] As used herein, the singular form "a", "a", and "the" include plural references unless the context clearly specifies otherwise. E.g., the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.
[00343] Throughout the present application, various applications of this invention may be presented in varied format. It is to be understood that the description in assorted format is merely for convenience and brevity and should not be regarded as an inflexible limitation of the scope of the invention. Therefore, the description of a variety should be considered to have specifically revealed all possible subvariations as well as the individual numerical values within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically revealed subvariations such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, eg, 1, 2, 3, 4, 5, and 6. This applies regardless of the range of the range.
[00344] Wherever a numerical range is indicated in this document, this means including any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/varies between” a first indicated number and a second indicated number and “ranging/varies from” a first indicated number “to” to a second indicated number are used interchangeably herein and are meant to include the first and second indicated numbers. and all fractional and integral numerals in between.
[00345] [00343] As used herein, the term "method" refers to ways, means, techniques and procedures for performing a particular task including, but not limited to, those ways, means, techniques and procedures that are known or readily developed. from ways, means, techniques and procedures known to professionals in the chemical, pharmacological, biological, biochemical and medical arts.
[00346] Where reference is made to listings of specific sequences, such reference shall be understood to also encompass sequences which substantially correspond to their complementary sequence, as if including minor sequence variations resulting from, e.g., sequencing errors, cloning errors or other changes resulting in base substitution, base deletion or base addition, provided the frequency of such variations is less than 1 in 50 nucleotides, alternatively less than 1000 nucleotides, alternatively less than 1 in 5000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
[00347] It should be noted that certain features of the invention, which are, for purposes of clarification, described in the context of separate applications, may also be presented in combination in a single application. Conversely, various features of the invention, which are, for purposes of brevity, described in the context of a single application, may also be presented separately or in any suitable sub-combination or in the appropriate form in any other described application of the invention. Certain features described in the context of various applications should not be considered essential features of those applications unless the application is inoperative without these elements.
[00348] Various applications and aspects of the present invention are outlined above and, as claimed in the claims section below, find experimental support in the following examples. EXAMPLES
[00349] 00347] Reference is now made to the following examples which, together with the above descriptions, illustrate some applications of the invention in a non-limiting manner.
[00350] Generally, the nomenclature used herein and the laboratory procedures used in the present invention include molecular, biochemical, microbiological and DNA and matching techniques. These techniques are explained in detail in the literature. See, e.g., "Molecular Cloning: A Laboratory Manual" Sambrook et al., (1989); “Current Protocols in Molecular Biology” Volumes I-III Ausubel, R.M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies, as defined in US Patent Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J.E., ed. (1994); “Current Protocols in Immunology” Volumes I-III Coligan J.E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W.H. Freeman and Co., New York (1980); Available immunoassays are extensively described in the scientific and patent literature, see, e.g., US Patent No. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M.J., ed. (1984); “Nucleic Acid Hybridization” Hames, B.D., and Higgins S.J., eds. (1985); “Transcription and Translation” Hames, B.D., and Higgins S.J., Eds. (1984); “Animal Cell Culture” Freshney, R.I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods and Applications”, Academic Press, San Diego, CA (1990); Marshak et al., “Strategies for Protein Purification and Characterization - A Laboratory Course Manual” CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are presented throughout this document. The procedures described in these works are believed to be well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference. EXPERIMENTAL AND BIOINFORMATICS METHODS IN GENERAL.
[00351] RNA Extraction - Tissues grown under various culture conditions (as described below) were sampled and RNA was extracted using Invitrogen's TRIzol Reagent [http://www (dot) invitrogen (dot) com/content (dot) cfm pageid=469]. Approximately 30-50 mg of tissue was collected from the samples. The weighed tissues were ground using a pestle and mortar in liquid nitrogen and resuspended in 500 μl of TRIzol Reagent. To the homogenized lysate, 100 µl of chloroform was added followed by precipitation using isopropanol and two washes with 75% ethanol. RNA was eluted in 30 μl of RNase-free water. RNA samples were cleaned using Qiagen's RNeasy minikit cleaning protocol according to the manufacturer's protocol (QIAGEN Inc., CA USA). For convenience, each tissue type with microarray expression information has been assigned an Expression Set ID.
[00352] Correlation analysis - was performed for selected genes, according to some applications of the invention, in which the characterized parameters (parameters measured according to Correlation Identities) were used as “X axis” for correlation with the transcriptome of the fabric that was used as the “Y axis”. For each gene and parameter measured, an “R” correlation coefficient was calculated using the Evilharson correlation along with a p-value for the significance of the correlation. When the correlation coefficient (R) between the levels of a gene expression in a given tissue and a phenotypic performance across ecotypes/variety/hybrid is high in absolute value (between 0.5-1), there is an association between the gene (specifically the level of expression of that gene) and phenotypic trait (eg, increased production, growth rate, nitrogen use efficiency, abiotic stress tolerance, and the like).EXAMPLE 1 IDENTIFICATION OF GENES THAT INCREASE EFFICIENCY IN NITROGEN USE (NUE), FERTILIZER USE EFFICIENCY (FUE), PRODUCTION, GROWTH RATE, VIGOR, BIOMASS, OIL CONTENT, ABIOTIC STRESS TOLERANCE (ABST) AND/OR WATER USE EFFICIENCY (WUE) IN PLANTS.
[00353] The present inventors have identified polynucleotides whose respective upregulation of expression in plants increases nitrogen use efficiency (NUE), fertilizer use efficiency (FUE), production (eg, seed production, oil production , biomass, grain quality and/or quantity), growth rate, vigor, biomass, oil content, fiber production, fiber quality, fiber length, abiotic stress tolerance (ABST) and/or efficiency in the use of water (WUE) of a plant.
[00354] All nucleotide sequence datasets used here were sourced from publicly available databases or from sequences obtained using Solexa technology (eg, Barley and Sorghum). Sequence data from 100 different plant species were entered into a single, comprehensive database. Other information on gene expression, protein annotation, enzymes and pathways was also incorporated. The main databases used include: Genomes:
[00355] Arabidopsis genome [TAIR genome, version 6 (Arabidopsis (dot) org/)]; Rice genome [IRGSP 4.0 framework (rgp (dot) dna (dot) affrc (dot) go (dot) jp/IRGSP/ )];Populus [Populus trichocarpa, JGI release 1.1 (joint release vl.0) (genome (dot) jgi-psf (dot) org/)];Brachypodium [set 4x JGI, brachpodium (dot) org)];Soybean [DOE-JGI SCP, versions Glyma0 or Glyma1 (phytozome (dot) net/)];Grape [Franco-Italian Public Consortium for the Characterization of the Grapevine Genome (genoscope (dot) ens (dot) fir /)];Castor beans [TIGR /J, Craig Venter Institute, 4x set [(msc (dot) jevi (dot) org/r_communis];Sorghum [DOE-JGI SCP, version Sbil [hytozome (dot) net/)];Corn [cornsequence (dot) org /];Cucumber [cucumber (dot) genomics (dot)org (dot) cn/page/cucumber/index (dot) jsp]Tomato [solgenomics (dot) net/tomato/]Cassava [hytozome (dot) net/cassava ( dot) php]Expressed EST and mRNA sequences were extracted from the following databases:GenBank (n cbi (dot) nlm (dot) nih (dot)gov/ Genbank/);RefSeq (ncbi (dot) nlm (dot) nih (dot)gov/RefSeq/); TAIR (Arabidopsis (dot) org/); Protein databases and pathways: Uniprot [uniprot (dot) org/].
[00356] AraCyc [Arabidopsis (dot) org/biocyc/index (dot) jsp].
[00357] ENZYME [expasy (dot) org/enzyme/]. The microarray datasets were downloaded from:
[00358] GEO (ncbi.nlm.nih.gov/geo/)TAIR (Arabidopsis.org/).
[00359] Unique Property Microarray Data (See WO2008/122980 and Examples 3-13 below). QTL and SNPs information:
[00360] Gramen [gramene (dot) org/qtl/].
[00361] Panzea [anzea (dot) org/index (dot) html].
[00362] Soybean QTL: [soybeanbreederstoolbox(dot) com/].
[00363] Database Set - is designed to provide a comprehensive, rich, detailed, reliable and easy-to-use database comprising publicly available mRNA, ESTs, DNA genomic sequences, multi-culture data as well as gene expression data , protein annotation and path, QTL data and other relevant information.
[00364] The set of databases comprises a toolbox of enhancement, structuring, genetic annotation and analysis tools that allow building a customized database for each genetic discovery project. Genetic enhancement and structuring tools allow reliably detecting pooled variants and antisense transcripts, generating understanding of multiple potential phenotypic outcomes of a single gene. The capabilities of Compugen LTD's “LEADS” platform to analyze the human genome have been confirmed and accepted by the scientific community [see, eg, “Widespread Antisense Transcription”, Yelin, et al. (2003) Nature Biotechnology 21, 379-85; “Splicing of Alu Sequences”, Lev-Maor, et al. (2003) Science 300 (5623),1288-91; “Computational analysis alternative splicing using EST tissueinformation”, Xie H etGenomics 2002] and proved effective in plant genomics as well. ofal.be more
[00365] Gene setEST - For and grouping the genetic grouping and the grouping of organisms with available genomic sequence data (Arabidopsis, rice, castor bean, grape, brachypodium, poplar, soybean, sorghum), the genomic version (GANG) of LEADS was used. This tool allows for more accurate clustering of ESTs and mRNA sequences in the genome and predicts genetic structure as well as alternative clustering and antisense transcription events.
[00366] For organisms without complete genomic sequence data available, the clustering software “expressed LEADS” was applied.
[00367] Genetic annotation - Predicted genes and proteins were annotated as follows: The sequence comparison search [blast (dot) ncbi (dot) nlm (dot) nih (dot) gov /Blast (dot) cgi] against all UniProt of the plant [uniprot (dot) org/] was performed. Open reading frames of each putative transcript were analyzed and the longest ORF with the highest number of homologs was selected as the predicted protein of the transcript. The predicted proteins were analyzed by InterPro [ebi (dot) ac (dot) uk/interpro/].
[00368] Comparison against proteins from the AraCyc and ENZYME databases was used to map predicted transcripts to AraCyc pathways.
[00369] Proteins were compared (dot) gov/Blast (dot) cgi] comparison algorithm for efficient sequence detection of orthologs.
[00370] Gene expression profiling - Several data sources were explored for gene expression profiling, namely microarray data and digital expression profiling (see below). According to the gene expression profile, a correlation analysis was performed to identify genes that are co-regulated developmental and environmental phenotypes.
[00371] Available database. The profile of important important for production.
[00372] A digital summary of the expression profile was compiled for each cluster according to all keywords included in the records of the sequence comprising the cluster. Digital expression, also known as electronic Northern Blot, is a tool that displays the virtual profile of expression based on the EST sequences that form the genetic cluster. The tool profiles the expression of a cluster in terms of plant anatomy (eg, the tissue/organ in which the gene is expressed), developmental stage (the developmental stages at which a gene can be found ) and treatment profile (presents the physiological conditions under which a gene is expressed, such as drought, cold, pathogen infection, etc.). Given the random distribution of ESTs in the different clusters, the digital expression has a probability value that describes the probability of a cluster having a total of N ESTs to contain X ESTs from a given library collection. For probability calculations, the following are taken into account: a) the number of ESTs in the cluster, b) the number of ESTs from the involved and related libraries, c) the general number of ESTs available representing the species. Thus, clusters with low probability values are highly enriched with ESTs from the library group of interest indicating a specialized expression.
[00373] Recently, the accuracy of this system was demonstrated by Portnoy et al., 2009 (Analysis Of The Melon Fruit Transcriptome Based On 454 Pyrosaquencing) at: XVII Conference on Plant and Animal Genomes, San Diego, CA. Transcriptomic analysis based on the relative abundance of ESTs in the data was performed by 454 pyrosequencing of cDNA representing the melon mRNA. Fourteen double-stranded cDNA samples obtained from two genotypes, two fruit tissues (pulp and peel) and four developmental stages were sequenced. Pyrosequencing by GS FLX (Roche/454 Life Sciences) of non-normalized and purified cDNA samples yielded 1,150,657 expressed sequence tags (ESTs) that clustered into 67,477 unigenes (32,357 singletons and 35,120 contiguous). Analysis of the data obtained against the Cucurbitaceae Genomic Database [icugi (dot) org/] confirmed the accuracy of sequencing and clustering. Expression patterns of selected genes well fitted their qRT-PCR (reverse transcription polymerase chain reaction) data.
[00374] Overall, 215 genes were identified as having a major impact on nitrogen use efficiency, fertilizer use efficiency, yield (eg, seed yield, oil yield, grain quality and/or quantity ), growth rate, vigor, biomass, oil content, fiber production, fiber quality, fiber length, abiotic stress tolerance and/or water use efficiency when their respective expression is increased in plants. The identical genes, their cured polynucleotides and polypeptide sequences, as well as their updated sequences, according to the GenBank database, are summarized in Table 1, below.








Table 1: Gene names, cluster names, organisms from which they are derived, and sequence identifiers of polypeptide and polynucleotide sequences are provided. “Poly.” = polypeptide; "Polin." = polynucleotide. EXAMPLE 2 IDENTIFICATION OF HOMOLOGOUS SEQUENCES (EG, ORTHOLOGY) THAT INCREASE NITROGEN USE EFFICIENCY, FERTILIZER USE EFFICIENCY, PRODUCTION, GROWTH RATE, VIGOR, BIOMASS, OIL CONTENT, TOLERANCE TO ABIOTIC STRESS AND/OR EFFICIENCY IN THE USE OF WATER IN PLANTS.
[00375] The concepts of orthology and paralogy have recently been applied to functional characterizations and classifications on the scale of whole genome comparisons. Orthologs and paralogs constitute two main types of homologs: The former evolved from a common ancestor by specialization and the latter are related by duplication events. It is assumed that paralogs arising from ancient duplication events tend to have diverged in function whereas true orthologs are more likely to retain identical function over evolutionary time.
[00376] For further investigation and identification of putative orthologs of genes that affect nitrogen use efficiency, fertilizer use efficiency, production (eg, seed production, oil production, biomass, quality and/or quantity of grain), growth rate, vigor, biomass, oil content, abiotic stress tolerance and/or water use efficiency, all sequences were aligned using the BLAST Basic Local Alignment Search Tool ]. Sufficiently similar sequences were grouped by trial. These putative orthologs were further organized under a Philogram - a branching (tree) diagram assumed to represent the evolutionary relationships between biological taxa. Putative orthologous groups were analyzed for their agreement with the phylogram and, in cases of divergence, these orthologous groups were divided accordingly. Expression data were analyzed and EST libraries were classified using a fixed vocabulary of custom terms, such as developmental stages (e.g. genes showing similar expression profile, through development with upregulation in the specific stage, such as in the storage system) and/or plant organ (e.g. genes that show similar expression profile in their organs with up-regulation in specific organs such as the seed). The annotations of all ESTs grouped to a gene were statistically analyzed by comparing their frequency in the set in relation to their abundance in the database, allowing the structure of a numerical and graphic expression profile of such gene, which is called “ digital expression”. The logic of using these two complementary methods with methods of phenotypic association studies of QTLs, SNPs and phenotypic expression is based on the assumption that true orthologs tend to retain identical function over evolutionary time. These methods provide different sets of indications about the functional similarities between two homologous genes, similarities at the amino acid sequence level - identical in protein domains, and similarity in expression profiles.
[00377] Research and identification of homologous genes involves screening for sequence information available, for example, in public databases including, but not limited to, the DNA Database of Japan (DDBJ | DNA Database of Japan) , Genbank and the Nuclear Biology Laboratory Nucleic Acid Sequence Database (EMBL | European Molecular Biology Laboratory) or their versions or the MIPS database. A number of different search algorithms have been developed, including but not limited to the set of programs referred to as BLAST programs. There are five implementations of BLAST, three designed for nucleotide sequence queries (BLASTN, BLASTX, and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology: 76-80, 1994; Birren et al. al., Genome Analysis, I: 543, 1997). Such methods involve sequence alignment and comparison. The BLAST algorithm calculates the percentage of sequence identity and performs a statistical analysis of similarity between the two sequences. Software for performing BLAST analysis is publicly available through the National Biotechnology Information Center. Other of these types of software or algorithms are GAP, BESTFIT, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps.
[00378] Homologous genes may belong to the same genetic family. Analysis of a genetic family can be performed using sequence similarity analysis. To perform this analysis, standard programs for multiple alignments can be used, for example, Clustal W. A Neighbor Joining tree of the homologous proteins of the genes of some applications of the invention can be used to provide an overview of the structural and ancestral relationships. Sequence identity can be calculated using an alignment program as described below. Other plants are expected to have a similar functional gene (ortholog) or a family of similar genes and such genes will provide the same preferred phenotype as the genes shown herein. Advantageously, these family members may be useful in the methods of some applications of the invention. Examples of other plants include, but are not limited to, Barley (Hordeum vulgare), Arabidopsis (Arabidopsis thaliana), Corn (Zea mays), Cotton (Gossypium), Canola (Brassica napus), Rice (Oryza sativa), Sugarcane Sugar (Saccharum officinarum), Sorghum (Sorghum bicolor), Soybean (Glycine max), Sunflower (Helianthus annuus), Tomato (Lycopersicon esculentum) and Wheat (Triticum aestivum).
[00379] The aforementioned analyzes for sequence homology are preferably performed on a full-length sequence, but may also be based on a comparison of certain regions, such as conserved domains. Identification of such domains would also be well within the skill of one skilled in the art and would involve, for example, a computer-readable format of the nucleic acids of some applications of the invention, the use of alignment software programs, and the use of publicly available information on protein domains, conserved motifs and boxes This information is available in the PRODOM database (biochem (dot) ucl (dot) ac (dot) uk/bsm/dbbrowser/protocol/prodomqry (dot) html), PIR (pir (dot) Georgetown (dot) edu/) or Pfam (sanger (dot) ac (dot) uk/Software/Pfam/). Sequence analysis programs developed for motif screening can be used to identify conserved fragments, regions and domains as mentioned above. Preferred computer programs include, but are not limited to, MEME, SIGNALSCAN, and GENESCAN.
[00380] One skilled in the art can use the homologous sequences provided herein to find similar sequences in other species and other organisms. Homologs of a protein encompass peptides, oligopeptides, polypeptides, proteins, and enzymes that have amino acid substitutions, deletions, and/or insertions relative to the unmodified protein in question and that have similar biological and functional activity to the unmodified protein from which are derived. To produce such homologs, the amino acids in the protein can be replaced with other amino acids that have similar properties (conservative modifications such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form a structure or break helical structures or 3-sheet structures). Conservative substitution tables are well known in the art (see, e.g., Creighton (1984) Proteins. W.H. Freeman and Company). Homologs of an amino acid encompass nucleic acids with nucleotide substitutions, deletions and/or insertions relative to the unmodified nucleic acid in question and which have similar biological and functional activity to the unmodified nucleic acid from which they are derived.
[00381] Polynucleotides and polypeptides with significant homology to the identified genes described in Table 1 (Example 1, above) were identified from the databases using the BLAST software with the Blastp and tBlastn algorithms as filters for the first stage and the needle (EMBOSS package) or FRAME+ alignment algorithm for the second phase. The local identity (Blast alignments) was defined with a very permissive cut - 60% Identity in a range of 60% of sequence lengths because it uses only one filter for the global alignment phase. The default Blast packet filtering was not used (setting the parameter “-F F”).
[00382] In the second stage, homologs were defined based on the overall identity of at least 80% to the main genetic polypeptide sequence.
[00383] Two distinct ways to find the optimal global alignment for protein or nucleotide sequences were used in this application.
[00384] 1. Between two proteins (after the blastp filter): Needleman-Wunsch EMBOSS-6.0.1 algorithm with the following modified parameters: gapopen=8 gapextend=2. The rest of the parameters remained unchanged from the default options described above.
[00385] 2. Between a protein sequence and a nucleotide sequence (after the rblastn filter): GenCore 6.0 OneModel application, using the Frame+ algorithm with the following parameters: model=frame+_p2n.model mode=qglobal -q=protein. sequence -db= nucleotide.sequence. The rest of the parameters remained unchanged from the default options described above.
[00386] The query polypeptide sequences were SEQ IDs. Nos. 496-794 and the query polynucleotides were SEQ IDs. Nos. 1-495 and identified orthologous and homologous sequences having at least 80% overall sequence identity are provided in Table 2, below. These homologous genes are expected to increase yield, seed yield, oil yield, oil content, growth rate, fiber yield, fiber quality, fiber length, photosynthetic capacity, biomass, vigor, ABST and/or NUE of a plant.Table 2Homologues (eg, orthologs) of genes/polypeptides identified for increased nitrogen use efficiency, fertilizer use efficiency, yield, seed yield, growth rate, vigor, biomass, content of oil, fiber production, fiber quality, fiber length, abiotic stress tolerance and/or a plant's water use efficiency.




































