• 专利附录
  • 专利说明
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    • 专利摘要:
    Use of compounds for the regulation of plant growth. The present invention relates to the use of compounds of formula (I) for the regulation of plant growth, preferably for the regulation of root growth, and by extension, the growth of the plant in general. The use of these compounds results in a significant increase in the radicular mass of the plants, as well as in the early induction of flowering and fruit formation. Additionally, the use of the compounds of formula (I) favors the growth of the cultures in nutrient-poor media. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2636363A1
    申请号:ES201630412
    申请日:2016-04-05
    公开日:2017-10-05
    发明作者:Juan Carlos Del Pozo Benito；Concepción MANZANO FERNANDEZ；Pilar HOYOS VIDAL；María Josefa HERNÁIZ；Stephan POLLMANN
    申请人:Universidad Politecnica de Madrid；Universidad Complutense de Madrid；Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria INIA；
    IPC主号:
    专利说明:

    5 The present invention relates to the technical field of agrochemicals andmethods used in agriculture for the regulation of plant growth.In particular, the present invention relates to the use of compounds of formula (I)for plant growth regulation, inducing regulatory responses ofgrowth resulting in superior growth of treated plants, of certain
    10 parts of the plants, or more generally, an increase in crop yield. Especially, the use of said compounds results in a significant increase in the root mass of the plants, as well as the early induction of flowering and fruit formation. Additionally, the use of the compounds of formula (I) favors the growth of crops in nutrient-poor media. STATE OF THE TECHNIQUE
    Demographic trends predict a doubling of the population in 2050, which will lead to a greater demand for food. This situation is a great scientific challenge, since it is urgent to increase crop production, but within a more sustainable agriculture. The last century has witnessed an extraordinary success in the improvement of plants and agronomic techniques, "the green revolution", which resulted in the duplication of crop productivity, achieving a significant global decline in hunger. In this green revolution, the increase in
    25 agricultural productivity was largely the result of increased use of inputs, water and fertilizers, in addition to the use of biotechnology for the genetic improvement and achievement of these new milestones.
    Plants require various macro- and mineral micro-nutrients for their
    30 growth In fact, one of the most relevant determinants of agricultural productivity is the nutritional status of crops, especially with regard to nitrogen (N), phosphorus (P) and potassium (K). Traditionally the problem of nutrition has been solved with massive contributions of nutrients in fertilizers. This practice involves important problems, such as the economic cost of
    35 raw materials and the environmental problem of excess nutrient intake, which is washed by the rains and pollutes the aquifers promoting eutrophication,


    that is, excessive nutrient enrichment of a particular ecosystem, in this particular case, of aquatic ecosystems, such as an aquifer (Ansari AA, et al. Springer Netherlands. 2014, pp 17-27; Lewis WM, et al. 2014, Environmental Science & Technology 45: 10300-10305).
    5 We are currently facing a fundamentally different challenge that consists in increasing crop yields within a sustainable agricultural system, through less use of fertilizers, water and pesticides. On the other hand, it is also recommended that crops be able to adapt to climate changes
    10 that are currently occurring (Tilman D, et al. Nature. 2002; 418: 671-677). Therefore, in order to demand these new challenges, science must make use of new biotechnological tools that allow increasing agricultural productivity within a more sustainable agriculture.
    15 In the improvement and increase of crop production, the study of the root system has had little relevance compared to the study of aerial organs, although it has been used in improvement programs through its use as a graft holder . But due to growing crop needs, soil nutritional deficiencies, water scarcity and climate change, they have made
    20 the manipulation of root systems to make crops more efficient and to develop more sustainable agriculture is a biotechnological option with great perspectives (from Dorlodot S, et al. Trends Plant Sci. 2007; 12: 474-481; Den Herder G , et al. Trends Plant Sci. 2010, 15: 600-607). In the past hormonal treatments have been used to increase crop productivity
    25 (US5614467A1; Tie Cai, et al. Field Crops Research. 2014; 155: 172-183). However, these treatments have pleiotropic effects (those phenomena for which a single gene is responsible for phenotypic effects or different and unrelated characters) that can alter many developmental processes to a different extent, which can affect the occurrence of side effects. not wanted. Plus
    30 Recently, the use of chemical compounds to increase crop yields has also been explored, however, among all the molecules tested, few have been marketed due to the side effects they produce in the crops themselves and in the medium in which they grow (De Rybel B. et al. Chemistry & Biology. 2009; 16 (6): 594-604; De Rybel B. et al. Nat Chem Biol.
    35 2012; 8 (9): 798-805).