Table 2: Homologous polypeptides and polynucleotides (eg, orthologs) of the genes identified in Table 1 and their cloned genes are provided, which can increase nitrogen use efficiency, fertilizer use efficiency, production, seed production , growth rate, vigor, biomass, oil content, fiber production, fiber quality, fiber length, abiotic stress tolerance and/or a plant's water use efficiency. Homology was calculated as % identity over alignment sequences. The query sequences were the polypeptide sequences of SEQ IDs. 496-794 and the polynucleotide sequences of SEQ IDs. No. 1-495 and the sequences in question are polypeptide sequences or polynucleotide sequences that have been dynamically transposed into all six reading frames identified in the database based on greater than 80% identity to the query polypeptide sequences . “Poly.” = polypeptide; “Polin” - polynucleotide; "Something." = Algorithm; “globlastp” - global homology using blastp; “glotblastn” - global homology using tblastn. “Man.” - homolog, “Id.” = identity.
[00387] The result of the functional genomics approach described here is a set of genes with high estimates of improved nitrogen use efficiency, fertilizer use efficiency, yield, seed yield, growth rate, vigor, biomass, oil, fiber production, fiber quality, fiber length, abiotic stress tolerance and/or water use efficiency of a plant, increasing its expression.
[00388] While each gene is estimated to have its own impact, modifying the mode of expression of more than one gene or gene product (RNA, polypeptide) is expected to provide an additive or synergistic effect on a desired trait (e.g. (eg, nitrogen use efficiency, fertilizer use efficiency, yield, growth rate, vigor, biomass, oil content, abiotic stress tolerance, and/or a plant's water use efficiency). By altering the expression of each gene described herein, the gene alone or the set of genes increases overall yield and/or other important agricultural traits, and thus is expected to increase agricultural productivity. EXAMPLE 3 BARLEY TRANSCRIPTOME PRODUCTION AND HIGH PRODUCTIVITY CORRELATION ANALYSIS USING 44K BARLEY OLIGONUCLEOTIDE MICROARRANGE
[00389] In order to produce a high throughput correlation analysis comparing plant phenotype and gene expression level, the present inventors used a barley oligonucleotide microarray, produced by Agilent Technologies [chem(dot) agilent ( dot) com/Scripts/PDS (dot) asp lPage=50879]. The array oligonucleotide represents about 47,500 barley genes and transcripts. To define the correlations between RNA expression levels and parameters related to yield or vigor components, several plant characteristics of 25 different barley accessions were analyzed. Among them, 13 accessions encompassing the observed variation were selected for analysis of RNA expression. Correlation between RNA levels and characterized parameters was analyzed using Pearson's correlation test [davidmlane (dot) com/hyperstat/A34739 (dot) html]. Experimental Procedures:
[00390] Barley tissues analyzed - Five tissues at different stages of development [meristem, flower, spikelet, stem and leaf] representing different plant characteristics were sampled and RNA was extracted as described above. Each tissue type of microarray expression information was assigned a Set ID, as summarized in Table 3 below.Table 3 Barley transcriptome expression sets
Table 3.
[00391] Evaluation of parameters related to the components of production and vigor of Barley - 25 accessions of barley in 4 repetitive blocks (called A, B, C and D), each containing 4 plants per lot were cultivated in a greenhouse with a net. Plants were phenotyped on a daily basis, following the standard barley descriptor (Table 4, below). Harvesting was conducted while 50% of the ears were dried to avoid spontaneous seed release. The plants were separated for the vegetative part and ears, of which 5 ears were threshed (grains were separated from the glumes) for further grain analysis such as size measurement, grain count per spike and grain yield per spike. All material was oven dried and seeds were manually threshed from the ears before measuring seed characteristics (weight and size) using scanning and image analysis. The image analysis system included a personal computer (Intel P43.0 GHz processor) and a public domain program - ImageJ 1.37 (Java-based image processing program, developed at the US National Institutes of Health and freely available on the internet [rsbweb (dot) nih (dot) gov/] Then the analyzed data were saved as text files and processed using the JMP software for statistical analysis (Instituto SAS). Barley standard descriptors
[00392] Table 4.

[00393] Grains per ear - At the end of the experiment (50% of the ears were dried), all the ears of the lots within blocks A-D were collected. The total number of grains from the 5 ears that were manually threshed was counted. The average grain per ear was calculated by dividing the total number of grain by the number of ears.
[00394] Average grain size (cm) - At the end of the experiment (50% of the ears were dried), all the ears of the lots within blocks A-D were collected. The total grains of 5 ears that were manually threshed were digitized and the images were analyzed using the digital imaging system. Grain scanning was performed using the Brother scanner (model DCP-135), at a resolution of 200 dpi and analyzed with the Image J software. The average grain size was calculated by dividing the total grain size by the total number of grains.
[00395] Average grain weight (mg) - At the end of the experiment (50% of the ears were dried), all the ears of the lots within blocks A-D were collected. The total grains of the 5 ears that were manually threshed were counted and weighed. The average weight was calculated by dividing the total weight by the total number of grains.
[00396] Grain production per ear (g) - At the end of the experiment (50% of the ears were dried), all ears of lots within blocks A-D were collected. The total grains of 5 ears that were manually threshed were weighed. Grain yield was calculated by dividing the total weight by the ear number.
[00397] Ear Length Analysis - At the end of the experiment (50% of the ears were dried), all ears from lots within blocks A-D were collected. The five ears chosen per plant were measured using a tape measure, excluding edges.
[00398] Number of ears analysis - At the end of the experiment (50% of the ears were dried), all the ears of the lots within blocks A-D were collected. The ears per plant were counted.
[00399] Cultivation Habit Score - In Cultivation Stage 10 (Initialization), each of the plants was scored for its Cultivation Habit Nature. The scale that was used was 1 for prostrate to upright nature.
[00400] Basal Leaf Hairiness - In cultivation stage 5 (leaf coat heavily erect; end of tillering), each of the plants was scored for its leaf hairiness nature before the last. The scale that was used was 1 for prostrate nature to 9 for upright.
[00401] Plant height - At the harvest stage (50% of the ears were dried), each plant was measured by its height using a tape measure. Height was measured from ground level to the top of the longest ear excluding edges.
[00402] Days to Flower - Each of the plants was tracked for flowering date. Flowering days were calculated from the sowing date to the flowering date.
[00403] Stem Pigmentation - In cultivation stage 10 (initialization), each of the plants was scored by its stem color. The scale that was used was 1 for green to 5 for full purple.
[00404] Dry plant weight and ear yield - At the end of the experiment (50% of the ears were dried), all ears and plant material from the lots within blocks A-D were collected. The weight of biomass and ears of each lot was separated, measured and divided by the number of plants.
[00405] Dry weight = total weight of aboveground vegetative part (excluding roots) after drying at 70°C in the oven for 48 hours.
[00406] Ear production per plant = total ear weight per plant (g) after drying at 30°C in the oven for 48 hours.
[00407] Harvest index (for barley) - the harvest index was calculated using Formula XVIII above.Table 5 Parameters correlated to Barley (vectors)
Table 5. Experimental Results:
[00408] 13 different barley accessions were cultivated and characterized with respect to 13 parameters as described above. The mean for each of the measured parameters was calculated using the JMP software and the values are summarized in Tables 6 and 7 below. Subsequent correlation analysis between the various transcriptome expression sets (Table 3) and mean parameters was conducted. Then, the results were integrated into the database.Table 6Measured parameters of correlation IDs in barley accessions

Table 6. Values are provided for each of the parameters measured in accessions of Barley, according to the correlation identifications (Correlation IDs from Table 5 above).Table 7
Table 7. Values are provided for each of the parameters measured in Barley accessions, according to the correlation identifications (Correlation IDs from Table 5 above).Table 8Correlation between the expression level of selected polynucleotides from the Correlation between the level of expression of selected polynucleotides of the invention and their homologs at developmental or tissue-specific stages and phenotypic performance in Barley accessions
Table 8. Correlations (R) and p-values are provided between the expression levels of selected genes from some applications of the invention at various developmental stages or tissues (Expression Sets) and phenotypic performance on various components of vigor, production ( seed yield, oil yield, oil content), biomass and/or growth rate [Correlation (Cor.), Vector (Vet.), Expression (Exp.)] Corr. = correlation vector specified in Table 5; set of Exp. = expression set specified in Table 3.EXAMPLE 4 BARLEY TRANSCRIPTOME PRODUCTION AND HIGH PRODUCTIVITY CORRELATION ANALYSIS USING 60K BARLEY OLIGONUCLEOTIDE MICROARRANGE
[00409] In order to produce a high throughput correlation analysis comparing plant phenotype and gene expression level, the present inventors used a Barley oligonucleotide microarray, produced by Agilent Technologies [chem. (dot) agilent (dot) com/Scripts/PDS (dot) asp lPage=50879]. The array oligonucleotide represents about 60,000 Barley genes and transcripts. To define the correlations between RNA expression levels and parameters related to production or vigor, several plant traits from 15 different Barley accessions were analyzed. Among them, accessions encompassing the observed variation were selected for the analysis of RNA expression. The correlation between RNA levels and the characterized parameters was analyzed using the Pearson correlation test [davidmlane (dot) com/hyperstat/A34739 (dot) html]. Experimental Procedures:
[00410] Barley tissues analyzed - Tissues at different stages of development, representing different plant characteristics, were sampled and RNA was extracted as described above. Each tissue type of microarray expression information was assigned a Set ID, as summarized in Tables 9-11 below.Table 9Barley transcriptome expression sets under normal and low N conditions (at the plant stage)
Table 9. Barley transcriptome expression sets under normal and low N (low nitrogen) conditions (at the plant stage) are provided.Table 10 Barley transcriptome expression sets under normal and low N conditions (at the reproductive stage)
Table 10. Barley transcriptome expression sets under normal and low N conditions (at the reproductive stage) are provided.Table 11 Barley transcriptome expression sets under dry conditions (at the plant stage)
Table 11. Barley transcriptome expression sets under dry conditions (at the plant stage) are provided.
[00411] Evaluation of parameters related to the components of production and vigor of Barley - 15 accessions of Barley in 5 repetitive blocks, each one containing 5 plants per lot, were cultivated in the net greenhouse. Three different treatments were applied: plants were fertilized and watered regularly during plant growth until harvest (as recommended for commercial growth, plants were watered 2-3 times a week, and fertilization was given in the first 1, 5 months of the growing period) or low Nitrogen (80% percent less Nitrogen) or under drought stress (drought and re-irrigation cycles were conducted throughout the entire experiment, total 40% less water given in dry treatment). Plants were phenotyped daily according to parameters listed in Table 12 below. Harvesting was carried out while all ears were dried. All material was oven dried and seeds were manually threshed from the ears before measuring seed characteristics (weight and size) using scanning and image analysis. The image analysis system includes a personal computer (Intel P4 3.0 Ghz processor) and a public domain program - ImageJ 1.37 (Java based image processing program, which was developed at the National Institutes of Health of the United States and freely available on the internet [rsbweb (dot) nih (dot) gov/] Then analysis data were saved to text files and processed using JMP statistical analysis software (SAS Institute).
[00412] Grain Production (g.) - At the end of the experiment, all the ears of the lots were collected. The total grain from all ears that were manually threshed was weighed. Grain production was calculated per batch or per plant.
[00413] Ear length and width analysis - At the end of the experiment, the length and width of the five ears chosen per plant were measured using a tape measure, excluding the edges.
[00414] Ear number analysis - Ears per plant were counted.
[00415] Height of the plant - Each of the plants was measured when its height, using tape measure. Height was measured from ground level to the furthest top of the ear excluding awns at two time points in plant growth (30 days after sowing) and at harvest.
[00416] Ear weight - The weight of biomass and ears of each unit was separated, measured and divided by the number of plants.
[00417] Dry weight = total weight of the aboveground plant part (excluding roots) after drying at 70°C in the oven for 48 hours at two time points in Plant growth (30 days after sowing) and at harvest.
[00418] Spikelet per ear = number of spikelets per ear was counted.
[00419] Root/Sprout Ratio - The Root/Sprout ratio is calculated using Formula XXII above.
[00420] Total No. of Tillers - all tillers were counted per unit at two time points in Plant growth (30 days after sowing) and at harvest.
[00421] Percentage of reproductive tillers - the number of reproductive tillers excluding one ear at harvest was divided by the total number of tillers.
[00422] SPAD - Chlorophyll content was determined using a Minolta SPAD 502 chlorophyll meter and measurement was taken at the time of flowering. SPAD measurement readings were taken on fully developed new sheet. Three measurements per sheet were taken per batch.
[00423] Root FW (g.), root length (cm) and No. of lateral roots - 3 plants per unit were selected to measure root weight, root length and to count the number of lateral roots formed.
[00424] Sprout FW (fresh weight) - weight of 3 plants per pot was recorded at different time points.
[00425] Average Grain Area (cm2) - At the end of the growing period, the grains were separated from the ear. A sample of ~200 grains was weighed, photographed and the images processed using the image processing system described below. The grain area was measured from those images and divided by the number of grains.
[00426] Average Grain Length and Width (cm) - At the end of the growing period, the grains were separated from the ear. A sample of ~200 grains was weighed, photographed and images processed using the image processing system described below. The sum of grain length and width (longest axis) was measured from those images and divided by the number of grains.
[00427] Average Grain Perimeter (cm) - At the end of the growing period, the grains were separated from the ear. A sample of ~200 grains was weighed, photographed and images processed using the image processing system described below. The sum of the grain perimeter was measured from those images and divided by the number of grains.
[00428] Description date - the day on which the start-up stage was observed was recorded and the number of days from seeding to description was calculated.
[00429] Relative water content - the fresh weight (FWIfresh weight) of three leaves from three plants of each of the different seed IDs was recorded immediately; then, the leaves were soaked for 8 hours in distilled water at room temperature in the dark and the turgid weight (TW|turgid weight) was recorded. The total dry weight (DW|dry weight) was recorded after drying the sheets at 60°C at constant weight. The relative water content (RWC|relative water content) was calculated according to Formula I above.
[00430] Crop Index (for barley) - The crop index was calculated using Formula XVIII above.
[00431] Relative growth rate: The relative growth rates (RGR | relative growth rate) of Plant Height (Formula III above), SPAD (Formula IV above) and Number of tillers (Formula V above) were calculated using the Formulas indicated.
[00432] Dry/Normal Ratio: Represents the ratio for the specific parameter of Drought condition results divided by the results of Normal conditions (maintenance of phenotype under Drought compared to normal conditions).Table 12 Parameters correlated to Barley (vectors) under conditions normal and low-N (at the plant stage)
Table 12. Barley correlated parameters are provided. “TP [time point]” = point in time; “DW” = dry weight; “FW” = fresh weight; “Low N” = Low nitrogen.Table 13 Parameters correlated to Barley (vectors) under normal and low N conditions (in the reproductive stage)
Table 13. Parameters correlated to Barley under normal and low N conditions (in the reproductive stage) are provided. “TP” = point in time; “DW” = dry weight; “FW” = fresh weight; “Low N” = Low nitrogen; “Relative water content [ercentage], Dry/Normal Ratio - maintenance of the phenotype under drought compared to normal conditions.Table 14 Parameters correlated to Barley (vectors) under drought conditions (at the plant stage)
Table 14. Parameters correlated to barley under dry conditions (at the vegetable stage) are provided. “RBiH/BiH” = root-shoot ratio. Experimental results:
[00433] 15 different accessions of Barley were cultivated and characterized with respect to different parameters, as described above. Tables 12-14 describe the parameters correlated to Barley. The average for each of the measured parameters was calculated using the JMP software and the values are summarized in Tables 15-24 below. Subsequent correlation analysis between the various transcriptome sets and the mean parameters was conducted. The results were then integrated into the database.Table 15Measured parameters of correlation IDs in Barley accessions under low N conditions (at the plant stage)
Table 15. Values for each of the parameters (as described above) measured in Barley accessions (row) under low N conditions are provided. The growing conditions are specified in the experimental procedure section.Table 16 Measured Parameters of Correlation IDs on additional accessions of Barley under low N conditions (at the plant stage)
Table 16. Values for each of the parameters (as described above) measured in Barley accessions (row) under low N conditions are provided. The growing conditions are specified in the Experimental Procedure section. Table 17 Measured Parameters of Correlation IDs in Barley accessions under normal conditions (at the plant stage)
Table 17. Values for each of the parameters (as described above) measured in Barley accessions (row) under normal conditions are provided. The growing conditions are specified in the experimental procedure section.Table 18Measured parameters of correlation IDs in additional accessions of Barley under normal conditions (at the plant stage)
Table 18. Values for each of the parameters (as described above) measured in Barley accessions (row) under normal conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 19 Measured parameters of correlation IDs in Barley accessions under low N conditions (in reproductive stage)
Barley accessions (row) under low N conditions (in the reproductive stage). Cultivation conditions are specified in the experimental procedure section. Table 20 Measured parameters of correlation IDs in additional accessions of Barley under low N conditions (in the reproductive stage)
Table 21 Measured parameters of correlation IDs in Barley accessions under normal conditions (in the reproductive stage)
Table 22 Measured parameters of correlation IDs in additional accessions of Barley under normal conditions (in the reproductive stage)
Table 22. Values for each of the parameters (as described above) measured in Barley accessions (row) under normal conditions (in the reproductive stage) are provided. The growing conditions are specified in the experimental procedure section.Table 23Additional parameters measured from correlation IDs in Barley accessions under Dry conditionsTable 23. Values of each of the parameters (as described above) measured in Barley accessions are provided ( row) under dry growing conditions. Cultivation conditions are specified in the experimental procedure section.
Table 24 Additional Measured Parameters of Correlation IDs in Additional Barley Accessions Under Drought Conditions
Table 24. Values for each of the parameters (as described above) measured in Barley accessions (row) under dry growing conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 25Correlation between the expression level of selected genes from some applications of the invention in various tissues and the phenotypic performance under normal and low nitrogen conditions (at the plant stage) in barley accessions

Table 25. Correlations (R) are provided between production of expression levels that enhance genes and their homologues in various tissues [Expression Sets (Exp.)] and phenotypic performance [components of vigor, production, biomass and/or or growth rate (Correlation vector (Cor.))] under normal and low nitrogen conditions in barley accessions. P = p-value.Table 26Correlation between the expression level of selected genes from some applications of the invention in various tissues and the phenotypic performance under normal and low nitrogen conditions (in the reproductive stage) in barley accessions.

Table 26. Correlations (R) are provided between production of expression levels that enhance genes and their homologues in various tissues [Expression Sets (Exp.)] and phenotypic performance [components of vigor, production, biomass and/or or growth rate (Correlation vector (Cor.))] under normal and low nitrogen conditions in barley accessions. P = p-value.Table 27Correlation between the expression level of selected genes from some applications of the invention in various tissues and the phenotypic performance under drought conditions in barley accessions.

Table 27. Correlations (R) are provided between production of expression levels that enhance genes and their homologues in various tissues [Expression Sets (Exp.)] and phenotypic performance [components of vigor, production, biomass and/or or growth rate (Correlation vector (Cor.))] under normal and low nitrogen conditions in barley accessions. P = p-value. EXAMPLE 5 PRODUCTION OF SORGHUM TRANSCRIPTOME AND HIGH PRODUCTIVITY CORRELATION ANALYSIS WITH PARAMETERS RELATED TO PRODUCTION, NUE AND ABST MEASURED IN FIELDS USING SORGHUM OLIGONUCLEOTIDE MICROARRANGES
[00434] In order to produce a high throughput correlation analysis between plant phenotype and gene expression level, the present inventors utilized a sorghum oligonucleotide microarray, produced by Agilent Technologies [chem(dot) agilent(dot ) com/Scripts/PDS (dot) asp lPage=50879]. The array oligonucleotide represents about 44,000 sorghum genes and transcripts. In order to define the correlations between the levels of RNA expression and the parameters related to vigor or production components, ABST and NUE, several plant characteristics of 17 different sorghum hybrids were analyzed. Among them, 10 hybrids encompassing the observed variation were selected for RNA expression analysis. Correlation between RNA levels and characterized parameters was analyzed using Pearson's correlation test [davidmlane (dot) com/hyperstat/A34739 (dot) html].
[00435] Correlation of Sorghum varieties in ecotypes cultivated under regular cultivation conditions, severe drought conditions and low nitrogen conditions. Experimental Procedures:
[00436] 17 varieties of Sorghum were cultivated in 3 repeated lots, in the field. Briefly, the cultivation protocol was as follows:1. Regular growing conditions: Sorghum plants were grown in the field using commercial fertilization and irrigation protocols (370 liters per m2, fertilization of 14 units of 21% urea per entire growing period).
[00437] 2. Drought Conditions: sorghum seeds were sown in soil and cultivated under normal conditions until about 35 days from sowing, around V8 stage (eight green leaves are fully expanded, start-up not yet started). At this point, irrigation was stopped, and severe drought stress developed.
[00438] 3. Low nitrogen fertilization conditions: sorghum plants were fertilized with 50% less amount of nitrogen in the field than the amount of nitrogen applied in the regular crop treatment. All fertilizers were applied before flowering.
[00439] Sorghum Tissues Analyzed - All 10 selected sorghum hybrids were sampled for each treatment. Tissues [leaf, flower meristem and flower] of plants grown under normal, severe drought stress and low nitrogen conditions were sampled and RNA was extracted as described above. Each tissue type of microarray expression information was assigned a Set ID, as summarized in Table 28 below.Table 28 Sorghum transcriptome expression sets in field experiments
Table 28: Sorghum transcriptome expression sets are provided. Flag leaf = the leaf below the flower; Flower meristem = apical meristem after panicle initiation; Flower = the flower on the day of anthesis.
[00440] The following parameters were collected using a digital imaging system: Average Grain Area (cm2) - At the end of the cultivation period, the grains were separated from the 'Head' of the Plant. A sample of ~200 grains was weighed, photographed and the images processed using the image processing system described below. The grain area was measured from those images and divided by the number of grains.
[00441] Average Grain Length (cm) - At the end of the cultivation period, the grains were separated from the 'Head' of the Plant. A sample of ~200 grains was weighed, photographed and the images processed using the image processing system described below. The sum of grain lengths (longest axis) was measured from those images and divided by the number of grains.
[00442] Average Head Area (cm2) - At the end of the cultivation period, 5 'Heads' were photographed and the images were processed using the image processing system described below. The 'head' area was measured from those images and divided by the number of 'Heads'. Average Head Length (cm) - At the end of the cultivation period, 5 'Heads' were photographed and the images were processed using the image processing system described below. The length of the 'Head' (longest axis) was measured from those images and was divided by the number of 'Heads'.
[00443] An image processing system was used, consisting of a personal computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.37, Java-based image processing software, developed at the National Institute of Health of the United States and freely available on the Internet at rsbweb (dot) nih (dot) gov/. The images were captured in 10 Mega Pixels resolution (3888x2592 pixels) and stored in a low-compression JPEG (Joint Photographic Experts Group standard | standard of the Joint Photographic Experts Group standard). Then the image processing output data for seed area and seed length were saved to text files and analyzed using JMP statistical analysis software (Instituto SAS).
[00444] Additional parameters were collected either by sampling 5 plants per batch or by measuring the parameter across all plants within the batch.
[00445] Total Seed Weight per Head (g.) - At the end of the experiment (plant 'Heads'), the heads of the lots within the AC blocks were collected. 5 heads were threshed separately and the grains were weighed, all additional heads were threshed together and also weighed. The average grain weight per head was calculated by dividing the total grain weight by the total number of heads per batch (batch-based). In the case of 5 heads, the total weight of grains from 5 heads was divided by 5.
[00446] Head FW per Gram of Plant - At the end of the experiment (when the heads were harvested), the total heads and 5 heads selected by lots within blocks A-C were collected separately. The heads (total and 5) were weighed (g.) separately and the average fresh weight per plant was calculated for the total heads (Head FW/Plant g. based on the lot) and for 5 heads (Head FW /g of Plant based on 5 plants).
[00447] Plant Height - Plants were characterized with respect to height during the growing season at 5 time points. In each measurement, the plants were measured in relation to their height using a measuring tape. Height was measured from ground level to the top of the longest leaf.
[00448] Number of Plant Leaves - Plants were characterized with respect to the number of leaves during the growing period at 5 time points. In each measurement, plants were measured in relation to their number of leaves by counting all leaves of 03 plants selected per lot.
[00449] Relative Growth Rate - was calculated using Formulas III (above) and VIII (above).
[00450] SPAD - Chlorophyll content was determined using a Minolta SPAD 502 chlorophyll meter and measurement was performed 64 days after sowing. SPAD meter readings were taken on fully developed new sheet. Three measurements per sheet were taken per batch.
[00451] Dry Plant Weight and Heads - At the end of the experiment (when the inflorescences were dry), all inflorescence and plant material from the lots within blocks A-C were collected. The weight of biomass and heads of each batch was separated, measured and divided by the number of heads.
[00452] Dry weight = total weight of the plant portion above the ground (excluding the roots) after drying at 70°C in an oven for 48 hours.
[00453] Crop Index (HI) (Sorghum) - The crop index was calculated using Formula XVI above.
[00454] Head FW / (Head FW + Plant FW) - The total fresh weight of the heads and their respective plant biomass were measured on the day of harvest. The weight of the heads was divided by the sum of the weights of the heads and plants. Experimental Results:
[00455] 17 different sorghum hybrids were grown and characterized with respect to different parameters (Table 29). The mean for each of the measured parameters was calculated using JMP software (Tables 30-35) and a subsequent correlation analysis was conducted (Table 36). The results were then integrated into the database.Table 29 Parameters correlated to Sorghum (vectors)
Table 29. Sorghum correlated parameters (vectors) are provided. "g." = grams; “SPAD” = chlorophyll levels; “FW” = Fresh Plant Weight; “DW” = Dry Plant Weight; “normal” = standard growing conditions; “DPS” = days after sowing; “Low N” = Low Nitrogen. Head - FW /Plan g. (based on 5 plants) = the fresh weight of the harvested heads was divided by the number of heads that were phenotyped; “Low N” = low nitrogen conditions; “Average Area of Lower Rate Grain” = grain area of the lower fraction of grains.Table 30Parameters measured in Sorghum accessions under normal conditions
Table 30: Values for each of the parameters (as described above) measured in Sorghum accessions (row ID) under normal conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 31Additional parameters measured in Sorghum accessions under normal conditions
Table 31: The values of each of the parameters (as described above) measured in Sorghum accessions (row ID) under normal conditions are provided. Cultivation conditions are specified in the experimental procedure section.VINICIUSTTable 32 Parameters measured in Sorghum accessions under low nitrogen conditions
Table 32: Values for each of the parameters (as described above) measured in Sorghum accessions (row ID) under low nitrogen conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 33Additional parameters measured in Sorghum accessions under low nitrogen cultivation conditions
Table 33: Values for each of the parameters (as described above) measured in Sorghum accessions (row ID) under low nitrogen conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 34 Parameters measured in Sorghum accessions under drought conditions
Table 34: Values for each of the parameters (as described above) measured in Sorghum accessions (row ID) under drought conditions are provided. Growing conditions are specified in the experimental procedure section.Table 35Additional parameters measured in Sorghum accessions under drought conditions
Table 35: Values for each of the parameters (as described above) measured in Sorghum accessions (row ID) under drought conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 36Correlation between the expression level of selected genes from some applications of the invention in various tissues and the phenotypic performance under abiotic or normal stress conditions in sorghum accessions





Table 36. Correlations (R) between expression levels of production-enhancing genes and their homologs in various tissues are provided [Flag leaf, flower meristem, stem and flower; Expression Sets (Exp.)] and phenotypic performance on various components of vigor, yield, biomass and/or growth rate [Correlation vector (Cor.)] under normal and stress conditions in sorghum accessions. P = p-value.EXAMPLE 6 SORGHUM TRANSCRIPTOME PRODUCTION AND HIGH PRODUCTIVITY CORRELATION ANALYSIS WITH PARAMETERS RELATED TO BIOMASS, NUE AND ABST MEASURED UNDER SEMI-HYDROPONIC CONDITIONS USING SORGHUM OLIGONUCLEOTIDE MICROARRANGES.
[00456] Parameters related to Sorghum vigor under conditions of low nitrogen, 100 mM NaCl, low temperature (10 ± 2°C) and normal cultivation conditions - Ten sorghum hybrids were cultivated in 3 repetitive lots, each containing 17 plants , in a net greenhouse under semi-hydroponic conditions. In summary, the cultivation protocol was as follows: Sorghum seeds were sown in trays filled with a mixture of vermiculite and peat in a 1:1 ratio. After germination, the trays were transferred to a high salinity solution (100 mM NaCl, in addition to Hogland's Complete solution) at low temperature (10 ± 2°C, in the presence of Hogland's Complete solution), a low nitrogen solution ( the amount of total nitrogen was reduced by 90% from the Complete Hogland solution (i.e., to a final concentration of 10% from the Complete Hogland solution, final amount of 1.2 mM N) or in a culture solution Normal [Hogland's Complete solution containing 16 mM N, at 28 ± 2°C.] Plants were grown at 28 ± 2°C.
[00457] Hogland's Complete Solution consists of: KNO3 - 0.808 grams/liter, MgSO4 - 0.12 grams/liter, KH2PO4 - 0.172 grams/liter and 0.01% (volume/volume) of the “Super coratin” microelements (Iron-EDDHA [ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid)] - 40.5 grams/liter; Mn - 20.2 grams/liter; Zn 10.1 grams/liter; Co 1.5 grams /liter, and Mo 1.1 grams/liter), the pH of the solution should be 6.5-6.8].
[00458] Sorghum Tissues Analyzed - All 10 selected Sorghum hybrids were sampled for each treatment. Three tissues [leaves, meristems and roots] cultured in 100 mM NaCl, at low temperature (10 ± 2°C), low nitrogen (1.2 mM N) or under Normal conditions were sampled and RNA was extracted as described above . Each tissue type of microarray expression information was assigned a Set ID, as summarized in Table 37 below.Table 37 Sorghum transcriptome expression sets under semi-hydroponic conditions
Table 37: Sorghum transcriptome expression sets are provided. Cold conditions = 10 ± 2°C; NaCl = 100 mM NaCl; low nitrogen = 1.2 mM Nitrogen; Normal conditions = 16 mM of Nitrogen. Experimental Results:
[00459] 10 different Sorghum hybrids were cultivated and characterized with respect to the following parameters: “Number of leaves” = number of leaves per plant (average of five plants), “Plant Height” = plant height [cm] (average of five plants), “DW Root/Plant” - root dry weight per plant (average of five plants); “DW shoot/plant” - shoot dry weight per plant (average of five plants) (Table 38); The mean for each of the measured parameters was calculated using the JMP software and the values are summarized in Tables 39-45 below. A subsequent correlation analysis was conducted (Table 46). The results were then integrated into the database.Table 38 Parameters correlated to Sorghum (vectors)

Table 38: Sorghum correlated parameters are provided. Cold conditions = 10 ± 2°C; NaCl = 100 mM NaCl; low nitrogen = 1.2 mM Nitrogen; Normal conditions = 16 mM Nitrogen; * TP1-2-3 refer to time periods 1, 2 and 3.Table 39 Sorghum accessions, parameters measured under low nitrogen cultivation conditions
Table 39: Values for each of the parameters (as described above) measured in Sorghum accessions (row ID) under low nitrogen conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 40Additional Sorghum accessions, parameters measured under
Table 40: Values for each of the parameters (as described above) measured in Sorghum accessions (row ID) under low nitrogen conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 41 Sorghum accessions, parameters measured under salinity conditions (100 mM NaC
Table 41: Values for each of the parameters (as described above) measured in Sorghum accessions (row ID) under cultivation conditions at 100 mM NaCl are provided. Cultivation conditions are specified in the experimental procedure section.Table 42 Additional Sorghum accessions, parameters measured under salinity conditions (100 mM NaCl)
Table 42: Values for each of the parameters (as described above) measured in Sorghum accessions (row ID) under cultivation conditions at 100 mM NaCl are provided. Growing conditions are specified in the experimental procedure section.Table 43 Sorghum accessions, parameters measured under cold conditions
Table 43: Values for each of the parameters (as described above) measured in Sorghum accessions (row ID) under cool growing conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 44 Sorghum accessions, parameters measured under regular cultivation conditions
Table 44: Values for each of the parameters (as described above) measured in Sorghum accessions (row ID) under regular cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 45 Additional Sorghum accessions, parameters measured under regular cultivation conditions
Table 44: Values for each of the parameters (as described above) measured in Sorghum accessions (row ID) under regular cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 46Correlation between the expression level of selected genes from some applications of the invention in roots and the phenotypic performance under abiotic or normal stress conditions in sorghum accessions



Table 46. Correlations (R) are provided between production of expression levels that enhance genes and their homologues in various tissues [Expression Sets (Exp.)] and phenotypic performance [components of vigor, production, biomass and/or or growth rate (Correlation vector (Cor.))] under abiotic stress conditions (salinity) or normal conditions in sorghum accessions. Color. - correlation vector, as described above (Table 38). P = p-value. EXAMPLE 7 CORN TRANSCRIPTOME PRODUCTION AND HIGH PRODUCTIVITY CORRELATION ANALYSIS WITH PRODUCTION-RELATED AND NUE PARAMETERS USING 60K CORN OLIGONUCLEOTIDE MICROARRANGE
[00460] In order to produce a high throughput correlation analysis between plant phenotype and gene expression level, the present inventors used a corn oligonucleotide microarray, produced by Agilent Technologies [chem(dot) agilent(dot) ) com/Scripts/PDS (dot) asp lPage=50879]. The oligonucleotide array represents about 60,000 maize genes and transcripts.Correlation of Maize hybrids in ecotypes cultivated under regular cultivation conditionsExperimental Procedures:
[00461] 12 corn hybrids were cultivated in 3 repetitive lots, in the field. Corn seeds were planted and the plants were grown in the field, using commercial fertilization and irrigation protocols (485 cubic meters of water per dunam, 30 units of uran, 21% fertilization per complete growing season). In order to define the correlations between RNA expression levels and parameters related to vigor or stress and yield components, the 12 different corn hybrids were analyzed. Among them, 10 hybrids, encompassing the observed variation, were selected for analysis of RNA expression. The correlation between RNA levels and the characterized parameters was analyzed using the Pearson correlation test [davidmlane (dot) com/hyperstat/A34739 (dot) html].
[00462] Corn Tissues Analyzed - All 10 corn hybrids were sampled with respect to 3 time points (TP2=V6-V8, TP5=R1-R2, TP6=R3-R4). Four types of plant tissues [Ear, flag leaf indicated in Table 47 as “leaf”, distal part of the grain, and internode] grown under normal conditions were sampled and RNA was extracted as described above. Each tissue type of microarray expression information was assigned a Set ID, as summarized in Table 47 below.Table 47 Maize transcriptome expression sets