    Another of the strategies to increase crop yield has been aimed at obtaining transgenic plants that have a modification of their root system via overexpression and / or inhibition of specific genes involved in root development (Comas LH. et al. Frontiers in Plant Science. 2013; 4: 442). However, obtaining these plants is carried out by means of an expensive and arduous methodology, in addition to knowing the genetic and molecular mechanisms that determine both the number and the location of the lateral roots before proceeding to the modification of a native plant to obtain the desired transgenic plant. Another additional limitation for the use of plants
    10 GM is that many countries prohibit their cultivation.
    In this regard, the need to increase crop yields within an environmentally sustainable agricultural system still exists in the state of the art.
    DESCRIPTION OF THE INVENTION
    To solve the deficiencies in the prior art mentioned above, the present invention describes the use of a compound of formula (I) or an agriculturally acceptable salt thereof, for the regulation of growth, induction of flowering, and / or fruit formation in plants, where said compound of formula (I) in addition to increasing the yield of crops treated with it, does not present toxicity either in the plants themselves, or in the soil on which they are grown, or on the subjects, humans and animals, that feed on these crops, due to
    25 which is a natural compound of plant origin, thus allowing a sustainable agriculture system, even in those soils that are deficient in nutrients, such as nitrates, phosphates, potassium, iron, and / or any combination thereof, those soils being preferred deficient in nitrates and / or phosphates.
    The compound of formula (I), or an agriculturally acceptable salt thereof is:


    where: R1 represents a carbohydrate; R2 and R4 independently represent
    5 hydrogen or (C1-C6) alkyl; R3 represents hydrogen or (C1-C6) alkyl; and R5 and R6 independently represent hydrogen or halogen; for growth regulation, flowering induction, and / or fruit formation in plants.
    As the inventors demonstrate, the use of compounds of formula (I), and more preferably the use of compound (Ia),
    applied by root and / or leaf, preferably in the form of an aqueous solution, induces the growth of the root system, in addition to inducing flowering, and / or formation of


    fruits in plants. Additionally, the application of said compounds to the cultures induces the expression of molecular markers of the auxin response, but only in the primordia of the lateral roots, without inhibiting the growth of the main root just as auxin does.
    5 Said regulation of growth, induction of flowering and / or fruit formation occurs preferentially through the increase in root mass, both at the level of the increase in lateral roots, as well as the increase in the length of the main root. , even in nutrient deficient soils, such as nitrates, phosphates,
    10 potassium, iron, and / or any combination thereof, those soils deficient in nitrates and / or phosphates being preferred. This increase, preferably at the root level, is also able to accelerate the formation of different organs of plants, including fruits.
    15 Auxins comprise a group of substances, both natural and synthetic, that participate in the regulation of multiple aspects of root development, including the growth of the main root and the formation of lateral roots and root hairs. It is interesting to note that compound (Ia) did not modulate all processes regulated by auxins, but that there are processes that were not modulated
    20 by the treatment with the compounds of formula (I), preferably with the compound (Ia), so it is possible that said compound (Ia) has a specific function within the auxin response pathway, regulating specific processes within of plant physiology.
    For the purposes of the present invention, the term "growth regulation" preferably refers to plants refers to a variety of plant responses that improve some characteristics of the plant, such as growth, flowering, fruit synthesis, etc.
    For the purposes of the present invention, the term "growth regulator (s) of the plant (s)" refers to the compounds, described in the present invention, which have activity in one or more regulatory processes of the growth of a plant. Said compounds are used in the practice of the present invention in amounts that are not phytotoxic with respect to the plant being treated, but which
    35 stimulate the growth thereof, or certain parts thereof. Therefore, these compounds can also be called "plant stimulants" or


    "Plant growth stimulants." Plant growth regulators are preferably, and not limited to, chemicals or natural products, also called plant hormones (such as auxins, gibberellins, cytokinins, ethylene, brassinosteroids, strigolactones, abscisic acid, and Salicylic acid; 5 lipooligosaccharides (such as Nod factors), peptides (e.g., systemin), fatty acid derivatives (e.g., jasmonates), and oligosaccharines, among others,
    or it can be synthetically produced compounds such as derivatives of natural origin plant growth hormones, such as ethephon. For the purposes of the present invention the growth regulators are preferably those
    10 compounds of formula (I) and their agriculturally acceptable salts.
    Thus, in a first aspect the present invention describes the use of a compound of formula (I) or an agriculturally acceptable salt thereof:
    15 (I)
    where: R1 represents a carbohydrate; R2 and R4 independently represent hydrogen or (C1-C6) alkyl; R3 represents hydrogen or (C1-C6) alkyl; and R5 and R6 independently represent hydrogen or halogen; for regulation of
    20 growth, induction of flowering, and / or fruit formation in plants.
    The invention also encompasses the use of any stereoisomer, enantiomer, geometric isomer or tautomer, and mixtures of the compounds of formula (I).