Table 47: Maize transcriptome expression sets are provided. Leaf = the leaf below the main spike Flower meristem = apical meristem after male-leaf initiation; Spike = the female flower on the day of anthesis. Distal Grain = corn developing grains from the external area of the cob, Basal Grain - corn developing grains from the basal area of the cob; Internodes = Internodes located above and below the main ear on the plant. TP= time point.
[00463] The following parameters were collected using a digital imaging system: Grain Area (cm2) - At the end of the growing period, the grains were separated from the ear. A sample of ~200 grains was weighed, photographed and the images processed using the image processing system described below. Grain area was measured from those images and divided by the number of grains.
[00464] Grain Length and Grain Width (cm) - At the end of the growing period, the grains were separated from the ear. A sample of ~200 grains was weighed, photographed and the images processed using the image processing system described below. The sum of grain length/or width (longest axis) was measured from those images and divided by the number of grains.
[00465] Ear Area (cm2) - At the end of the growing period, 5 ears were photographed and the images were processed using the image processing system described below. The ear area was measured from those images and divided by the number of ears.
[00466] Ear Length and Width (cm) - At the end of the growing period, 5 ears were photographed and the images were processed using the image processing system described below. The length and width of the spike (longest axis) were measured from those images and divided by the number of spikes.
[00467] An image analysis system was used, consisting of a personal computer (Intel P4 3.0 Ghz processor) and a public domain program - ImageJ 1.37, an image processing program based on Java, developed at the National Institute of Health of the United States and freely available on the Internet at rsbweb (dot) nih (dot) gov/. The images were captured in 10 Mega Pixels resolution (3888x2592 pixels) and stored in a low-compression JPEG format (standard of the Joint Group of Experts in Photography). Then, image processing output data for seed area and seed length were saved to text files and analyzed using JMP statistical analysis software (SAS Institute).
[00468] Additional parameters were collected by sampling 6 plants per batch or by measuring the parameter on all plants within the batch.
[00469] Normalized Grain Weight per plant (gr.) - At the end of the experiment, all the ears of the lots within the blocks from A to C were harvested. Six ears were separated and the beans were weighed, all additional ears were threshed together and weighed in the same way. The average grain weight per ear was calculated by dividing the total grain weight by the total number of ears per lot (based on the lot). In the case of 6 ears, the total grain weight of 6 ears was divided by 6.
[00470] Ear FW (g.) - At the end of the experiment (when the ears were harvested), the total and 6 ears selected per lot within blocks A to C were harvested separately. Plants with total and 6 were weighed (g.) separately and the average number of ears per plant was calculated for the total [Ear FW (fresh weight) per lot] and for 6 (Ear FW per plant).
[00471] Plant weight and ear height - The plants were characterized with respect to height at harvest. In each measurement, 6 plants were measured for height using a tape measure. Height was measured from ground level to the top of the plant below the tassel. The height of the spikes was measured from the ground level to the place where the main spike was located.
[00472] Number of leaves per plant - The plants were characterized with respect to the number of leaves during the growing period at 5 time points. In each measurement, plants were measured in relation to their number of leaves, counting all leaves of 3 selected plants per lot.
[00473] Relative Growth Rate - was calculated using Formulas II to XIII (described above).
[00474] SPAD - The chlorophyll content was determined using a Minolta SPAD 502 chlorophyll meter and the measurement was performed 64 days after sowing. SPAD meter readings were taken on a fully developed young leaf. Three measurements per leaf were taken per ear. Data were collected after 46 and 54 days after sowing (DPS|days after sowing).
[00475] Dry weight per plant - At the end of the experiment (when the inflorescence was dry), all plant material from the plots within blocks A to C was harvested.
[00476] Dry weight - total weight of the aboveground plant part (excluding roots) after drying at 70°C in an oven for 48 hours.
[00477] Crop Index (HI) (Corn) - The crop index was calculated using Formula XVII described above.
[00478] Percentage of Ears Filled [%] - was calculated as the percentage of the ear area with grains outside the total ear.
[00479] Cob Diameter [cm] - The diameter of the cob without the grains was measured using a ruler.
[00480] Number of kernel rows per ear - The number of rows in each ear was counted. Experimental Results:
[00481] 12 different corn hybrids were cultivated and characterized with respect to different parameters. The correlated parameters are described in Table 48 below. The mean for each of the measured parameters was calculated using the JMP software (Tables 49-50) and a subsequent correlation analysis was performed. The results were then integrated into the database.Table 48Correlated parameters to Maize (vectors)
Table 48. SPAD 46DPS and SPAD 54DPS: Chlorophyll levels after 46 and 54 days after sowing (DPS). “FW” = fresh weight; “DW” = dry weight.Table 49 Parameters measured in accessions of Corn under normal conditions

Table 49. Values for each of the parameters (as described above) measured in maize accessions (row ID) under regular cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 50Additional parameters measured in corn accessions under normal cultivation conditions
Table 50. Values for each of the parameters (as described above) measured in maize accessions (row ID) under regular cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 51Correlation between the expression level of selected genes from some applications of the invention in various tissues and the phenotypic performance under normal conditions in maize varieties


Table 51. Correlations (R) between expression levels of production-enhancing genes and their homologues in various tissues are provided [Expression sets (exp.)] and phenotypic performance [roduction components, biomass, growth rate and/or vigor (Correlation vector (corr)] under normal conditions in maize varieties. P = p-value. EXAMPLE 8 CORN TRANSCRIPTOME PRODUCTION AND HIGH PRODUCTIVITY CORRELATION ANALYSIS WITH PRODUCTION-RELATED PARAMETERS AND NUE WHEN CULTIVATED UNDER REDUCED NITROGEN FERTILIZATION USING 60K CORN OLIGONUCLEOTIDE MICROARRANGE
[00482] In order to produce a high throughput correlation analysis between plant phenotype and gene expression level, the present inventors used a corn oligonucleotide microarray, produced by Agilent Technologies [chem(dot) agilent(dot) ) com/Scripts/PDS (dot) asp lPage=50879]. The oligonucleotide array represents about 44,000 of maize genes and transcripts.Correlation of Maize hybrids in ecotypes grown under low nitrogen conditions.Experimental procedures:
[00483] 12 corn hybrids were cultivated in 3 repetitive lots, in the field. Corn seeds were planted and the plants were grown in the field, using commercial fertilization and irrigation protocols (485 cubic meters of water per dunam, 30 units of uran, 21% fertilization per complete growing season). In order to define the correlation between RNA expression levels with parameters related to vigor or NUE components and yield, 12 different maize hybrids were analyzed. Among them, 11 hybrids encompassing the observed variation were selected for RNA expression analysis. The correlation between RNA levels and the characterized parameters was analyzed using the Pearson correlation test [davidmlane (dot) com/hyperstat/A34739 (dot) html].
[00484] Corn Tissues Analyzed - All 10 corn hybrids were sampled for each treatment (low N conditions and normal conditions), at three time points: TP2 = V6-V8 (six to eight leaves of the collar are visible, the rapid growth phase and the determination of core rows are started), TP5 = R1-R2 (satin - bubble), TP6 = R3-R4 (milk - paste). Four types of plant tissues [Ears, flag leaf indicated in Tables 52 to 53 as leaf, distal part of the grain and internode] were sampled and RNA was extracted as described above. Each tissue type of microarray expression information was assigned a Set ID, as summarized in Tables 52 to 53 below.Table 52 Maize transcriptome expression sets under low N conditions
Table 52: Maize transcriptome expression sets are provided. Leaf = the leaf below the main ear; Flower meristem = apical meristem after male-leaf initiation; Spike = the female flower on the day of anthesis. Distal Grain = corn developing grains from the external area of the cob, Basal Grain = corn developing grains from the basal area of the cob; Internode = Internodes located above and below the main ear on the plant.Table 53 Maize transcriptome expression sets under normal conditions
Table 53: Maize transcriptome expression sets are provided. Leaf = the leaf below the main ear; Flower meristem = apical meristem after male-leaf initiation; Spike = the female flower on the day of anthesis. Distal Grain = corn developing grains from the external area of the cob, Basal Grain = corn developing grains from the basal area of the cob; Entrenode = Entrenodes located above and below the main ear on the plant.
[00485] The following parameters were collected either by sampling 6 plants per batch or by measuring the parameter across all plants within the batch.
[00486] Seed Production per plant (Kg.) - At the end of the experiment, all ears of lots within blocks A-C were collected. 6 ears were threshed separately and the beans were weighed, all additional ears were threshed together and also weighed. The average grain weight per ear was calculated by dividing the total grain weight by the total number of ears per lot (based on unit). In the case of 6 ears, the total weight of grains from 6 ears was divided by 6.
[00487] Ear Weight per lot (g.) - At the end of the experiment (when the ears were harvested) total and 6 ears selected by lots within blocks A-C were collected separately. Plants with (total and 6) were weighed (g.) separately and the average ear per plant was calculated for Ear Weight per lot (total of 42 plants per lot).
[00488] Plant height and ear height - Plants were characterized by height at harvest. In each measurement, 6 plants were measured for their height using a tape measure. Height was measured from ground level to the top of the plant below the corn hair. The height of the ear was measured from the ground level to the place where the main ear is located.
[00489] Number of leaf per plant - The plants were characterized by the number of leaf during the growing period in 5 times in time. In each measurement, plants were measured by their leaf number by counting all leaves of 3 selected plants per lot.
[00490] SPAD - Chlorophyll content was determined using a Minolta SPAD 502 chlorophyll meter and measurement was performed 64 days after sowing. SPAD measurement readings were taken on fully developed new sheet. Seven measurements per sheet were taken per batch. Data were taken after once a week after sowing.
[00491] Dry weight per plant - At the end of the experiment (when the inflorescence is dry) all plant material from the lots within blocks A-C was collected.
[00492] Dry weight = total weight of the plant part above the ground (excluding the roots) after drying at 70°C in a drying chamber for 48 hours; Ear Length of the Filled Ear [cm] - was calculated as the length of the Cob with kernels out of full cob.
[00493] Ear Length and Width [cm] - was calculated as the unfilled ear length and width. The measurement was performed on 6 plants per lot.
[00494] Number of Core Row per Spike - The number of rows in each spike has been counted.
[00495] Stem Width [cm] - The stem diameter was measured at the internode located below the main ear. The measurement was performed on 6 plants per lot.
[00496] Sheet Area Index [LAI | leaf area index] = total leaf area of all plants in a plot. Measurement was performed using a leaf area meter.
[00497] NUE [kg/kg]- is the ratio of total grain production per total N applied to the soil.
[00498] NUpE [kg/kg]- is the ratio of total plant biomass to total N applied to the soil.
[00499] Yield/stem width [kg/cm] - is the ratio between total grain yields and stem width.
[00500] Production/LAI [kg] - is the ratio between the total grain yields and the total leaf area index. Experimental Results:
[00501] 11 different corn hybrids were grown and characterized with respect to different parameters. Tables 54-55 describe the parameters correlated to Maize. The mean for each of the measured parameters was calculated using JMP software (Tables 5659) and a subsequent correction analysis was performed (Tables 60-61). The results were integrated into the database.Table 54Correlated parameters to Maize (vectors) under Low N conditions
Table 54. “cm” = centimeters' “mm” = millimeters; “kg” = kilograms ; SPAD in R1-R2 and SPAD R3-R4: Chlorophyll level after early and late stages of grain filling, “NUE” = nitrogen use efficiency; “NUpE” = Efficiency in nitrogen uptake; “LAI” = Area of the sheet; “N” = nitrogen; Low N - under low nitrogen conditions; “Normal” = under normal conditions, “dunam” = 1000 m2.Table 55 Parameters correlated to Maize (vectors) under normal conditions
Table 55. “cm” = centimeters' “mm” = millimeters; “kg” = kilograms ; SPAD in R1-R2 and SPAD R3-R4: Chlorophyll level after early and late stages of grain filling, “NUE” = nitrogen use efficiency; “NUpE” = Efficiency in nitrogen uptake; “LAI” = Area of the sheet; “N” = nitrogen; Low N - under low Nitrogen conditions; “Normal” = under normal conditions, “dunam” = 1000 m2.Table 56 Parameters measured in corn accessions under normal conditions
Table 56: Values for each of the parameters (as described above) measured in maize accessions (row ID) under normal conditions are provided. Growing conditions are specified in the experimental procedure section.Table 57Additional parameters measured in corn accessions under
Table 57: Values for each of the parameters (as described above) measured in maize accessions (row ID) under normal conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 58 Parameters Measured in Corn Accessions Under Low Nitrogen Conditions

Table 58: Values for each of the parameters (as described above) measured in maize accessions (row ID) under low nitrogen conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 59Additional parameters measured in Corn accessions under Low Nitrogen conditions
Table 59. Values for each of the parameters (as described above) measured in maize accessions (row ID) under low nitrogen conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 60Correlation between the expression level of selected genes from some applications of the invention in various tissues and the phenotypic performance under normal conditions in maize varieties


Table 60: Correlations (R) between expression levels of production-enhancing genes and their homologues in various tissues are provided [Expression sets (exp.)] and phenotypic performance [roduction components, biomass, growth rate and/or vigor (Correlation vector (corr)] under normal conditions in maize varieties. P = p-value.Table 61Correlation between the expression level of selected genes from some applications of the invention in various tissues and the phenotypic performance under low N conditions in maize varietiesName dc*



Table 61. Correlations (R) between expression levels of production-enhancing genes and their homologues in various tissues are provided [Expression sets (exp.)] and phenotypic performance [roduction components, biomass, growth rate and/or vigor (Correlation vector (corr)] under low nitrogen conditions in maize varieties. P = p-value. EXAMPLE 9 TOMATO TRANSCRIPTOME PRODUCTION AND HIGH PRODUCTIVITY CORRELATION ANALYSIS USING 44K TOMATO OLIGONUCLEOTIDE MICROARRANGE
[00502] In order to produce a high-throughput correlation analysis between NUE-related phenotype and gene expression, the present inventors utilized a Tomato oligonucleotide microarray, produced by Agilent Technologies [chem(dot) agilent(dot) com/Scripts/PDS (dot) asp lPage=50879]. The oligonucleotide array represents about 44,000 Tomato genes and transcripts. In order to define the correlation between RNA expression levels and parameters related to vigor or production components NUE, and ABST, several plant characteristics of 18 different tomato varieties were analyzed. Among them, 10 varieties encompassing the observed variation were selected for analysis of RNA expression. The correlation between RNA levels and the characterized parameters was analyzed using the Pearson correlation test [davidmlane (dot) com/hyperstat/A34739 (dot) html]. Correlation of Tomato varieties in ecotypes cultivated under low nitrogen conditions, drought conditions and regular cultivation conditions Experimental Procedures:
[00503] 10 varieties of Tomatoes were grown in 3 repetitive blocks, each containing 6 plants per lot, in a greenhouse with a net. In short, the cultivation protocol was as follows:1. Regular growing conditions: Tomato varieties were grown under normal conditions (4 to 6 Liters/m2 of water per day and fertilized with NPK, as recommended in the protocols for commercial tomato production).
[00504] 2. Low Nitrogen Fertilization Conditions: Tomato varieties were grown under normal conditions (4 to 6 Liters/m2 per day and fertilized with NPK, as recommended in the protocols for commercial tomato production) until the flowering stage. At this time, nitrogen fertilization was stopped.
[00505] 3. Drought stress: The Tomato variety was grown under normal conditions (4 to 6 Liters/m2 per day) until the flowering stage. At this time, irrigation was reduced to 50% compared to normal conditions.
[00506] Plants were phenotyped daily, following standard tomato descriptors (Table 63). Harvesting was carried out while 50% of the fruits were red (ripe). The plants were separated into vegetable and fruit parts; of these, 2 nodes were analyzed for additional inflorescence parameters such as size, number of flowers and inflorescence weight. The fresh weight of all plant material was measured. The fruits were separated by color (red vs. green) and according to the size of the fruit (small, medium and large). Then, the analyzed data were saved as text files and processed using the JMP statistical analysis software (SAS Institute). The data parameters collected are summarized in Tables 64 to 70 below.
[00507] Analyzed Tomato Tissues- Two tissues at different stages of development [leaf flower], representing different plant characteristics, were sampled and RNA extracted as described above. For convenience, each type of microarray expression information has been assigned Set, as summarized in Table 62 below. Table 62 Tomato Transcriptome Expression Sets
Table 62: Identification (ID) numbers of each tomato expression set are provided.
[00508] Table 63 provides the parameters correlated to tomato (Vectors). The average for each of the measured parameters was calculated using the JMP software and the values are summarized in Tables 64 to 70 below. A subsequent correlation analysis was conducted (Table 71). The results were integrated into the database.Table 63Tomato correlated parameters (vectors)

Table 63. Parameters correlated to tomato are provided. "g." = grams, "FW" = fresh weight; “NUE” = nitrogen use efficiency; “RWC” = relative water content; “NUpE” = nitrogen uptake efficiency, “SPAD” = chlorophyll levels; “HI” = harvest index (vegetable weight divided by production); “SLA” = specific sheet area (sheet area divided by dry sheet weight).
[00509] Fruit Yield (grams) - At the end of the experiment [when 50% of the fruits were ripe (red)], all the fruits from the lots within blocks A to C were harvested. The total number of fruits was counted and weighed. The average fruit weight was calculated by dividing the total fruit weight by the number of fruits.
[00510] Yield/SLA and Yield/Total Leaf Area - Fruit yield divided by specific leaf area or total leaf area provides a measure of a balance between reproductive and plant processes.
[00511] Fresh Fruit Weight (grams) - At the end of the experiment [when 50% of the fruits were ripe (red)], all plants from the lots within blocks A to C were harvested. Fresh weight was measured (grams).
[00512] Inflorescence weight (grams) - At the end of the experiment [when 50% of the fruits were ripe (red)], two inflorescences from the lots within blocks A to C were harvested. Inflorescence weight (gr.) and number of flowers per inflorescence were counted.
[00513] SPAD - The chlorophyll content was determined using a Minolta SPAD 502 chlorophyll meter and the measurement was performed at the time of flowering. SPAD meter readings were taken on a fully developed young leaf. Three measurements were taken per leaf per ear.
[00514] Water Use Efficiency (WUE) - can be determined as the biomass produced per unit transpiration. To analyze the WUE, the water content relative to the leaf was measured in control and transgenic plants. Fresh weight (FW) was immediately recorded; then, the leaves were soaked for 8 hours in distilled water at room temperature in the dark and the turgid weight (TW | turgid weight) was recorded. The total dry weight (DW) was recorded after drying the leaves at 60°C at a constant weight. The relative water content (RWC) was calculated according to Formula I below [(FW - DW/TW - DW) x 100], as described above.
[00515] The plants that maintained a high relative water content (RWC) compared to the control lines were considered more drought tolerant than those that exhibited a reduced relative water content.Experimental Results:Table 64 Parameters measured in Tomato accessions under conditions of drought
Table 64: Values for each of the parameters (as described above) measured in Tomato accessions (row ID) under drought conditions are provided. Growing conditions are specified in the experimental procedure section.Table 65 Additional parameters measured in Tomato accessions under drought conditions
Table 65: Values for each of the parameters (as described above) measured in Tomato accessions (row ID) under drought conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 66 Parameters measured in Tomato accessions under low nitrogen conditions
Table 66: The values of each of the parameters (as described above) measured in Tomato accessions (Seed ID) under low Nitrogen cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 67Additional parameters measured in Tomato accessions under Low Nitrogen conditions
Table 67: Values of each of the parameters (as described above) measured in Tomato accessions (Seed ID) under low nitrogen cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section. Table 68 Additional parameters measured in Tomato accessions under Low Nitrogen conditions
Table 68: Values for each of the parameters (as described above) measured in Tomato accessions (Seed ID) under low Nitrogen cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 69 Parameters measured in Tomato accessions under normal conditions
Table 69: The values of each of the parameters (as described above) measured in Tomato accessions (row ID) under normal cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 70Additional parameters measured in Tomato accessions under normal conditions
Table 70: The values of each of the parameters (as described above) measured in Tomato accessions (row ID) under normal cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 71Correlation between the expression level of selected genes from some applications of the invention in various tissues and the phenotypic performance under normal and stress conditions in tomato ecotypes
Table 71. Correlations (R) between expression levels of production-enhancing genes and their homologs in various tissues are provided [Expression sets (exp.)] and phenotypic performance [roduction components, biomass, growth rate and/or vigor (Correlation vector (corr)] under normal and low nitrogen conditions in all tomato ecotypes. P = p value.
[00516] Correlation of vigor traits in collections of Tomato ecotypes under Low nitrogen conditions, 300 mM NaCl and normal cultivation conditions - Ten tomato hybrids were cultivated in 3 repetitive lots, each containing 17 plants, in a greenhouse with net under semi-hydroponic conditions. In summary, the cultivation protocol was as follows: Tomato seeds were sown in trays filled with a mixture of vermiculite and peat in a 1:1 ratio. After germination, the trays were transferred to the high salinity solution (300 mM NaCl, plus Hoagland's complete solution), low nitrogen ("low N") solution (the amount of total nitrogen was reduced by 90% of the complete Hoagland solution, final amount 0.8 nM N), or in Normal culture solution (Hoagland's Complete, containing 8 mM N solution, culture at 28 ± 2°C). Plants were grown at 28 ± 2°C.
[00517] Hoagland's complete solution consists of: KNO3 - 0.808 grams/liter, MgSO4 - 0.12 grams/liter, KH2PO4 - 0.172 grams/liter and 0.01% (volume/volume) of 'Super coratin' microelements (Iron-EDDHA [ethylenediamine-N,N'-bis(2-hydroxyphenylacetic acid)]- 40.5 grams/liter; Mn - 20.2 grams/liter; Zn 10.1 grams/liter; Co 1.5 grams /liter; and Mo 1.1 grams/liter), the pH of the solution should be 6.5 - 6.8].
[00518] Tomato Tissues Analyzed - All 10 selected tomato varieties were sampled for each treatment. Three tissues [leaves, meristems and flowers] were sampled and RNA was extracted as described below. For convenience, each tissue type of microarray expression information was assigned a Set ID, as summarized in Table 72 below.Table 72 Tomato Transcriptome Experimental Sets

Table 72. Tomato transcriptome experimental sets are provided.
[00519] Parameters related to tomato vigor - After 5 weeks of cultivation, the plants were harvested and analyzed regarding the number of leaves, plant height, chlorophyll levels (SPAD units), different indices of nitrogen use efficiency ( NUE) and plant biomass. Next, the analyzed data were saved as text files and processed using the JMP statistical analysis software (Instituto SAS). The data parameters collected are summarized in Table 73 below. Table 73 Parameters correlated to Tomato (vectors)
Table 73. Parameters correlated with tomato are provided. “FW” = fresh weight; “cm” = centimeter. “Sheet No.” = number of leaves. Experimental Results:
[00520] 10 different varieties of Tomato were cultivated and characterized with respect to parameters, as described below. The average for each of the measured parameters was calculated using the JMP software and the values are summarized in Tables 74 to 77 below. A subsequent correlation analysis was conducted (Table 78). Subsequently, the results were integrated into the database.Table 74Parameters measured in Tomato accessions under low nitrogen conditions
Table 74. Values for each of the parameters (as described above) measured in Tomato (Line) accessions are provided under low nitrogen cultivation conditions. Cultivation conditions are specified in the experimental procedure section.Table 75Additional parameters measured in Tomato accessions under Low Nitrogen conditions
Table 75. Values for each of the parameters (as described above) measured in Tomato (Line) accessions are provided under low nitrogen cultivation conditions. Cultivation conditions are specified in the experimental procedure section.Table 76 Parameters measured in Tomato accessions under normal conditions

Table 76. Values for each of the parameters (as described above) measured in Tomato (Line) accessions are provided under normal growing conditions. Cultivation conditions are specified in the experimental procedure section.Table 77Parameters measured in Tomato accessions under salinity conditions
Table 77. Values for each of the parameters (as described above) measured in Tomato (Line) accessions are provided under salinity cultivation conditions. Cultivation conditions are specified in the experimental procedure section.Table 78Correlation between the expression level of selected genes from some applications of the invention in various tissues and the phenotypic performance under normal and stress conditions in tomato ecotypes
Table 78. Correlations (R) between expression levels of production-enhancing genes and their homologues in various tissues [Expression sets (exp.)] and phenotypic performance [roduction components, biomass, growth rate] are provided. and/or vigor (Correlation vector (Corr))] under normal conditions and under conditions of low nitrogen in all tomato ecotypes. P = p value. EXAMPLE 10 CORN TRANSCRIPTOME PRODUCTION AND HIGH PRODUCTIVITY CORRELATION ANALYSIS WHEN GROWN UNDER NORMAL AND DEFOLIATION CONDITIONS USING 60K CORN OLIGONUCLEOTIDE MICROARRANGE
[00521] In order to produce a high throughput correlation analysis, the present inventors used a corn oligonucleotide microarray, produced by Agilent Technologies [chem(dot) agilent(dot)com/Scripts/PDS(dot)asp lPage =50879]. The oligonucleotide array represents about 60K of Maize genes and transcripts, designed based on data from public databases (Example 1). In order to define the correlation between RNA expression levels and parameters related to vigor or biomass components and yield, several plant characteristics of 13 different corn varieties were analyzed under normal conditions and defoliation treatment. Some varieties were subjected to RNA expression analysis. The correlation between RNA levels and the characterized parameters was analyzed using the Pearson correlation test [davidmlane (dot) com/hyperstat/A34739 (dot) html]. Experimental Procedures:
[00522] 13 lines of corn varieties were cultivated in 6 repeated lots, in the field. Corn seeds were planted and the plants were cultivated in the field, using commercial fertilization and irrigation protocols. After satining, 3 lots in all variety lines were subjected to defoliation treatment. In this treatment, all leaves above the ear were removed. After treatment, all plants were cultivated according to the same commercial fertilization and irrigation protocols.
[00523] Three tissues in flowering developmental stage (R1), including leaf (blooming - R1), stem (blooming - R1), and flowering meristem (blooming - R1), representing different plant characteristics, were sampled from plants treated and untreated. RNA was extracted as described in “GENERAL EXPERIMENTAL AND BIOINFORMATICS METHODS”. For convenience, each tissue type of microarray expression information was assigned a Set ID, as summarized in Tables 79 to 80 below.Table 79 Tissues used for Maize transcriptome expression sets (under normal conditions)
Table 79: Identification numbers (ID) of each Maize expression set are provided.Table 80 Tissues used for Maize transcriptome expression sets (under defoliation conditions)
Table 80: Identification (ID) numbers of each Maize expression set are provided.
[00524] The following parameters were collected by imaging.
[00525] The image processing system used consists of a personal computer (Intel P4 3.0 Ghz processor) and a public domain program - ImageJ 1.37 (Image processing program based on Java), developed at the National Institutes of Health in United States and is freely available on the internet at rsbweb (dot) nih (dot) gov/. The images were captured in a resolution of 10 Mega Pixels (3888x2592 pixels) and stored in a low-compression JPEG format (standard of the Joint Group of 5 Specialists in Photography). Then, the analyzed data were saved in text files and processed using the JMP statistical analysis software (SAS institute).
[00526] Weight of 1000 Grains - At the end of the experiment, all seeds from all lots were harvested and weighed and the weight of 1000 was calculated.
[00527] Ear Area (cm2)- At the end of the cultivation period, 5 ears were photographed and the images were processed using the image processing system described below. The ear area was measured from these images and divided by the number of ears.
[00528] Ear Length and Ear Width (cm) - At the end of the cultivation period, 6 ears were photographed and the images were processed using the image processing system described below. The length and width of the spikes (longest axis) were measured from these images and divided by the number of spikes.
[00529] Grain Area (cm2) - At the end of the growing period, the grains were separated from the ear. A sample of ~200 grains was weighed, photographed and the images processed using the image processing system described below. Grain area was measured from these images and divided by the number of grains.
[00530] Grain Length and Grain Width (cm) - At the end of the growing period, the grains were separated from the ear. A sample of ~200 grains was weighed, photographed and the images processed using the image processing system described below. The sum of grain lengths/or width (longest axis) was measured from these images and divided by the number of grains.
[00531] Grain Perimeter (cm) - At the end of the growing period, the grains were separated from the ear. A sample of ~200 grains was weighed, photographed and the images processed using the image processing system described below. The sum of the grain perimeter was measured from these images and divided by the number of grains.
[00532] Area filled by ear grains (cm2)
[00533] - At the end of the cultivation period, 5 ears were photographed and the images were processed using the image processing system described below. The area of the ear filled with nuclei was measured from these images and was divided by the number of ears.
[00534] Filled by Whole Ear [%] - was calculated as the length of the ear with grains outside the full ear.
[00535] Additional parameters were collected by sampling 6 plants per batch or by measuring the parameter on all plants within the batch.
[00536] Cob Width [cm] - The diameter of the cob without the grains was measured using a ruler.
[00537] Average Ear Weight (Kg.) - At the end of the experiment (when the ears were harvested), the total and 6 selected ears per lot were harvested. The ears were weighed and the average number of ears per plant was calculated. The average ear weight was normalized using the relative humidity as 0%.
[00538] Plant weight and ear height - Plants were characterized with respect to height at harvest. In each measurement, 6 plants were measured for height using a tape measure. Height was measured from ground level to the top of the plant below the tassel. The height of the spikes was measured from the ground level to the place where the main spike was located.
[00539] Number of ear rows - The number of rows per ear has been counted.
[00540] Fresh ear weight per plant (GF) - During the grain filling period (GF | grain filling), a total of 6 selected ears per lot were harvested. The ears were weighed and the average ear weight per plant was calculated.
[00541] Ear Dry Weight - At the end of the experiment (when the ears were harvested), the total and 6 selected ears per lot were harvested and weighed. The average ear weight was normalized using the relative humidity as 0%.
[00542] Fresh Ear Weight - At the end of the experiment (when the ears were harvested), the total and 6 selected ears per lot were harvested and weighed.
[00543] Ears per plant - the number of ears per plant has been counted.
[00544] Grain Weight (Kg.) - At the end of the experiment, all ears were harvested. Ears of 6 plants from each lot were threshed separately and the grains were weighed.
[00545] Dry weight of grains (Kg.) - At the end of the experiment, all ears were harvested. Ears of 6 plants from each lot were threshed separately and the grains were weighed. The grain weight was normalized using the relative humidity as 0%.
[00546] Grain weight per ear (Kg.) - At the end of the experiment, all the ears were harvested. 5 ears of each lot were threshed separately and the grains were weighed. The average weight of grains per ear was calculated by dividing the total weight of grains by the number of ears.
[00547] Leaf area per plant (GF) and (HD) [LAI] = Total leaf area of 6 plants in a lot; its parameter was measured at two time points during the course of the experiment; in the flowering stage (HD | heading) and during the grain filling period (GF). Measurement was performed using a leaf area meter at two time points in the course of the experiment; during the grain filling period and in the flowering stage (VT).
[00548] Fresh leaf weight (GF) and (HD) - This parameter was measured at two time points during the course of the experiment; in the flowering stage (HD) and during the grain filling period (GF). The sheets used for the measurement of LAI were weighed.
[00549] Lower Stem Fresh Weight (GF) (HD) and (H) - This parameter was measured at three time points during the course of the experiment: at the flowering stage (HD), during the grain filling period ( GF) and at harvest (H). The lower internodes of at least 4 plants per batch were separated from the plant and weighed. The average internode weight per plant was calculated by dividing the total grain weight by the number of plants.
[00550] Lower Stem Length (GF) (HD) and (H) - This parameter was measured at three time points during the course of the experiment; in the flowering stage (HD), during the grain filling period (GF) and at harvest (H). The lower internodes of at least 4 plants per batch were separated from the plant and their lengths were measured using a rule. The average length of internodes per plant was calculated by dividing the total weight of the grain by the number of plants.
[00551] Bottom stem width (GF) (HD) and (H)- This parameter was measured at three time points during the course of the experiment; at the flowering stage (HD), during the grain filling period (GF) and at harvest (H). The lower internodes of at least 4 plants per batch were separated from the plant and their diameters were measured using a gauge. The average width of internodes per plant was calculated by dividing the total weight of the grain by the number of plants.
[00552] Plant Height Growth: The Relative Growth Rate (RGR) of Plant Height was calculated using Formula III above.
[00553] SPAD - The chlorophyll content was determined using a Minolta SPAD 502 chlorophyll meter and the measurement was performed 64 days after sowing. SPAD meter readings were taken on a fully developed young leaf. Three measurements were taken per leaf per ear. Data were collected after 46 and 54 days after sowing (DPS|days after sowing)
[00554] Fresh Stem Weight (GF) and (HD) - This parameter was measured at two time points during the course of the experiment; in the flowering stage (HD) and during the grain filling period (GF). The plant stems used for the measurement of LAI were weighed.
[00555] Total dry matter was calculated using Formula XXXV.
[00556] Top Stem Fresh Weight (GF) (HD) and (H) - This parameter was measured at three time points during the course of the experiment; at the flowering stage (HD), during the grain filling period (GF) and at harvest (H). The upper internodes of at least 4 plants per batch were separated from the plant and weighed. The average weight of internodes per plant was calculated by dividing the total weight of the grain by the number of plants.
[00557] Upper stem height (GF) (HD) and (H)
[00558] - This parameter was measured at three time points during the course of the experiment; at the flowering stage (HD), during the grain filling period (GF) and at harvest (H). The upper internodes of at least 4 plants per lot were separated from the plant and their lengths were measured with a ruler. The average length of internodes per plant was calculated by dividing the total weight of the grain by the number of plants.
[00559] Upper Stem Width (GF) (HD) and (H) (mm) - This parameter was measured at three time points during the course of the experiment; at the flowering stage (HD), during the grain filling period (GF) and at harvest (H). The upper internodes of at least 4 plants per batch were separated from the plant and their diameters were measured using a gauge. The average width of internodes per plant was calculated by dividing the total weight of the grain by the number of plants.
[00560] Dry plant weight (Kg.)- Total weight of the plant part above ground (excluding roots) after drying at 70°C in the oven for 48 hours; weight per number of plants.
[00561] Fresh vegetable weight (Kg.)- Total weight of the vegetable part of 6 plants (above the ground, excluding the roots).
[00562] Number of nodes - The nodes on the stem were counted in the flowering stage of the plant development.Table 81 Parameters correlated to Maize (vectors) under normal conditions and under defoliation conditions.

Table 81. Parameters correlated to maize are provided. “NUE” = nitrogen use efficiency; “DW” = dry weight; “cm” = centimeter, “GF” = grain filling, “PP” = per plant, “h”= harvest, “avg.” = average, “N°” = number. “mm” = millimeter; “g” = grams; “kg” = kilograms; “cm” = centimeters.
[00563] Thirteen maize varieties were cultivated and characterized with respect to parameters as described below. The average for each of the parameters was calculated using the JMP software and the values are summarized in Tables 82 to 85 below. A subsequent correlation between the various transcriptome sets for all sets or subsets of lines was made by the bioinformatics unit and the results were integrated into the database (Tables 86 to 87 below).Table 82 Parameters measured in Maize varieties under normal conditions

Table 82.Table 83 Parameters measured in Maize varieties under normal conditions, additional maize lines.

Table 83.Table 84 Parameters measured in Maize varieties under defoliation

Table 84.Table 85 Parameters measured in Maize varieties under defoliation, additional maize rows
Table 85.
[00564] Tables 86 and 87 below provide correlations (R) between expression levels of production-enhancing genes and their homologues in various tissues [Expression sets (exp.)] and phenotypic performance [roduction components, biomass, growth rate and/or vigor (Correlation vector (corr)] under normal and defoliation conditions in maize varieties. P = p-value.Table 86Correlation between the expression level of selected genes from some applications of the invention in various tissues and the phenotypic performance under normal conditions in maize varieties

Table 86.Table 87Correlation between the expression level of selected genes from some applications of the invention in various tissues and the phenotypic performance under defoliation in maize varieties