    For the purposes of the present invention, the term "agriculturally acceptable salt (s)" refers to salts whose anions or cations are known and accepted in the art for the formation of salts for agricultural use. Suitable salts with bases, for example formed by compounds of formula (I) containing a carboxylic acid group, include alkali metal (for example sodium and potassium), alkaline earth metals (for example calcium and magnesium) and ammonium salts. Ammonium salts include ammonium salts (NH +) and ammonium salts of organic amines, (for example, diethanolamine, triethanolamine, octylamine, morpholine and dioctylmethylamine
    10 salts), and quaternary ammonium salts (NR +), for example, tetramethylammonium salts. Suitable salts for the addition of acid, for example, those formed by compounds of formula (I) containing an amino group, include salts with inorganic acids, for example hydrochlorides, sulfates, phosphates and nitrates and salts with organic acids, for example acetic acid.
    The term "alkyl" refers to a saturated, linear or branched hydrocarbon chain, consisting of carbon and hydrogen atoms and having one to six carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, sec-butyl, n-pentyl, n-hexyl.
    Preferably the alkyl group has between 1 and 3 carbon atoms, more preferably the alkyl is a methyl.
    By "halogen" is meant in the present invention a bromine, chlorine or fluorine atom.
    The substituent R1 of the formula (I) represents a carbohydrate. By carbohydrates we also refer to any hydrophobic derivative, and as a derivative we consider a carbohydrate where at least one hydroxyl group is substituted with a hydrophobic moiety, including, but not limited to, esters and ethers,
    The derivatives are preferably acetylated. In a preferred embodiment, the carbohydrate is selected from the list consisting of: glucose, mannose, galactose, xylose, fructose, lactose, and any of its derivatives, preferably acetylated derivatives, for example and not limited to N-acetylactosamine, N-lactobiosa, Nacethylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc) or tetraacetate
    35 glucose In a more preferred embodiment, the R1 substituent is glucose or its derivatives, more preferably glucose tetraacetate.


    In another more preferred embodiment of the first aspect of the invention, the substituents R2 and R4 of the formula (I) are the same and represent a methyl or a hydrogen. In a more preferred embodiment, the substituents R2 and R4 are the same and represent a
    5 methyl
    In another more preferred embodiment of the first aspect of the invention, the R3 substituent of the formula (I) represents hydrogen or methyl. In a more preferred embodiment, the R3 substituent is hydrogen.
    In another preferred embodiment, R5 and / or R6 are hydrogen.
    In another more preferred embodiment of the first aspect of the invention, the compound of formula (I) is preferably compound (Ia):
    Compound (Ia)
    where R1 is glucose tetraacetate, R2 and R4 are the same and represent a methyl and R3, R5 and R6 are hydrogen.
    In a still more preferred embodiment, the use of the compounds of formula (I) is characterized in that they are administered to the plants in the form of an aqueous root solution, although they can also be administered via foliar via,
    25 preferably spray. In yet another more preferred embodiment, the aqueous solution is preferably irrigation water.
    In another more preferred embodiment, the use of the compounds of formula (I) is characterized in that the concentration administered thereof on the plants is


    is in the range of 0.05 to 5 μM in an aqueous solution, preferably 2.5 μM, or alternatively at a concentration of between 700-3500 μg / L, preferably 1800 μg / L, or alternatively between 0.001-0.0001% with respect to the volume of the aqueous solution. In another even more preferred embodiment, the concentration administered
    5 of compound of formula (I) will depend on each specific species or crop, and aaverage expert in the field is able to determine the concentration of saidcompound to be administered.
    For the purposes of the present invention, the aqueous solution in addition to comprising the
    10 minus one of the compounds of formula (I), preferably the compound of formula (Ia), at the indicated concentrations, may comprise other compounds, such as for example nutrients, fertilizers, pesticides, insecticides, acaricides, herbicides, fungicides, as well like other growth regulators.
    In another more preferred embodiment, the use of the compounds of formula (I) is characterized in that it induces an increase of at least 20% of the plant root mass in those plants treated with said compounds with respect to the control plants not treated with they.
    In another preferred embodiment, the use of the compounds of formula (I) is characterized in that it induces growth even in nutrient deficient soils, preferably in soils deficient in nitrates, phosphates, potassium, iron, or any combination thereof, and more preferably deficient in nitrates and / or phosphates.
    In another preferred embodiment, the use of the compounds of formula (I) is characterized in that the plant to which it is applied is an ornamental and / or crop plant.
    In yet another more preferred embodiment, the plant is selected from any of
    30 the list consisting of: tomato, lettuce, wheat, barley, rye, rice, corn, sugar beet, cotton or soy.
    Another aspect described in the present invention relates to a composition, preferably an aqueous composition comprising at least one compound of
    Formula (I) as defined throughout this document, or an agriculturally acceptable salt.