Table 87.EXAMPLE 11 PRODUCTION OF MILLET CORN TRANSCRIPTOME AND HIGH PRODUCTIVITY CORRELATION ANALYSIS USING 60K MILLET CORN OLIGONUCLEOTIDE MICROARRANGE
[00565] In order to produce a high throughput correlation analysis comparing plant phenotype and gene expression level, the present inventors utilized a corn-millet oligonucleotide microarray, produced by Agilent Technologies [chem (dot) agilent (dot) com/Scripts/PDS (dot) asp lPage=50879]. The oligonucleotide array represents about 60K of millet genes and transcripts. In order to define the correlation between RNA expression levels and parameters related to yield or vigor, several plant characteristics of 14 different accessions of millet were analyzed. Among them, 11 accessions encompassing the observed variation were selected for analysis of RNA expression. The correlation between RNA levels and the characterized parameters was analyzed using the Pearson correlation test [davidmlane (dot) com/hyperstat/A34739 (dot) html]. Experimental Procedures:
[00566] 14 varieties of millet were cultivated in 5 repeated lots, in the field. In short, the cultivation protocol was as follows:1. Regular Growing Conditions - Millet corn plants were grown in the field using commercial fertilization and irrigation protocols, which include 283 m3 of water per dunam (100 square meters) for full growing period and fertilization of 16 units of URAN® 32% (Nitrogen Fertilizer Solution; PCS Sales, Northbrook, IL, USA) (normal growing conditions).
[00567] 2. Drought conditions: millet seeds were sown in soil and cultivated under normal conditions until the flowering stage (22 days from sowing); a drought treatment was imposed by irrigating the plants with 50% water relative to the normal treatment from this stage onwards (171 m3 per dunam (100 square meters) for full growing season).
[00568] Millet Fabrics Analyzed - All 14 millet lines were sampled against each treatment. Three tissues [leaf, flower, and stem], in 2 different developmental stages [flowering and grain filling], representing different plant characteristics, were sampled and RNA was extracted as described above. Each tissue type of microarray expression information was assigned a Set ID, as summarized in Tables 88 to 89 below.Table 88 Millet transcriptome expression sets under drought conditions
Table 88. Maize-millet transcriptome expression sets under drought conditions are provided.Table 89 Maize-millet transcriptome expression sets under normal conditions
Table 89. Maize millet transcriptome expression sets under normal conditions are provided
[00569] Evaluation of parameters related to vigor and production components of millet corn - The plants were continuously phenotyped during the cultivation period and at harvest. (Table 102, below). The image analysis system included a personal computer (Intel P4 3.0 Ghz processor) and a public domain program - ImageJ 1.37 (Java-based Image Processing Program), developed at the United States National Institutes of Health and freely available. on the internet [http://rsbweb (dot) nih (dot) gov/]. Then, the analyzed data were saved as text files and processed using the JMP statistical analysis software (SAS institute).
[00570] The following parameters were collected using the digital imaging system: At the end of the cultivation period, the grains were separated from the “Head” of the Plant and the following parameters were measured and collected: Average Grain Area (cm2) - A ~200 grain sample was weighed, photographed, and the images were processed using the image processing system described below. Grain area was measured from these images and divided by the number of grains.
[00571] Mean Grain Length and Width (cm) - A sample of ~200 grains was weighed, photographed and the images were processed using the image processing system described below. The sum of the grain length and width (longest axis) was measured at from these images and was divided by the number of grains. [00554] At the end of the cultivation period,14 “Heads” were photographed and the images were processed using the image processing system described below.
[00572] Average Grain Perimeter (cm) - At the end of the cultivation period, the grains were separated from the “Head” of the Plant. A sample of ~200 grains was weighed, photographed and the images processed using the image processing system described below. The sum of the grain perimeter was measured from these images and divided by the number of grains.
[00573] Average Head Area (cm2) - The “Head” area was measured from these images and divided by the number of “Heads”.
[00574] Average Head Length and Width (mm) - The length and width of the “Heads” (longest axis) were measured from these images and were divided by the number of “Heads”.
[00575] The image processing system used consists of a personal computer (Intel P4 3.0 Ghz processor) and a public domain program - ImageJ 1.37 (Image processing program based on Java), developed at the National Institutes of Health in United States and freely available on the Internet [http://rsbweb(dot)nih(dot)gov/]. The images were captured in a resolution of 10 Mega Pixels (3888x2592 pixels) and stored in a low-compression JPEG format (standard of the Joint Group of 5 Specialists in Photography). Then, the analyzed data were saved in text files and processed using the JMP statistical analysis software (SAS Institute).
[00576] Additional parameters were collected by sampling 5 plants per batch or by measuring the parameter on all plants within the batch.
[00577] Head Weight (g.) and number of Heads (n°)- At the end of the experiment, the heads were harvested from each ear and were counted and weighed.
[00578] Total Grain Production (g.) - At the end of the experiment ("Heads" of the Plants), the heads of the ears were collected, threshed and the grains were weighed. In addition, the average grain weight per head was calculated by dividing the total grain weight by the total number of heads per batch (batch-based).
[00579] Weight of 1000 Seeds [g.] - The weight of 1000 seeds per lot.
[00580] Biomass at Harvest - At the end of the experiment, the aboveground plant part (excluding the roots) of the plots was weighed.
[00581] Total dry matter per batch - Calculated as aboveground plant plus all head dry weight per batch.
[00582] Number of days until anthesis - Calculated as the number of days from sowing until 50% of the lot reaches anthesis.
[00583] Maintenance of performance under drought conditions: Represents the ratio for the specified parameter of the results of drought conditions divided by the results of normal conditions (maintenance of phenotype under drought conditions compared to normal conditions).
[00584] The data parameters collected are summarized in Table 90, below. Table 90
Table 90. Parameters correlated to millet are provided. Experimental Results:
[00585] 14 different millet accessions were cultivated and characterized with respect to different parameters as described above (Table 90). The mean for each of the measured parameters was calculated using the JMP software and the values were summarized in Tables 91-96 below. A subsequent correlation analysis between the various transcriptome sets and the mean parameters (Tables 91-96) was conducted (Tables 97-99). Subsequently, the results were integrated into the database.Table 91Measured parameters of correlation IDs in maize-millet accessions under drought conditions
Table 91: The values of each of the parameters (as described above) measured in millet accessions (row) under drought conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 92Additional parameters measured from correlation IDs in millet accessions under drought conditions
Table 92: The values of each of the parameters (as described above) measured in millet accessions (row) under drought conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 93Measured Parameters of Correlation IDs in Corn-Mill Accessions for Maintaining Performance Under Drought Conditions
Table 93: Values for each of the parameters (as described above) measured in millet accessions (row) are provided for maintaining performance under drought conditions (calculated as % change under drought vs. normal growing conditions ). Growing conditions are specified in the experimental procedure section.Table 94Additional Measured Parameters of Correlation IDs in Millet Accessions for Maintenance of Performance Under Drought Conditions
Table 94: Values for each of the parameters (as described above) measured in millet accessions (row) are provided for maintaining performance under drought conditions (calculated as % change under drought vs. normal growing conditions ). Cultivation conditions are specified in the experimental procedure section.Table 95Measured Parameters of Correlation IDs in Maize-Millet Accessions Under Normal Conditions
Table 95: Values for each of the parameters (as described above) measured in millet accessions (row) under normal growing conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 96Additional parameters measured from correlation IDs in millet accessions under normal conditions
Table 96: Values for each of the parameters (as described above) measured in millet accessions (row) under normal growing conditions are provided. Cultivation conditions are specified in the experimental procedure section.Table 97Correlation between the expression level of selected genes from some applications of the invention in various tissues and the phenotypic performance under drought conditions in millet varieties
Table 97. Correlations (R) between gene expression levels in various tissues and phenotypic performance. “Color ID.” - Correlation set ID, according to the parameters correlated in the Table above. “Assembly of Exp.” - Expression set. “R” = Pearson's correlation coefficient; “P” = p-value. Table 98Correlation between the expression level of selected genes from some applications of the invention in various tissues and the phenotypic performance of maintaining performance under drought conditions in maize varieties.
Table 98. Correlations (R) between gene expression levels in various tissues and phenotypic performance. “Color ID.” - Correlation set ID, according to the parameters correlated in the Table above. “Assembly of Exp.” - Expression set. “R” = Pearson's correlation coefficient; “P” = p-value.Table 99Correlation between the expression level of selected genes from some applications of the invention in various tissues and the phenotypic performance under normal conditions in millet varieties
Table 99. Correlations (R) between gene expression levels in various tissues and phenotypic performance. "Color ID." - Correlation set ID, according to the parameters correlated in the Table above. “Assembly of Exp.” - Expression set. “R” = Pearson's correlation coefficient; “P” = p value. EXAMPLE 12 GENE CLONING AND BINARY VECTOR GENERATION FOR PLANT EXPRESSION
[00586] To validate their role in increasing production, the selected genes were overexpressed in plants, as follows.Cloning Strategy
[00587] The genes selected from those presented in Examples 1-13 above were cloned into binary vectors for the generation of transgenic plants. For cloning, full-length open reading frames (ORF's | open reading frame) were identified. EST groups and, in some cases, mRNA sequences, were analyzed to identify the total open reading frame by comparing the results of various translation algorithms for known proteins from other plant species.
[00588] In order to clone the full length cDNA's, a reverse transcription (RT|reverse transcription) followed by the polymerase chain reaction (PCR; RT-PCR) was performed on the total RNA extracted from the leaves, roots or other tissues of the plant. , grown under stress or normal/limiting conditions. Total RNA extraction, cDNA production, and PCR amplification were performed using standard protocols described elsewhere (Sambrook J., EF Fritsch and T. Maniatis. 1989. Molecular Cloning. A Laboratory Manual, 2nd Ed. Frio Spring Harbor Laboratory. Press, New York.) which are well known to those skilled in the art. PCR products were purified using a PCR purification kit (Qiagen).
[00589] Generally, 2 sets of primers were prepared for the amplification of each gene, via nested PCR (if necessary). Both sets of primers were used for cDNA amplification. In case no product was obtained, a nested PCR reaction was performed. Nested PCR was performed by amplifying the gene using external primers and then using the PCR product produced as a template for a second PCR reaction, where the internal set of primers was used. Alternatively, one or two of the nested primers were used for amplification of 25 genes, both in the first and second PCR reactions (meaning that only 2-3 primers were assigned to a gene). To facilitate further cloning of cDNA's, an 8-12 base pair extension (bp | base pairs) was added to the 5' of each nested primer. The primer extension includes an endonuclease restriction site. Restriction sites were selected using two parameters: (a) the restriction site does not exist in the cDNA sequence; and (b) the restriction sites on the forward and reverse primers were designed so that the digested cDNA is inserted in the sense direction into the binary vector used for transformation.
[00590] PCR products were digested with restriction endonucleases (New England BioLabs Inc.) according to designated sites on primers. Each digested/undigested PCR product was inserted into a pUC19 high copy vector (New England BioLabs Inc.) or into plasmids originating from this vector. In some cases, the undigested PCR product was inserted into pCR-Blunt II-TOPO (Invitrogen) or pJET1.2 (CloneJET PCR Cloning Kit, Thermo Scientific) or directly into the binary vector. The digested/undigested products and the linearized plasmid vector were ligated using a T4 DNA ligase enzyme (Roche, Switzerland, or other manufacturers). In cases where pCR-Blunt II-TOPO was used, no T4 ligase was required.
[00591] The sequencing of the inserted genes was performed using the ABI 377 sequencer (Applied Biosystems). In some cases, after confirming the sequences of the cloned genes, the cloned cDNA was introduced into a modified pGI binary vector containing the At6669 promoter (eg, pQFNc) and the NOS terminator (SEQ ID NO: 4891) via digestion with appropriate restriction endonucleases.
[00592] In cases of Brachypodium transformation, after confirming the sequences of the cloned genes, the cloned cDNAs were introduced into pEBbVNi (Figure 9A), containing the 35S promoter (SEQ ID NO: 4892) and the NOS terminator (SEQ ID No.: 4891) via digestion with suitable restriction endonucleases. These genes were cloned downstream of the 35S promoter and upstream of the NOS terminator.
[00593] Several DNA sequences of selected genes were synthesized via GeneArt (Life Technologies, Grand Island, NY, USA). Synthetic DNA was engineered on silica. Suitable restriction enzyme sites were added to the cloned sequences at the 5' end and 3' end to allow further cloning into the desired binary vector.
[00594] Binary vectors - The pPI plasmid vector was constructed by inserting a synthetic poly(A) signal sequence derived from the pGL3 basic plasmid vector (Promega, GenBank Accession No. U47295; nucleotides 4658-4811) into the HindIII restriction site of the binary vector pBI101.3 (Clontech, GenBank Accession No. U12640). pGI is similar to pPI, but the original gene in the spine is the GUS-Intron gene and not the GUS.
[00595] The modified pGI vector [eg, pQFN, pQFNc, pQYN_6669, pQNa_RP, pQFYN 25 or pQXNc] is a modified version of the pGI vector in which the cassette is flipped between the left and right edges so that the gene and its corresponding promoter are near the right edge and the NPTII gene is near the left edge.
[00596] At6669, the new Arabidopsis thaliana promoter sequence (SEQ ID NO: 4880) was inserted into the modified pGI binary vector, upstream of the cloned genes, followed by DNA ligation and extraction of the binary plasmid from E. coli colonies positive, as described above. Colonies were analyzed by PCR, using insert-covering primers, which are designed to span the promoter and introduced gene. Positive plasmids were identified, isolated and sequenced.
[00597] pEBbVNi (Figure 9A) is a modified version of pJJ2LB, in which the Hygromycin resistance gene has been replaced by a BAR gene conferring resistance to the herbicide BASTA [the coding sequence for the BAR gene is provided in GenBank Accession No. JQ293091.1 (SEQ ID NO: 5436); an additional description is provided in Akama K, et al. “Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker”, Plant Cell Rep. 1995, 14(7):450-4; Christiansen P, et al. “A rapid and efficient transformation protocol for the grass Brachypodium distachyon”, Plant Cell Rep. 2005 Mar; 23(10-11):751-8. Epub 2004 Oct 19; and Pacurar DI, et al. “A high-throughput Agrobacterium-mediated transformation system for the grass model species Brachypodium distachyon L”, Transgenic Res. 2008 17(5):965-75; each of which is fully incorporated herein by reference in their entirety]. The pEBbVNi structure contains the 35S promoter (SEQ ID NO: 4892). pJJ2LB is a modified version of pCambia0305.2 (Cambia).
[00598] In cases where genomic DNA was cloned, the genes were amplified by direct PCR on genomic DNA extracted from leaf tissue, using the DNAeasy kit (Qiagen Cat. No. 69104).
[00599] The selected genes cloned by the present inventors are given in Table 100 below. Table 100




Table 100. Names of cloned genes, plasmids with high copy numbers, the organism from which the gene was cloned, the primers used for cloning, and the polynucleotide and polypeptide sequence identifiers of the cloned genes are provided. EXAMPLE 13 TRANSFORMATION OF AGROBACTERIUMTUMEFACIENS CELLS WITH BINARY VECTORS HOSTING THE POLYNUCLEOTIDES OF THE INVENTION
[00600] The binary vectors described above were used to transform Agrobacterium cells. Two additional binary structures, having only the At6669 promoter or the 35S promoter or no additional promoter, were used as negative controls.
[00601] The binary vectors were introduced into competent cells Agrobacterium tumefaciens GV301 or LB4404 (for Arabidopsis) or AGL1 (for Brachypodium) (about 109 cells/mL), by electroporation. Electroporation was performed using a MicroPulser electroporator (Biorad), 0.2 cm crucibles (Biorad) and EC-2 electroporation program (Biorad). Treated cells were cultured in LB broth at 28°C for 3 hours, then placed on LB agar supplemented with gentamicin (for Arabidopsis; 50 mg/L; for Agrobacterium GV301 strains) or streptomycin (for Arabidopsis; 300 mg/L; for Agrobacterium strain LB4404); or with Carbenicillin (for Brachypodium; 50 mg/L) and kanamycin (for Arabidopsis and Brachypodium; 50 mg/L) at 28°C for 48 hours. Abrobacterium colonies, which are grown on selective media, were further analyzed by PCR, using primers designed to encompass the sequence inserted into plasmid pPI. The resulting PCR products were isolated and sequenced to verify that the correct polynucleotide sequences of the invention are properly introduced into Agrobacterium cells. EXAMPLE 14 TRANSFORMATION OF ARABIDOPSIS THALIANA PLANTS WITH THE POLYNUCLEOTIDES OF THE INVENTION
[00602] Arabidopsis thaliana Columbia plants (T0 plants) were transformed using the Floral Immersion procedure described by Clough and Bent, 1998 (Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16(6) : 735-43) and by Desfeux et al., 2000 (Female Reproductive Tissues Are the Primary Target of Agrobacterium-Mediated Transformation by the Arabidopsis FloralDip Method. Plant Physiol, July 2000, Vol. 123, pp. 895-904), with minor modifications. Briefly, T0 plants were sown in 250 ml pots filled with a wet peat-based culture mix. The pots were covered with aluminum foil and a plastic bell jar, kept at 4°C for 3-4 days, then uncovered and incubated in a culture chamber at 18-24°C in 16/8 hr light/ dark. T0 plants were ready for transformation six days before anthesis.
[00603] Single colonies of Agrobacterium carrying the binary structures were generated as described in Examples 12-13 above. Colonies were grown in LB medium supplemented with kanamycin (50 mg/L) and gentamicin (50 mg/L). The cultures were incubated at 28°C for 48 hours under vigorous shaking and then centrifuged at 4000 rpm for 5 minutes. Pellets comprising Agrobacterium cells were resuspended in a transformation medium containing medium concentration (2.15 g/L) Murashige-Skoog culture medium (Duchefa); 0.044 µM benzylamino purine (Sigma); 112 μg/L of vitamins B5 Gambourg (Sigma); 5% sucrose and 0.2 ml/L Silwet L-77 (OSI Specialists, CT) in double distilled water with pH 5.7.
[00604] The transformation of T0 plants was carried out by inverting each plant in an Agrobacterium suspension, so that the above-ground plant tissue was submerged for 3-5 seconds. Each inoculated T0 plant was immediately placed on a plastic tray, then covered with a clean plastic dome to maintain moisture and kept in the dark at room temperature for 18 hours to facilitate infection and transformation. The transformed (transgenic) plants were then covered and transferred to a greenhouse for recovery and maturation. The transgenic T0 plants were grown in the greenhouse for 3-5 weeks until the siliques turned brown and dried. Seeds were harvested from the plants and kept at room temperature until sowing.
[00605] For generation of T1 and T2 transgenic plants harboring the genes, the seeds collected from the transgenic T0 plants were surface sterilized by immersion in 70% ethanol for 1 minute, followed by immersion in 5% sodium hypochlorite and 0, 05% triton for 5 minutes. The surface-sterilized seeds were thoroughly washed in sterile distilled water and then placed in culture plates containing Murashige-Skoog medium concentration culture medium (Duchefa); 2% sucrose; 0.8% plant agar; 50 mM kanamycin and 200 mM carbenicillin (Duchefa). Culture plates were incubated at 4°C for 48 hours, then transferred to a 25°C culture room for an additional week of incubation. Vital T1 Arabidopsis plants were transferred to fresh culture plates for another week of incubation. Following incubation, the T1 plants were removed from the culture plates and culture plates and planted in the culture mixture contained in 250 ml pots. The transgenic plants were allowed to grow in a greenhouse to maturity. Seeds harvested from the T1 plants were cultivated and grown to maturity as the T2 plants under the same conditions as used for culture and growth of the T1 plants. EXAMPLE 15 TRANSFORMATION OF BRACHYPODIUM DISTACHYON PLANTS WITH THE INVENTION POLYNUCLEOTIDES
[00606] Similar to the Arabidopsis plant model, Brachypodium distachyon has several characteristics that make it recommended as a model plant for functional genomics studies, especially in grasses. Traits that make it an ideal model include its small genome (~160 Mbp for a diploid genome and 355 Mbp for a polyploid genome), small physical stature, short life cycle, and few cultivation requirements. Brachypodium is related to the main cereal grain species, but is understood to be more closely related to Triticeae (wheat, barley) than to other cereals. Brachypodium, with its polyploid accessions, may serve as an ideal model for these grains (whose size and genomic complexity are a major barrier to improving biotechnology).
[00607] Embryogenic calli of Brachypodium distachyon were transformed using the procedure described by Vogel and Hill (2008) [High-efficiency Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21-3. Plant Cell Rep 27:471-478], Vain et al (2008) [Agrobacterium-mediated transformation of the temperate grass Brachypodium distachyon (genotypeBd21) for T-DNA insertional mutagenesis. Plant Biotechnology J 6: 236-245] and Vogel J, et al. (2006) [Agrobacterium mediated transformation and inbred line development in the model grass Brachypodium distachyon. Plant Cell Tiss Org. Cult 85:199211], each of which is fully incorporated herein by reference, with some minor modifications, which are briefly summarized below.
[00608] Beginning of calluses - Immature ears (about 2 months after sowing) were harvested at the beginning of seed filling. The ears were then peeled and surface sterilized with 3% NaClO containing 0.1% Tween 20, shaken with a low speed rotary shaker for 20 minutes. After three washes with sterile distilled water, the embryos were extracted under a dissecting microscope in a laminar flow chamber using fine forceps.
[00609] The extracted embryos (size ~0.3 mm, bell-shaped) were placed in callus induction medium (CIM|callus induction medium) [LS salts (Linsmaier, EM & Skoog, F. 1965. Physiol. Plantarum 18, 100) and vitamins plus 3% sucrose, 6mg/L of CuSO4, 2.5mg/L of 2,4-dichlorophenoxyacetic acid, pH 5.8 and 0.25% of Phytagel (Sigma)] side down, 100 embryos on a plate, and incubated at 28°C in the dark. One week later, embryonic calluses were cleaned of emerging roots, shoots and somatic calluses and subcultured in fresh CIM medium. During culture, yellowish embryogenic calluses (EC | embryogenic callus) appeared and were further selected (eg, harvested and transferred) for further incubation under the same conditions for a further 2 weeks. Twenty-five pieces of subcultured callus were then placed separately in 90 X 15 mm Petri dishes and incubated as before for a further three weeks.
[00610] Transformation - As described in Vogel and Hill (2008, Supra), Agrobacterium was scraped off with 2 day old MGL plates (plates with the MGL medium containing: Tryptone 5 g/l, Yeast Extract 2.5 g/l, NaCl 5 g/l, D-mannitol 5 g/l, MgSO4*7H2O 0.204 g/l, K2HPO4 0.25 g/l, Glutamic Acid 1.2 g/l, Plant Agar 7.5 g) and resuspended in MS liquid medium supplemented with 200 μM acetosyringone at an optical density (OD | optical density) at 600 nm (OD600) of 0.6. Once the desired optical density was reached, 1 ml of 10% Synperonic PE/F68 (Sigma) was added per 100 ml of inoculation medium.
[00611] To begin inoculation, 300 pieces of callus were placed on about 12 plates (25 pieces of callus on each plate) and covered with the Agrobacterium suspension (8-8.5 ml). The callus was incubated in the Agrobacterium suspension for 15 minutes with occasional gentle shaking. After incubation, the Agrobacterium suspension was removed by aspiration and the calluses were then transferred to coculture plates, prepared by placing a 7 cm diameter sterile filter paper in an empty 90 X 15 mm Petri dish. The callus pieces were then gently distributed over the filter paper. One coculture plate was used for two callus starter plates (50 callus starter pieces). The coculture plates were then sealed with parafilm and incubated at 22°C in the dark for 3 days.
[00612] The callus pieces were then transferred individually into MIC medium as described above, which was further supplemented with 200 mg/L ticarcillin (to kill Agrobacterium) and Bialaphos (5 mg/L) (to kill the Agrobacterium). selection of one of the transformed resistant embryogenic callus sections) and incubated at 28°C, in the dark, for 14 days.
[00613] The callus pieces were then transferred to shoot induction media (SIM | shoot induction media; LS salts and vitamins plus 3% maltose monohydrate) supplemented with 200 mg/l ticarcillin, Bialaphos (5 mg/L), indole-3-acetic acid (IAA | indole-3-acetic acid) (0.25 mg/L) and 6-benzylaminopurine (BAP | Benzylaminopurine) (1 mg/L), and were subcultured in light for the same medium after 10 days (total of 20 days). In each subculture, all parts of a single callus were kept together to maintain their independence and were incubated under the following conditions: at an illumination level of 60 lE m-2 s-1, a 16-hour light, 8-hour dark photoperiod and a constant temperature of 24°C. The seedlings emerged from the transformed calluses.
[00614] When the seedlings were large enough to be handled without damage, they were transferred to plates containing the above mentioned shoot induction media (SIM) without Bialaphos. Each seedling was considered as a different event. The seedlings sprouted axillary tillers and eventually became thick. Each branch of the same plant (event ID) was then divided into tissue culture boxes (“Humus”) containing basal salts of “rooting medium” [basal salts MS, 3% sucrose, 3 g/L of Phytagel, 2 mg/l of acetic acid (x-naphthalene (NAA|naphthalene Acetic Acid) and 1 mg/l of IAA and 200mg/L Ticarcillin, pH 5.8). All the plants in a “Humus box” were different plants from the same transformation event.
[00615] When the plants established roots, they were transplanted into the soil and transferred to a greenhouse. To verify the status of transgenic plants containing the other structures, T0 plants were subjected to PCR, as previously described by Vogel et al. 2006 [Agrobacterium mediated transformation and inbred line development in the model grass Brachypodium distachyon. Plant Cell Tiss Org. Cult 85:199211]. EXAMPLE 16 EVALUATION OF TRANSGENIC ARABIDOSIS NUE UNDER NORMAL OR LOW NITROGEN CONDITIONS USING SEEDLINGS ASSAYS.Assay 1: Plant cultivation under favorable low nitrogen concentration levels
[00616] Surface sterilized seeds were sown in basal medium [50% Murashige-Skoog (MS) medium supplemented with 0.8% plant agar as a solidifying agent] in the presence of Kanamycin (used as a selection agent). After seeding, the plates were transferred for 2 to 3 days for stratification at 4°C and then grown at 25°C in daily cycles of 12 hours light and 12 hours dark for 7 to 10 days. At this time point, randomly chosen seedlings were carefully transferred to plates containing ^ MS medium (15 mM N) for normal nitrogen concentration treatment and 0.30 mM nitrogen for low nitrogen concentration treatment. For experiments performed in T2 lines, each plate contained 5 seedlings from the same transgenic event and 3 to 4 different plates (replicas) for each event. For each polynucleotide of each invention, at least four to five independent transformation events were analyzed from each framework. For experiments performed in T1 lines, each plate contained 5 seedlings from the same transgenic event and 3 to 4 different plates (replicas) were planted. In total, for T1 lines, 20 independent events were evaluated. Plants expressing polynucleotides of the invention were compared to the average measurement of control plants (empty vector or GUS reporter gene in the same promoter) used in the same experiment.
[00617] Digital Imaging - A laboratory imaging system, consisting of a digital reflex camera (Canon EOS 300D) attached to a 55mm focal length lens (Canon EF-S series), mounted on a reproduction device (Kaiser RS), which includes 4 light units (4 x 150 Watt illumination lamp) and located in a dark room, was used to capture images of seedlings seen on agar plates.
[00618] The image capture process was repeated every 3 to 4 days starting on day 1 to day 10 (see eg images in Figures 3A-B). An image analysis system was used, which consists of a personal computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.39 [image processing program based on Java, developed at the National Institutes of Health in United States and freely available on the Internet at rsbweb (dot) nih (dot) gov/]. Images were captured at 10 Mega Pixels resolution (3888 x 2592 pixels) and stored in a low-compression JPEG format. Then, the analysis data were saved in text files and processed using the JMP statistical analysis software (SAS Institute).
[00619] Seedling Analysis - Using digital analysis, seedling data was calculated including leaf area, root coverage and root length.
[00620] The relative growth rate for the various seedling parameters was calculated according to the following formulas, Formulas XIII (relative growth rate of leaf area) and VI (relative growth rate of root length).
[00621] At the end of the experiment, the seedlings were removed from the medium and weighed to determine the fresh weight of the plant. The seedlings were then dried for 24 hours at 60°C, and weighed again to measure the dry weight of the plant for further statistical analysis. The growth rate was determined by comparing leaf area coverage, root coverage and root length, between each pair of sequential photos, and the results were used to resolve the effect of the introduced gene on plant vigor under ideal conditions. Similarly, the effect of the introduced gene on biomass accumulation, under ideal conditions, was determined by comparing the dry and fresh weight of plants and that of control plants (containing an empty vector or GUS reporter gene in the same promoter). From each structure created, 3 to 5 independent transformation events were examined in replicates.
[00622] Statistical analysis - To identify the genes that confer improved vigor to the plant or improved architecture to the root, the results obtained from the transgenic plants were compared to those obtained from the control plants. To identify the high performing genes and structures, the results of the independent transformation events tested were analyzed separately. To assess the effect of a gene event on a control, data were analyzed by Student's t test and the p-value was calculated. Results were considered significant if p^0.1. The JMP statistical software package was used (Version 5.2.1, Instituto SAS Inc., Cary, NC, USA).Experimental Results:
[00623] The genes shown in the following Tables were cloned in the regulation of a constitutive promoter (At6669). The evaluation of the effect of transformation in a plant of each gene was performed by the performance test of different number of transformation events. Some of the genes were evaluated in more than one molt assay. The results obtained in these second experiments were also significantly positive. The event with p value <0.1 was considered statistically significant.
[00624] The genes shown in Tables 101-104 showed a significant improvement in plant NUE as they produced higher plant biomass (fresh and dry plant weight; leaf area, root length and root coverage) in T2 generation (Tables 101-102) or T1 generation (Tables 103-104) when grown under nitrogen-limiting growing conditions, compared to control plants that were grown under identical growing conditions. Plants that produce larger root biomass are more likely to absorb more nitrogen from the soil.Table 101Genes showing improved plant performance under nitrogen-deficient conditions (T2 generation)


Table 101: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means the p-value is less than 0.01, p<0.1 was considered to be significant.Table 102Genes showing improved plant performance under nitrogen deficient conditions (T2 generation)


Table 102: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means that the p-value is less than 0.01, p<0.1 was considered to be significant.Table 103Genes showing improved plant performance under nitrogen deficient conditions (T1 generation)
Table 103: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means that the p-value is less than 0.01, p<0.1 was considered significant. Table 104Genes showing improved plant performance under nitrogen deficient conditions (T1 generation)
Table 104: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means p-value is less than 0.01, p<0.1 was considered significant.
[00625] The genes listed in Tables 105-106 improved relative plant growth rate (relative growth rate of leaf area, root coverage, and root length) when grown under nitrogen-limiting growing conditions compared to control (T2 and T1 generations) that were grown under identical culture conditions. Plants that show rapid growth rate show better plant establishment in soil under nitrogen deficient conditions. The fastest growth was observed when the growth rate of leaf area, root length and root coverage was measured, Table 105Genes showing improved plant growth rate under nitrogen deficient conditions (T2 generation)

Table 105: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means p-value is less than 0.01, p<0.1 was considered significant. Table 106Genes showing improved plant growth rate under nitrogen deficient conditions (T2 generation)
Table 106: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means p-value is less than 0.01, p<0.1 was considered significant.
[00626] The genes listed in Tables 107-110 improved plant NUE when grown at standard nitrogen concentration levels. These genes produced greater plant biomass (fresh and dry plant weight; leaf area, root coverage, and root length) when grown under standard nitrogen growing conditions, compared to those control plants that were grown under identical growing conditions in T2 generation (Tables 107-108) or T1 generation (Tables 109-110). The higher plant biomass under these cultivation conditions indicates the high capacity of the plant to better metabolize the nitrogen present in the medium. Plants that produce higher root biomass are more likely to absorb more nitrogen from the soil.Table 107 Genes showing improved plant performance under standard nitrogen growing conditions (T2 generation)
Table 107: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means that the p-value is less than 0.01, p<0.1 was considered to be significant.Table 108Genes showing improved plant performance under standard nitrogen cultivation conditions (T2 generation)
Table 108: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means that the p-value is less than 0.01, p<0.1 was considered to be significant.Table 109Genes showing improved plant performance under standard nitrogen cultivation conditions (T1 generation)
Table 109: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means p-value is less than 0.01, p<0.1 was considered significant.Table 110Genes showing improved plant performance under standard nitrogen cultivation conditions (T1 generation)
Table 110: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means that the p-value is less than 0.01, p<0.1 was considered significant.
[00627] The genes listed in Tables 111-112 improved the relative plant growth rate (RGR of leaf area, root length and root coverage) when grown at standard nitrogen concentration levels. These genes produced plants that grew faster than control plants when grown under standard nitrogen growing conditions. The fastest growth was observed when the growth rate of leaf area, root length and root coverage were measured. Table 111Genes showing improved growth rate under standard nitrogen culture conditions (T2 generation)
Table 111: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means the p-value is less than 0.01, p<0.1 was considered to be significant.Table 112Genes showing improved growth rate under standard nitrogen culture conditions (T1 generation)
Table 112: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means p-value is less than 0.01, p<0.1 was considered significant. EXAMPLE 17 ASSESSMENT OF NUE, PRODUCTION AND GROWTH RATE OF TRANSGENIC ARABIDOPSIS PLANT IN LOW NITROGEN OR NORMAL FERTILIZATION IN GREENHOUSE TEST
[00628] Test 1: Efficiency in the use of nitrogen: Seed production, plant biomass and plant growth rate in ideal and limited nitrogen concentration under greenhouse conditions - This test tracks seed production, biomass formation and the growth of rosette area of plants grown in the greenhouse under non-limiting and limiting nitrogen growth conditions. Transgenic Arabidopsis seeds were sown on agar media supplemented with ^ MS medium and selection agent (Kanamycin). T2 transgenic seeds were then transplanted into 1.7 trays filled with peat and perlite in a ratio of 1: 1. The trays were irrigated with a solution containing nitrogen-limiting conditions, which were achieved by irrigating the plants with a solution containing 1.5 mM of inorganic nitrogen in the form of KNO3, supplemented with 1 mM KH2PO4, 1 mM MgSO4, 3.6 mM KCl, 2 mM CaCl2 and microelements, while normal nitrogen levels were achieved by applying a solution of 6 mM inorganic nitrogen also in the form of KNO3, with 1mM KH2PO4, 1mM MgSO4, CaCl2 and microelements. All plants were grown in the greenhouse until the seeds were mature. The seeds were harvested, extracted and weighed. The remaining plant biomass (the above-ground tissue) was also harvested, and weighed immediately or following oven drying at 50°C for 24 hours.
[00629] Each structure was validated in its T2 generation. The transgenic plants transformed with a structure formed by an empty vector carrying the 35S promoter and the selectable marker were used as a control.
[00630] The plants were analyzed for their total size, growth rate, flowering time, seed production, weight of 1,000 seeds, dry matter and harvest index (HI - seed production/dry matter). The performance of transgenic plants was compared to control plants grown in parallel under the same conditions. False transgenic plants expressing the uidA reporter gene (GUS-Intron) or without any gene in the same promoter were used as controls.
[00631] The experiment was designed in the nested random lot distribution. For each gene of the invention, three to five independent transformation events were analyzed from each structure.
[00632] Digital Imaging - A laboratory imaging system, consisting of a digital reflex camera (Canon EOS 300D) attached with a 55mm focal length lens (Canon EF-S series), mounted on a reproduction device (Kaiser RS), which includes 4 light units (lamp of 4 x 150 Watts) was used to capture images of the plant samples.
[00633] The image capture process was repeated every 2 days starting from day 1 post-transplant to day 15. The same camera, placed in a custom iron mount, was used to capture the images of larger plants seen in white vats in an environmentally controlled greenhouse. The vats are square in shape and include 1.7 liter trays. During the capture process, the vats were placed below the iron mount, while avoiding direct sunlight and casting shadows.
[00634] An image analysis system was used, consisting of a computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.39 [image processing program based on Java, developed at the National Institutes of Health of United States and freely available on the Internet at rsbweb (dot) nih (dot) gov/]. Images were captured at 10 Mega Pixels resolution (3888 x 2592 pixels) and stored in a low-compression JPEG format (Joint Photographic Experts Group standard). Then, analysis data were saved to text files and processed using the JMP statistical analysis software (SAS institute).
[00635] Leaf Analysis - Using digital analysis, leaf data were calculated, including leaf number, rosette area, rosette diameter, and leaf blade area.
[00636] Plant growth rate: the relative growth rate (RGR) of leaf number [Formula VIII (described above)], rosette area [Formula IX, described above], plot coverage [Formula XI, described above] and harvest index [Formula XV] were calculated as follows: Average seed weight - At the end of the experiment, all seeds were collected. The seeds were spread out on a glass tray and a photo was taken. Using digital analysis, the number of seeds in each sample was calculated.
[00637] Dry weight and seed production - Around the 80th day, the plants were harvested and left to dry at 30°C in a drying chamber. The biomass and seed weight of each batch were measured and divided by the number of plants in each batch. Dry weight = total weight of the aboveground plant part (excluding the roots) after drying at 30°C in a drying chamber;^ Seed production per plant = total seed weight per plant (gr.), weight of 1000 seeds (the weight of 1000 seeds) (gr.).
[00638] The Harvest Index (HI) was calculated using Formula XV as described above.
[00639] Percentage of oil in seeds - At the end of the experiment, all seeds from each batch were collected. Seeds from 3 batches were mixed with soil and then mounted in the extraction chamber. 210 ml of n-Hexane (Cat No. 080951 Biolab Ltd.) was used as solvent. Extraction was carried out for 30 hours on medium heat at 50°C. Once the extraction was complete, the n-Hexane was evaporated using the evaporator at 35°C and vacuum conditions. The process was repeated twice. Information obtained from the Soxhlet extractor (Soxhlet, F, Die gewichtsanalytische Bestimmung des Milchfettes, Polytechnisches J, (Dingler's) 1879, 232, 461) was used to create a calibration curve for Low Resonance NMR. The oil content of all samples was determined using Low Resonance NMR (MARAN Ultra - Oxford Instrument) and its MultiQuant software package.
[00640] Silique length analysis - On day 50 of sowing, 30 siliques from different plants in each lot were sampled in block A. The selected siliques were green-yellow and were collected from the lower parts of a stem of cultivated plant. A digital photograph was taken to determine the silique's length.
[00641] Statistical analysis - To identify genes that confer significantly improved tolerance to abiotic stresses, the results obtained from transgenic plants were compared to those obtained from control plants. To identify the high performing genes and structures, the results of the independent transformation events tested were analyzed separately. Data were analyzed by Student's t test and the results were considered significant if the p-value was less than 0.1. The JMP statistical software package was used (Version 5.2.1, Instituto SAS Inc., Cary, NC, USA).
[00642] Tables 113-122 summarize the observed phenotypes of transgenic plants that exogenously express gene structures using greenhouse seed maturation assays (GH-SM) under low nitrogen (Tables 113-117) or low nitrogen conditions. normal nitrogen (Tables 118-122). The evaluation of each gene was performed by the performance test of different numbers of events. The event with p value <0.1 was considered statistically significant.Table 113Genes showing improved plant performance under low nitrogen cultivation conditions under regulation of the At6669 promoter
Table 113: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means that the p-value is less than 0.01, p<0.1 was considered significant. The transgenes were under transcriptional regulation of the new At6669 promoter (SEQ ID NO: 4880). It should be noted that a negative increase (in percentage) when found at flowering or inflorescence emergence indicates water evasion from the plant.Table 114Genes showing improved plant performance under low nitrogen growing conditions under regulation of the At6669 promoter
Table 114: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means that the p-value is less than 0.01, p<0.1 was considered significant. The transgenes were under transcriptional regulation of the new At6669 promoter (SEQ ID NO: 4880).Table 115Genes showing improved plant performance under low nitrogen cultivation conditions under regulation of the At6669 promoter
Table 115: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means that the p-value is less than 0.01, p<0.1 was considered significant. The transgenes were under transcriptional regulation of the novel At6669 promoter (SEQ ID NO: 4880).Table 116Genes showing improved plant performance under low nitrogen cultivation conditions under regulation of the At6669 promoter
Table 116: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means that the p-value is less than 0.01, p<0.1 was considered significant. The transgenes were under transcriptional regulation of the new At6669 promoter (SEQ ID NO: 4880).Table 117Genes showing improved plant performance under low nitrogen cultivation conditions under regulation of the At6669 promoter

Table 117: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means that the p-value is less than 0.01, p<0.1 was considered significant. The transgenes were under transcriptional regulation of the novel At6669 promoter (SEQ ID NO: 4880).Table 118Genes showing improved plant performance under normal cultivation conditions under regulation of the At6669 promoter

Table 118: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means that the p-value is less than 0.01, p<0.1 was considered significant. The transgenes were under transcriptional regulation of the new At6669 promoter (SEQ ID NO: 4880). It should be noted that a negative increase (in percentage) when found at flowering or inflorescence emergence indicates water evasion from the plant.Table 119Genes showing improved plant performance under normal growing conditions under regulation of the At6669 promoter
Table 119: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means that the p-value is less than 0.01, p<0.1 was considered significant. The transgenes were under transcriptional regulation of the new At6669 promoter (SEQ ID NO: 4880).Table 120Genes showing improved plant performance under normal cultivation conditions under regulation of the At6669 promoter
Table 120: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means that the p-value is less than 0.01, p<0.1 was considered significant. The transgenes were under transcriptional regulation of the novel At6669 promoter (SEQ ID NO: 4880).Table 121Genes showing improved plant performance under normal cultivation conditions under regulation of the At6669 promoter

Table 121: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means that the p-value is less than 0.01, p<0.1 was considered significant. The transgenes were under transcriptional regulation of the novel At6669 promoter (SEQ ID NO: 4880).Table 122Genes showing improved plant performance under normal cultivation conditions under regulation of the At6669 promoter