    In a preferred embodiment, the aqueous composition of the invention is an aqueous solution preferably comprising irrigation water. In another more preferred embodiment, the aqueous composition of the invention is characterized in that the compound 5 of formula (I), more preferably the compound of formula (Ia), is in said composition in the range of 0.05 to 5 μM , preferably 2.5 μM, or alternatively at a concentration of between 700-3500 μg / L, preferably 1800 μg / L, or alternatively between 0.001-0.0001% with respect to the volume of the aqueous composition. In yet another more preferred embodiment, the concentration is going to
    It depends on each specific species or crop, and a person skilled in the art is able to determine the concentration of said compound to be administered.
    In another more preferred embodiment, the composition of the invention further comprises agriculturally acceptable vehicles, useful for the regulation of growth of the
    15 floor
    The term "vehicle" refers to a diluent, adjuvant, excipient or substance or combination of substances that can be used in the agricultural sector, and includes any agriculturally acceptable liquid or solid material, which may be added and / or
    20 to be mixed with the compound of formula (I) of the invention, to put it in a simpler or improved application form, or with an applicable or desirable intensity of activation. Due to the nature of the active ingredient of the agricultural composition, (compound of formula (I), said agriculturally acceptable vehicle must allow, or not harm or compromise, the activity of said compound.
    On the basis of these compositions, it is also possible to add other active ingredients such as pesticides, insecticides, acaricides, herbicides, fungicides, and with antidotes, fertilizers and / or growth regulators.
    Another aspect of the present invention relates to the use of the composition, preferably aqueous composition, for the regulation of growth, induction of flowering and / or formation of fruits in plants, by applying an effective amount of the compound of formula ( I), more preferably of the compound (Ia), or of a composition comprising it, by root route, as previously defined.


    Another aspect described in the present invention relates to a method for the regulation of growth, induction of flowering and / or fruit formation in plants, which comprises the administration of an effective amount of a compound of formula (I), more preferably of the compound (Ia), or of a composition,
    5 preferably aqueous, comprising it, as previously defined inthis document.
    For the purposes of the present invention, the term "effective amount" or "effective amount" refers to the amount necessary to alleviate, improve, stabilize, prevent, delay or
    10 delay the progression of the development stages of the phytophagous agent in question in the plant and thus obtain beneficial or desired results. Said effective amount can be applied in a single administration or in several administrations. Although the effective amount of the compound of formula (I), more preferably of the compound (Ia), or of a composition, preferably aqueous
    15 which includes it, may vary within the range of 0.05 to 5 μM, preferably 2.5 μM, or alternatively between 700-3500 μg / L, preferably 1800 μg / L, or alternatively between 0.001 to 0.0001% with respect to the volume of the aqueous solution. In yet another more preferred embodiment, the concentration will depend on each specific species or crop, and an average expert in the field is capable of
    20 determine the concentration of said compound to be administered.
    In a more preferred embodiment, the method described in the present invention is characterized in that the compound of formula (I), preferably the compound (Ia), is administered in the form of an aqueous solution, preferably root and / or route
    25 foliar, where the aqueous solution is preferably irrigation water. In another more preferred embodiment, foliar administration is carried out by spraying.
    In another more preferred embodiment of the method of the present invention, this is characterized in that the compounds of formula (I), more preferably the compound
    30 (Ia), is administered in an amount ranging from 0.05-5 μM, preferably 2.5 μM, or alternatively between 700-3500 μg / L, preferably 1800 μg / L, or alternatively between 0.001 to 0, 0001% with respect to the volume of the aqueous solution.
    In another more preferred embodiment of the method of the present invention, this is characterized in that the compound of formula (I), more preferably the compound


    (Ia), induces an increase of at least 20% of the plant root mass in those plants treated with said compounds with respect to the control plants not treated with them.
    In another more preferred embodiment, the method of the invention is characterized in that thecompound of formula (I), more preferably compound (Ia), induces thePlant growth even in nutrient deficient soils. In othermost preferred embodiment, the compound (Ia) induces the growth of the plants innutrient deficient soils selected from any of the following
    10 list: nitrate, phosphate, potassium, iron, or any combination thereof, those soils deficient in nitrates and / or phosphates being preferred.
    In another more preferred embodiment, the method of the invention is characterized in that the plant is an ornamental and / or crop plant.
    In another more preferred embodiment, the method of the invention is characterized in that the plant is selected from any of the list consisting of: tomato, lettuce, wheat, barley, rye, rice, corn, sugar beet, cotton or soy .
    Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and figures are provided by way of illustration, and
    25 are not intended to be limiting of the present invention. DESCRIPTION OF THE FIGURES
    FIG. 1. Root development of Arabidopsis thaliana plants grown in presence
    30 of the compound of formula (Ia). A) Photographs of Arabidopsis plants grown for 12 days in MS1 / 2 medium in the absence or presence of 5 μM of the compound of formula (Ia). An increase in the number of emerged lateral roots and their length in the plants treated with the compound (Ia) is appreciated. The graphs show the quantification of the
    Increase in the relative length of the main root or increase in the growth of the length of the lateral roots of Arabidopsis plants treated with the compound