Table 122: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means p-value is less than 0.01, p<0.1 was considered significant. The transgenes were under transcriptional regulation of the new At6669 promoter (SEQ ID NO: 4880). EXAMPLE 18 EVALUATION OF NUE, PRODUCTION AND GROWTH RATE OF TRANSGENIC ARABIDOPSIS PLANT UNDER NORMAL AND LOW NITROGEN FERTILIZATION IN GREENHOUSE TESTS
[00643] Test 2: Nitrogen Use Efficiency measured up to the sieving stage: plant biomass and plant growth rate at optimal and limited nitrogen concentration under greenhouse conditions - This test follows the formation of plant biomass and the growth of the rosette area of greenhouse-grown plants under nitrogen-limiting and non-nitrogen-limiting growing conditions. Transgenic Arabidopsis seeds were sown on agar medium supplemented with ^ of MS medium and a selection agent (Kanamycin). The T2 transgenic seedlings were then transplanted into 1.7 liter trays filled with peat and perlite in a 1:1 ratio. The trays were irrigated with a solution containing nitrogen limiting conditions that were obtained by irrigating the plants with a solution containing 1.5 mM of inorganic nitrogen in the form of KNO3, supplemented with 1 mM of KH2PO4, 1 mM of MgSO4, 3.6 mM KCl, 2 mM CaCl2 and microelements, while normal nitrogen levels were obtained by applying a solution of 6 mM inorganic nitrogen also in the form of KNO3 with 1 mM KH2PO4, 1 mM MgSO4, 2 mM CaCl2 and microelements. All plants were grown in a greenhouse until the sieving stage. Plant biomass (the tissue above ground) was indirectly weighed after harvesting the rosette (fresh plant weight [FW]). Then, the plants were dried in an oven at 50°C for 48 hours and weighed (dry plant weight [DW]).
[00644] Each structure has been validated in its
[00645] T2 generation. Transgenic plants transformed with a structure formed by an empty vector carrying the 35S promoter and the selectable marker were used as controls.
[00646] The plants were analyzed with respect to their total size, growth rate, fresh weight and dry matter. The performance of transgenic plants was compared to control plants grown in parallel under the same conditions. Mock transgenic plants expressing the uidA reporter gene (GUS-Intron) or without any gene in the same promoter were used as controls.
[00647] The experiment was designed in the nested random lot distribution. For each gene of the invention, three to five independent transformation events were analyzed from each construct.
[00648] Digital Imaging - A laboratory imaging system, consisting of a digital reflex camera (Canon EOS 300D) attached with a 55mm focal length lens (Canon EF-S series), mounted on a reproduction device (Kaiser RS), which includes 4 light units (lamps 4 x 150 Watts) was used to capture images of plant samples.
[00649] The image capture process was repeated every 2 days, starting on day 1 after transplanting until day 15. The same camera, placed in a custom iron mount, was used to capture images of larger plants encased in vats. white in an environmentally controlled greenhouse. The vats were square in shape and included 1.7 liter trays. During the capture process, the vats were placed below the iron mount, while avoiding direct sunlight and casting shadows.
[00650] An image analysis system was used, consisting of a personal computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.39 [image processing program based on Java, developed at the National Institutes of Health of the United States and freely available on the Internet at rsbweb (dot) nih (dot) gov/]. The images were captured in 10 Mega Pixels resolution (3888 x 2592 pixels) and stored in a low-compression JPEG format (Joint Photographic Experts Group standard). Then, the analyzed data were saved in text files and processed using the JMP statistical analysis software (SAS Institute).
[00651] Leaf Analysis - Using digital analysis, leaf data were calculated, including leaf number, rosette area, rosette diameter, and leaf blade area.
[00652] Plant growth rate: the relative growth rate (RGR) of leaf number (Formula VIII, described above), rosette area (Formula IX described above) and plot coverage (Formula XI, described below) were calculated using the formulas indicated.
[00653] Dry and fresh plant weight - Around day 80 from sowing, the plants were harvested and directly weighed to determine the plant fresh weight (FW) and allowed to dry at 50°C in a drying chamber for about 48 hours before pressing to determine the plant dry weight (DW).
[00654] Statistical Analysis - To identify the genes that confer a significant improvement in tolerance to abiotic stress, the results obtained from transgenic plants were compared to those obtained from control plants. To identify the high performing genes and structures, the results of the tested independent transformation events were analyzed separately. Data were analyzed using Student's t test and results are considered significant if the p value is less than 0.1. The JMP statistical software package was used (Version 5.2.1, Instituto SAS Inc., Cary, NC, USA).
[00655] The genes listed in Tables 123-124 improved plant NUE when grown at limiting nitrogen concentration levels. These genes produced larger plants with a larger photosynthetic area and biomass (fresh weight, dry weight, number of leaves, rosette diameter, rosette area and plot cover) when grown under nitrogen-limiting conditions (nutrient deficiency stress), compared to control plants grown under identical growing conditions.Table 123Genes showing improved plant biomass production under nitrogen-limiting growing conditions







Table 123: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means that the p-value is less than 0.01, p<0.1 was considered to be significant.Table 124 Genes showing improved plant biomass production under nitrogen-limiting cultivation conditions





Table 124: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means that the p-value is less than 0.01, p<0.1 was considered significant.
[00656] The genes listed in Table 125 improved plant NUE when grown at limiting nitrogen concentration levels. These genes produced faster growing plants when grown under nitrogen-limiting growing conditions, compared to control plants grown under identical conditions, as measured by growth rate of leaf number, rosette diameter, and plot coverage.Table 125Genes showing improved rosette growth performance under nitrogen-limiting growing conditions





Table 125: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means p-value is less than 0.01, p<0.1 was considered significant.
[00657] The genes listed in Tables 126-127 improved plant NUE when grown at standard nitrogen concentration levels. These genes produced larger plants with a larger photosynthetic area and improved biomass (fresh weight, dry weight, number of leaves, rosette diameter, rosette area, and plot coverage) when grown under standard nitrogen conditions compared to cultivated control plants. under identical growing conditions.Table 126Genes showing improved plant biomass production under standard nitrogen growing conditions





Table 126: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means that the p-value is less than 0.01, p<0.1 was considered to be significant.Table 127Genes showing improved plant biomass production under standard nitrogen cultivation conditions



Table 127: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means that the p-value is less than 0.01, p<0.1 was considered significant.
[00658] The genes listed in Table 128 improved plant NUE when grown at standard nitrogen concentration levels. These genes produced faster growing plants when grown under nitrogen-limiting growing conditions, compared to control plants grown under identical conditions, as measured by growth rate of leaf number, rosette diameter, and plot coverage.Table 128Genes showing improved rosette growth performance under standard nitrogen growing conditions



Table 128: “CONT” - Control; “Avg.” - Average; “% Aum.” = % increase; “p-Val” - p-value; L means p-value is less than 0.01, p<0.1 was considered significant. EXAMPLE 19 EVALUATION OF NUE AND TRANSGENIC BRACHYPODIUM PRODUCTION UNDER NORMAL AND LOW NITROGEN FERTILIZATION IN GREENHOUSE TESTS
[00659] Test 1: Efficiency in Nitrogen Use Measured from plant biomass and production at limited and optimal nitrogen concentration under greenhouse conditions until flowering - This test follows biomass formation and plant growth (measured by height ) of plants that are grown in the greenhouse under limiting and non-limiting (eg, normal) nitrogen conditions. Transgenic Brachypodium seeds were sown in peat plugs. The T1 transgenic seedlings were then transplanted into 27.8 X 11.8 X 8.5 cm trays filled with peat and perlite in a 1:1 ratio. The trays were irrigated with a solution containing nitrogen limiting conditions, which were achieved by irrigating the plants with a solution containing 3 mM of inorganic nitrogen in the form of NH4NO3, supplemented with 1 mM KH2PO4, 1 mM MgSO4, 3.6 mM KCl, 2 mM CaCl2 and microelements, while normal nitrogen levels were achieved by applying a solution of 6 mM inorganic nitrogen, also in the form of NH4NO3 with 1 mM KH2PO4, 1 mM MgSO4 , 2 mM CaCl 2 , 3.6 mM KCl and microelements. All plants were grown in the greenhouse until they sprouted. Plant biomass (the above-ground tissue) was weighed shortly after the shoots were harvested (plant fresh weight [FW]). Then, the plants were dried in an oven at 70°C for 48 hours and weighed (dry weight [DW] of the plant).
[00660] Each structure has been validated in its T1 generation. Transgenic plants transformed with a structure formed by an empty vector carrying the selectable marker BASTA were used as controls (Figure 9B).
[00661] The plants were analyzed in terms of their total size, fresh weight and dry matter. The performance of transgenic plants was compared to control plants grown in parallel under the same conditions. Mock transgenic plants with no gene and no promoter were used as controls (Figure 9B).
[00662] The experiment was designed in blocks and with distribution of randomized nested lots within them. For each gene of the invention, five independent transformation events were analyzed from each structure.Phenotyping
[00663] Fresh and Dry Weight of Plant and Sprout - In flowering trials, when the flowering stage was completed (about 30 days after sowing), the plants were harvested and directly weighed to determine the fresh plant weight on semi-analytical scales (0.01 g.) (FW) and allowed to dry at 70°C in a drying chamber for about 48 hours before weighing to determine the plant dry weight (DW).
[00664] Time to Flowering - In both the Seed Maturation and Flowering assays, flowering was defined as the total appearance of the first spikelet on the plant. The time until flowering occurs is defined by the date when flowering is fully visible. The time to flowering occurrence date was documented for all plants and then the time from planting to flowering was calculated.
[00665] Leaf Thickness - In Flowering trials, when a minimum of 5 plants per batch in at least 90% of the batches in an experiment was documented at flowering, leaf thickness measurement was performed using a micrometer on the second leaf below the flag leaf.
[00666] Plant Height - In both the Seed Maturation and Flowering assays, once flowering was fully visible, the height of the first spikelet was measured from ground level to the bottom of the spikelet.
[00667] Number of Tillers - In Flowering trials, manual tiller count was performed per plant after harvest, before weighting. EXAMPLE 20 EVALUATION OF NUE AND TRANSGENIC BRACHYPODIUM PRODUCTION UNDER NORMAL AND LOW NITROGEN FERTILIZATION IN GREENHOUSE TESTS
[00668] Test 2: Efficiency in the Use of Nitrogen Measured plant biomass and production at limited and optimal nitrogen concentration under greenhouse conditions until Seed Maturation - This test follows the biomass production and production of plants that have been cultivated in a greenhouse under nitrogen-limiting and non-nitrogen-limiting cultivation conditions. Transgenic Brachypodium seeds were sown in peat plugs. The T1 transgenic seedlings were then transplanted into 27.8 X 11.8 X 8.5 cm trays filled with peat and perlite in a 1:1 ratio. The trays were irrigated with a solution containing nitrogen limiting conditions, which were achieved by irrigating the plants with a solution containing 3 mM of inorganic nitrogen in the form of NH4NO3, supplemented with 1 mM of KH2PO4, 1 mM of MgSO4, 3, 6 mM of KCl, 2 mM of CaCl2 and microelements, while normal nitrogen levels were reached by applying a solution of 6 mM of inorganic nitrogen, also in the form of NH4NO3 with 1 mM of KH2PO4, 1 mM of MgSO4, 2 mM CaCl2, 3.6 mM KCl and microelements. All plants were grown in the greenhouse until seed maturation. Each structure was validated in its T1 generation. Transgenic plants transformed with a structure formed by an empty vector carrying the selectable marker BASTA were used as controls (Figure 9B).
[00669] The plants were analyzed in terms of their total biomass, fresh weight and dry matter, as well as a large number of parameters related to production and yield components. The performance of transgenic plants was compared to control plants grown in parallel under the same conditions. Fake transgenic plants with no gene and no promoter (Figure 9B). The experiment was designed in blocks with distribution of randomized nested lots within them. For each gene of the invention, five independent transformation events were analyzed from each structure.Phenotyping.
[00670] Fresh and Dry Weight of the Plant and Vegetable - In Seed Maturation trials, when the maturity stage was completed (about 80 days after sowing), the plants were harvested and directly weighed to determine the fresh weight of the plant. plant (FW) and allowed to dry at 70°C in a drying chamber for about 48 hours before weighing to determine the dry weight (DW) of the plant.
[00671] Dry weight of the spikelet (SDWISpikelets Dry weight) - In Seed Maturation trials, when the maturity stage was completed (about 80 days after sowing), spikelets were separated from the biomass, allowed to dry at 70°C in a drying chamber for approx.
[00672] 48 hours before weighting to determine spikelet dry weight (SDW).
[00673] Grain Production per Plant - In Seed Maturation trials, after drying the spikelets for SDW, the spikelets were spread through a production machine, then through a cleaning machine, until the seeds were produced per batch, then they were weighed and the Grain Production per plant was calculated.
[00674] Number of Grains - In Seed Maturation trials, after the seeds per batch were produced and cleaned, the seeds were spread through a counting machine and counted.
[00675] Weight of 1000 Seeds - In Seed Maturation trials, after seed production, a fraction was collected from each sample (seeds per lot; ~0.5 g.), counted and photographed. The weight of 1000 seeds was calculated.
[00676] Harvest Index - In Seed Maturation trials, after seed production, the harvest index was calculated by dividing grain yield and plant dry weight.
[00677] Time to Flowering - In both tests, Seed Maturation and Flowering, flowering was defined as the total appearance of the first spikelet on the plant. The time for flowering to occur is defined by the date when flowering is fully visible. The time to flowering occurrence date was documented for all plants and then the time from planting to flowering was calculated.
[00678] Leaf Thickness - In Flowering trials, when at least 5 plants per batch in at least 90% of the batches in an experiment were documented at flowering, leaf thickness measurement was performed using a micrometer in the second sheet below the flag sheet.
[00679] Grain filling period - In Seed Maturation trials, maturation was defined by the first color change of the spikelet + stem in the plant from green to yellow/brown.
[00680] Plant Height - In both the Seed Maturation and Flowering assays, once flowering was fully visible, the height of the first spikelet was measured from ground level to the bottom of the spikelet.
[00681] Number of tillers - In Flowering trials, manual tiller count was performed per plant after harvest, before weighing.
[00682] Number of reproductive heads per plant - In Flowering trials, manual head count per plant was performed.
[00683] Statistical analysis - To identify genes that confer significantly improved tolerance to abiotic stress, the results obtained from the transgenic plants were compared with those obtained from control plants. To identify genes and structures above performance, the results of the independent transformation events tested were analyzed separately. Data were analyzed using Student's t test and results were considered significant if the p-value was less than 0.1. The JMP statistical software package was used (version 5.2.1, SAS Institute Inc., Cary, NC, USA).
[00684] While the invention has been described in conjunction with specific applications thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to cover all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.
[00685] All publications, patents and patent applications mentioned in this specification are incorporated in their entirety by reference in said specification, to the same extent as if each individual publication, patent or patent application were specifically and individually indicated as being incorporated herein by reference. Furthermore, the citation or identification of any reference in the present patent application should not be interpreted as an admission that such reference is available as prior art to the present invention. To the extent that section titles are used, they should not be construed as necessarily limiting.

权利要求:
Claims (8)
[0001]
1. Method for increasing the growth rate, biomass, seed yield, photosynthetic capacity and/or tolerance to abiotic stress of a plant compared to a control plant of the same species that is grown under the same growth conditions, the method characterized by fact of overexpressing in the plant a polynucleotide having the nucleic acid sequence as established by: (i) SEQ ID NO: 315 or 29, which encodes the polypeptide established by SEQ ID NO: 524, or its degenerate sequences which encode said established polypeptide by SEQ ID NO: 524,(ii) SEQ ID NO: 960, encoding the polypeptide set forth by SEQ ID NO: 3057, or degenerate sequences thereof encoding said polypeptide set forth by SEQ ID NO: 3057,(iii) SEQ ID NO: 961, which encodes the polypeptide set forth by SEQ ID NO: 3058, or degenerate sequences thereof encoding said polypeptide set forth by SEQ ID NO: 3058,(iv) SEQ ID NO: 962, which co encodes the polypeptide set forth by SEQ ID NO: 3059, or degenerate sequences thereof which encode said polypeptide set forth by SEQ ID NO: 3059,(v) SEQ ID NO: 964, which encodes the polypeptide set forth by SEQ ID NO: 3061, or degenerate sequences thereof encoding said polypeptide set forth by SEQ ID NO: 3061,(vi) SEQ ID NO: 966 encoding said polypeptide set forth by SEQ ID NO: 3063, or degenerate sequences thereof encoding said polypeptide set forth by SEQ ID NO: 3063,(vii) SEQ ID NO: 970, encoding the polypeptide set forth by SEQ ID NO: 3067, or degenerate sequences thereof encoding said polypeptide set forth by SEQ ID NO: 3067, (viii) SEQ ID NO: 971 , which encodes the polypeptide set forth by SEQ ID NO: 3068, or degenerate sequences thereof encoding said polypeptide set forth by SEQ ID NO: 3068, or (ix) SEQ ID NO: 973, which encodes the polypeptide set forth by SEQ ID NO: 3070, or its degenerate sequences encoding said polypeptide set forth by SEQ ID NO: 3070, wherein said polynucleotide is operably linked to a heterologous promoter to direct expression of said nucleic acid sequence in a plant cell, wherein the plant is a plant monocot or dicot.
[0002]
2. Method according to claim 1, characterized in that said nucleic acid sequence is established by SEQ ID NO: 315, 29, 960, 961, 962, 964, 966, 970, 971 or 973.
[0003]
3. Method according to claim 1, characterized in that said polynucleotide is established by: (i) SEQ ID NO: 315 or 29, which encodes the polypeptide established by SEQ ID NO: 524, or degenerate sequences of same ones encoding said polypeptide set forth by SEQ ID NO: 524,(ii) SEQ ID NO: 960 encoding said polypeptide set forth by SEQ ID NO: 3057, or degenerate sequences thereof encoding said polypeptide set forth by SEQ ID NO : 3057, or (iii) SEQ ID NO: 961, which encodes the polypeptide set forth by SEQ ID NO: 3058, or degenerate sequences thereof which encode said polypeptide set forth by SEQ ID NO: 3058.
[0004]
4. Method according to claim 1, characterized in that said polypeptide is expressed from a polynucleotide having the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 315, 29 and 960-961
[0005]
5. Method according to claim 1, characterized in that said polynucleotide is established by SEQ ID NO: 315 or 29, which encode the polypeptide established by SEQ ID NO: 524, or degenerate sequences thereof that encode the said polypeptide set forth by SEQ ID NO: 524.
[0006]
6. Method according to claim 1, characterized in that said polynucleotide is as established by SEQ ID NO: 315 or 29.
[0007]
7. Method according to any one of claims 1 to 6, characterized in that it also selects plants that overexpress said polynucleotide for an increased growth rate, biomass, seed yield, photosynthetic capacity and/or tolerance to abiotic stress of a plant in compared to a control plant of the same species that is grown under the same growth conditions, in which the referred abiotic stress is nitrogen deficiency.
[0008]
Method according to any one of claims 1 to 6, further characterized by the growth of the plant that overexpresses said polynucleotide under abiotic stress, wherein said abiotic stress is nitrogen deficiency.

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法律状态:
2018-03-06| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-13| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-03-20| B06I| Publication of requirement cancelled [chapter 6.9 patent gazette]|Free format text: ANULADA A PUBLICACAO CODIGO 6.6.1 NA RPI NO 2462 DE 13/03/2018 POR TER SIDO INDEVIDA. |

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2019-10-08| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-08-17| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-12-14| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2022-01-25| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 19/12/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201261745877P| true| 2012-12-26|2012-12-26|

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US61/745,877|2012-12-26|

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US201361827801P| true| 2013-05-28|2013-05-28|

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US61/827,801|2013-05-28|

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PCT/IL2013/051043|WO2014102774A1|2012-12-26|2013-12-19|Isolated polynucleotides and polypeptides, construct and plants comprising same and methods of using same for increasing nitrogen use efficiency of plants|

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