    (Ia) with respect to the control plants (treated with the methanol solvent), showing an amplification of the total root system. B) The treatment of Arabidopsis plants with compound (Ia) (5μM) induces the expression of lateral root formation markers (SKP2B :: GUS) and
    5 response to auxins (DR5 :: GUS) only at the emergency points of the lateral roots (RL). The left and central panels show an increase in the number of lateral root primordia (SKP2B :: GUS) demonstrating that the compound (Ia) induces the formation of primordia (arrows) and in root zones between lateral primordia. The panel on the right shows how the treatment with the compound
    10 (Ia) induces the auxin response marker (DR5 :: GUS) in the lateral root primordia. However, treatment with compound (Ia) does not induce the expression of said markers in the main root meristems (asterisk), avoiding unwanted side effects such as inhibition of root meristem. C) Treatment with compound (Ia) (5μM) induces a greater number of primordia
    15 lateral roots that treatment with endogenous indole acetic acid (IAA) at concentrations of 0.25, 0.5 or 1 μM for 6, 24, 48 or 120 hours. In the photographs they are shown as already at 3h of culture in the presence of the compound (Ia), the plants show an increase of the lateral roots with respect to the control plants. The quantification of the expression of the
    20 DR5 marker: LUC (side root primordia marker; LUC: luciferase).
    FIG. 2. Effect of the treatment with compound (Ia) during the growth of Arabidopsis thaliana plants on medium surfaces in (-Pi) or in nitrate (-N) for 12 days.
    25 A) and C). Photographs of plants grown in MS1 / 2 medium (Murashige-Skoog), nitrate deficient MS1 / 2 medium (-N), or phosphate deficient MS1 / 2 medium (-Pi) and whether or not treated with compound (Ia) a a concentration of 5μM. B) Quantification of the length of the main root, of the lateral roots, of the total sum (length of the root system) and of the number of lateral roots emerged in
    30 MS1 / 2 medium or MS1 / 2 medium without nitrate (-N). D) Quantification of the length of the main root, of the lateral roots, of the total sum (length of the root system) and of the number of lateral roots emerged in MS1 / 2 medium or in MS1 / 2 medium without phosphate (-Pi) . E) Phosphate deficiency response marker expression pattern
    35 IPS1 :: GUS. Arabidospis plants expressing the IPS1 :: GUS marker were grown for 12 days in MS1 / 2 medium with only 30 μM phosphate and analyzed for


    GUS activity showing that the treatment with the compound (Ia) at different concentrations reduced the intention and expression of IPS1, which suggests a higher uptake of Pi in media that contain little phosphate.
    5 FIG. 3. Quantification of fruit production in variety tomato plantsMoneymaker grown in a greenhouse for 20 weeks and treated once a yearweek with the compound (Ia) dissolved in the irrigation water (10 mL of watercontaining 2 μM of compound (Ia)). After the appearance of the first fruits,the number of tomatoes produced in groups of 10 plants was counted every week. EXAMPLES
    The invention will now be illustrated by tests carried out by the inventors, which demonstrates the effectiveness of the product of the invention.
    Example 1. Effect of treatment with compound (Ia) on root growth of plants.
    Arabidopsis plants used have lateral root markers and 20 primordial lateral roots pSKP2B: GUS (Manzano C, et al. Plant Physiol. 2012,
    160: 749-762) or the auxin response marker DR5: GUS (Ulmasov T, et al. 1997. Plant Cell 9: 1963-1971) and DR5: LUC (Moreno-Risueno MA, et al. Science. 2010, 329: 1306-1311).
    25 For in vitro Arabidopsis cultures, 12x12 cm square Petri dishes were used and grown following the D-Root system (Silva-Navas J, et al. The Plant Journal. 2015, 84: 244-255). This system allows plants to grow in vitro with the root system in darkness and the aerial part in the presence of light, simulating what happens in nature and therefore, providing more real results. The middle of
    The culture used was the modified MS culture medium to contain half the salts. We will call said modified medium from here, MS1 / 2 medium (Murashige T and Skoog F. Physiology Plantarum. 1962, 15: 473-497). The medium is adjusted to a pH = 5.7 and gelled with Plant Agar (1%) (Duchefa). The MS1 / 2 medium contains as macronutrients: ammonium nitrate (NH4NO3) 1,650 mg / l; chloride
    Calcium (CaCl2 / 2H2O) 220 mg / l; magnesium sulfate (MgSO4 / 7H2O) 185 mg / l, potassium phosphate (KH2PO4) 85 mg / l; potassium nitrate (KNO3) 950 mg / l and as micronutrients:


    boric acid (H3BO3) 3.1 mg / l; cobalt chloride (CoCl2 / 6H2O) 0.0125 mg / l; copper sulfate (CuSO4 / 5H2O) 0.0625 mg / l; ferrous sulfate (FeSO4 / 7H2O) 13.9 mg; manganese sulfate (MnSO4 / 4H2O) 11.15 mg / l; potassium iodide (KI) 0.415 mg / l; sodium molybdate (Na2MoO4 / 2H2O) 0.125 mg / l; zinc sulfate (ZnSO4 / 7H2O) 4.3 mg / l; EDTA of
    5 sodium / 2H2O 18.1 mg / l and Vitamins; I-Inositol 50 mg / l; Niacin 0.25 mg / l; Pyridoxine / HCl 0.25 mg / l; Thiamine / HCl 0.05 mg / l; and glycine 1 mg / l.
    The compound (Ia) has been described through the synthetic process shown in the
    Scheme 1, by 5 reaction steps from the acid methyl ester
    10 Indo-acetic (IAA, 1) as detailed in Chisholm and Vranken, J. Org. Chem., 1995, 60, 6672-6673; Gilbert et al., Tetrahedron 1997, 53, 16553-16564; Bergman et al., J. Chem. Soc., Perkin Trans I, 2000, 2609-2614. This route includes a glycosylation step of intermediate 2, through methodologies of classical sugar chemistry, responsible for obtaining low yields of the final product Ia.


    Where: TFA is trifluoroacetic acid, rt is room temperature, DDQ is 3-dichloro5,6-dicyano-p-benzoquinone, Py is pyridine and Ac2O is acetic anhydride.
    Said compound is dissolved in methanol at a final concentration of 50 mM. For
    Adding it to the culture medium is previously diluted in 200 μl of methanol to which 800 μl of water is subsequently added to facilitate dilution. Finally, this milliliter comprising water: methanol: compound of formula (Ia) is added to the culture medium before gelation, which occurs at an approximate temperature of 50 ° C.
    10 Arabidopsis plants carrying the lateral root and lateral root markers pSKP2B: GUS or the auxin response marker DR5: GUS and DR5: LUC were grown on MS1 / 2 media in the presence or absence of 5 μM of the compound. formula (Ia), for 12 days. Crops that were not treated with the
    The compound of formula (Ia) was added methanol, which is the solvent in which the compound of formula (Ia) is dissolved, to ensure that the crops grow under the same conditions. After this time, the morphological analysis of the root system was carried out. First, photographs of the crops were taken and then the plants were stained to determine GUS activity and thus detect
    20 and quantify the expression of the lateral root and lateral root marker genes pSKP2B :: GUS and the DR5 auxin response marker: GUS, following the protocol described in del Pozo JC, et al. Plant Cell 2002, 14: 3057-3071. To determine GUS activity, the plants are stained by introducing them into a medium containing 50 mM of sodium phosphate buffer pH = 7, 0.1% Triton X100 and 0.1% of
    25 acetone They are kept in said solution for 6 hours at a temperature of 37 ° C. After this time the plants are extracted from said solution and incubated for another 3 hours in a 90% ethanol solution. Subsequently, the ethanol is replaced by a solution of distilled water and photographs of the dyed plants are taken using a camera attached to a Leica z9 magnifying glass. The lengths of
    30 lateral roots were quantified with the Fiji / ImagenJ program.
    As shown in Fig. 1A, Arabidopsis plants treated with compound (Ia) show an increase in the number of lateral roots emerged, both at the level of the relative length of the main root, and the growth of the root. length of the lateral roots with respect to Arabidopsis plants that were not treated with said compound. These results show the amplification


    of the total root system of the plants treated with the compound (Ia) with respect to the untreated control plants.
    The quantification of the lateral root primordia was carried out analyzing the
    5 expression of the DR5 :: LUC auxin response marker that allows quantification ofprimordia over time. To do this, Arabidopsis plants that comprisesaid marker was grown for 5 days in MS1 / 2 medium. Subsequentlytransplanted into media containing the control solvent (methanol), or 5 μM ofcompound (Ia). After these times, photographs of the marker were taken
    10 LUC (as described in Moreno-Risueno MA, et al. Science. 2010. 329: 13061311) and its number was quantified. For this, the seedlings treated with or without compound of formula (Ia) were grown with 100 μM of luciferin, which emits light in the presence of the enzyme Luciferase. This emission is captured with a luminescence magnifier (coupled high sensitivity CCD camera). How luciferase enzyme expression is
    15 under the control of the DR5 promoter that marks lateral root primordia, the luminescent point count allows the number of lateral primordias developed in vivo to be calculated in a given time per plant.
    As can be seen in Fig. 1B, treatment with compound (Ia) induces the
    20 expression of lateral root formation markers (pSKP2B :: GUS) and response to auxins, specifically at those root points of the plant from which lateral roots emerge.
    Since indole acetic acid (IAA) is a natural auxin, an assay was carried out
    25 comparing the induction capacity of the expression of the lateral root formation markers indicated above in the presence of the compound (Ia) at the concentration of 5μM or in the presence of the natural auxin IAA at different concentrations: 0.25, 0.5 or 1 μM, for 6, 24, 48 or 120 hours. After these times, the number of points that expressed the marker was analyzed
    30 DR5 :: LUC. As observed in Fig. 1C, compound (Ia) induces more number of lateral root primordia than endogenous auxin IAA.
    All these results show that the treatment with the compound (Ia) described in the present invention induces the expression of molecular markers of
    The response to auxins, such as the expression of DR5 :: GUS in the lateral root formation zone (Fig. 1B), but does not induce its expression in meristems. Without


    However, other processes regulated by auxins, and in principle, negative for the root system, such as inhibition of root elongation, are not affected by the treatment of cultures with compound (Ia) (Fig. 1B ). Therefore, it is possible that the compound (Ia) functions, at least partially, as an auxinic type hormone, regulating only certain determined processes. Additionally, it has been shown that the compound (Ia) is more effective than the natural endogenous auxin IAA inducing the formation of a greater number of lateral root primordia (Fig. 1C). Therefore, all these results indicate that the compound (Ia) is capable of increasing the number of lateral root primordia and the length of
    10 the same, thus increasing the size of the root system in general of the plants treated with said compound.
    Example 2. Effect of the treatment with compound (Ia) on the root growth of plants grown in phosphate or nitrate deficient soils.
    15 To demonstrate the ability of compound (Ia) to induce the growth of the root system of plants that grow in phosphate or nitrate-free media, tests were conducted with Arabidopsis plants that were grown and germinated in MS1 / culture medium. 2 with a low concentration of nitrogen (-N), or MS1 / 2 medium
    20 with a low phosphate concentration (-Pi). Said culture medium was adjusted to a pH = 5.7 and gelled with Plant Agar (1%) (Duchefa).
    To analyze the effect of compound (Ia) on root development in nutrient deficiency situations, Arabidopsis plants were grown in a complete MS1 / 2 medium in the absence of nitrate (-N) and phosphate (-Pi), and with and without the compound (Ia) at the concentration of 5 μM (Figure 2A and C, respectively). At 12 days of growth the plants were photographed and the growth in millimeters (mm) of the main root, that of all the secondary side roots emerged and the sum of both (total root system growth) was calculated. On the other hand, the
    30 number of lateral roots emerged in each condition (Figure 2B and D).
    To analyze the molecular response to phosphate deficiency (-Pi) in the culture medium, Arabidopsis plants expressing the IPS1 :: GUS marker were grown in an MS1 / 2 medium with only 30 μM Pi, and with and without the compound (Ia) at different concentrations (0.1; 2.5 or 5 μM) (Figure 2E). Subsequently, said plants were stained, as indicated above, to analyze GUS activity and were


    observed that treatment with compound (Ia) reduces the induction of IPS1 and significantly shortens the root system of plants grown in phosphate deficient media. This result therefore suggests that treatment with compound (Ia) favors the absorption of Pi when this nutrient is in low
    5 concentrations in the medium.
    Therefore, the results shown in Fig. 2 as a whole show that the treatment with the compound (Ia) during plant growth in phosphate deficient medium (-Pi) (Fig. 2C and D) or in nitrate (-N) (Fig. 2A and B) for 12
    10 days significantly shortens the main root system of plants grown in phosphate deficient media (FIG. 2D), but significantly stimulates the lateral root system of plants grown in nitrate and phosphate deficient media (Fig. 2B and D).
    15 Example 3. Induction of fruit formation in tomato plants grown in the presence of compound (Ia).
    In addition to quantifying the induction of root system growth, fruit formation was quantified in Moneymaker tomato plants grown in
    20 presence of the compound (Ia).
    Cultures of tomato plants of the Moneymaker variety were grown in a greenhouse with controlled conditions and photoperiod of 18 h light / 8 h dark for 20 weeks. These plants are grown in pots with a soil substrate: 25 vermiculite 3: 1 and irrigated 3 days per week by adding water on the lower trays, not in the pot itself. The treatment of tomato plants was carried out by applying the compound (Ia) in the irrigated water and adding said water to the soil that is next to the plant stem. Of the three days of irrigation, only one of the days the compound (Ia) was added to said irrigation water, using
    30 for said irrigation 10 mL of water containing 2 μM of the compound (Ia). The formation of fruits is quantified every week, in groups of ten plants, from the day the first tomato is quantified.
    The results shown in Fig. 3 demonstrate that the treatment of the plants of
    35 tomato with the compound (Ia) in the irrigated water accelerates the production of tomato fruits, observing that as of week 7 a number of fruits of


    tomato significantly higher in plants treated with said compound (Ia) than in control, untreated plants. These results show that compound (Ia) promotes growth and accelerates the formation of different organs of plants, including fruits.

    权利要求:
    Claims (20)
    [1]
    1. Use of a compound of formula (I) or an agriculturally acceptable salt thereof
    5 (I)
    where: R1 represents a carbohydrate; R2 and R4 independently represent hydrogen or (C1-C6) alkyl; R3 represents hydrogen or (C1-C6) alkyl; and R5 and R6 independently represent hydrogen or halogen; for the
    10 growth regulation, induction of flowering, and / or fruit formation in plants.
    [2]
    2. Use according to claim 1 wherein R1 is glucose tetraacetate.
    Use according to any one of claims 1 or 2, wherein R2 and R4 are the same and represent a methyl or hydrogen.
    [4]
    4. Use according to claim 3, wherein R2 and R4 are methyl.
    Use according to any one of claims 1 to 4, wherein R 3 represents hydrogen or methyl.
    [6]
    6. Use according to claim 5, wherein R3 is hydrogen.
    Use according to any one of claims 1 to 6, wherein R5 and / or R6 are hydrogen.

    [8]
    8. Use according to any of claims 1 to 7, wherein the compound of formula (I) is the compound (Ia)
    5 Compound (Ia)
    [9]
    9. Use according to any of claims 1 to 8 characterized in that the compound of formula (I) is applied in an amount of 0.05 to 5 μM, preferably at a concentration of 2.5 μM.
    [10]
    10. Use according to any of claims 1 to 8 characterized in that the compound of formula (I) is applied in an amount of between 700 to 3500 μg / L, preferably at a concentration of 1800 μg / L.
    Use according to any one of claims 1 to 10 characterized in that it is administered to the plants in the form of an aqueous solution.
    [12]
    12. Use according to claim 11 wherein the aqueous solution is preferably water
    of irrigation. twenty
    [13]
    13. Use according to any of claims 11 to 12 wherein the aqueous solution is administered by root and / or foliar route.
    [14]
    14. Use according to claim 13 characterized in that the foliar administration is carried out by spraying.
    [15]
    15. Use according to any of claims 1 to 14 characterized in that it produces an increase of at least 20% of the plant root mass of the treated plants with respect to the control plants.

    [16]
    16. Use according to any one of claims 1 to 15 characterized in that the growth of the plant also occurs in nutrient deficient soils.
    Use according to claim 16 characterized in that the soil is deficient in any of the nutrients selected from the list consisting of: nitrates, phosphates, potassium, iron, or any combination thereof.
    [18]
    18. Use according to any of claims 16 to 17 characterized in that the soil is deficient in nitrates and / or phosphates.
    [19]
    19. Use according to any of claims 1 to 18 characterized in that the plant is an ornamental and / or crop plant.
    Use according to claim 19, characterized in that the plants are selected from the list consisting of: tomato, lettuce, wheat, barley, rye, rice, corn, sugar beet, cotton or soy.
    [21]
    21. Aqueous composition comprising at least one compound of formula (I) according to
    20 have been defined in any one of claims 1 to 8 or an agriculturally acceptable salt.
    [22]
    22. Aqueous composition according to claim 21 further comprising vehicles
    and / or agriculturally acceptable excipients useful for the regulation of plant growth.
    [23]
    23. Aqueous composition according to any of claims 21 to 22 characterized in that the concentration of compound of formula (I) ranges from 0.05 to 5 μM, preferably 2.5 μM.
    [24]
    24. Aqueous composition according to any of claims 21 to 22 characterized in that the concentration of compound of formula (I) ranges from 700 to 3500 μg / L, preferably 1800 μg / L.
    25. Aqueous composition according to any of claims 21 to 24 characterized in that it is administered to the plants by root and / or foliar route.

    [26]
    26. Aqueous composition according to claim 25 wherein administration via foliar is done by spraying.
    [27]
    27. Aqueous composition according to any of claims 21 to 26 which further comprises at least one other active ingredient selected from the list consisting of: nutrients, fertilizers, pesticides, insecticides, acaricides, herbicides, fungicides, growth regulators, or any combination thereof .

     DRAWINGS
    Fig. 1A

     Fig. 1B

     Fig. 1C

    Fig. 2A
    Fig. 2B

    Fig. 2C
    Fig. 2D

     Fig. 2E

    Fig. 3
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    同族专利:
    公开号 | 公开日
    ES2636363B2|2018-05-07|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO1997020034A1|1995-11-30|1997-06-05|Life Technologies, Inc.|Auxinic analogues of indole-3-acetic acid|
    US20080227640A1|2004-05-12|2008-09-18|Bayer Cropscience Gmbh|Plant Growth Regulation|
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