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    • 专利摘要:
    IQ 2G SAM MAN ÜRAG A method of sorting somatic plant embryos ai the after-growth pottery is described, comprising the steps of: (a) filling a newly formed somatic plant embryo and a liquid in a vessel (i2); (b) agitating the liquid and the sorrel plant plants at a constant rate to suspend the somatic plant embryos in the liquid and sorting the somatic plant embryos according to the germination potential, the liquid and the sorriatic plant embryos being stirred in a vortex arrangement; (o) distributing the sornatic plant neonates (s) from the vessel in a liquid stream over a period of time, the somatic plant fluids being digested later in the time period having a germination potential is greater than the germination potential of somatic plant nebrons digested earlier in the time period; and (d) depositing the somatic plant embryos on a substrate (ast
    公开号:SE538644C2
    申请号:SE1351518
    申请日:2013-12-18
    公开日:2016-10-11
    发明作者:N Cootsona Craig
    申请人:Weyerhaeuser Nr Co；
    IPC主号:
    专利说明:

    THE HAZARDOUS DEFINITION OF PLANT EMBROIDERY ëäkgtmd Modern silver culture often requires the planting of a large number of genetically identical plants that have been selected to have beneficial properties. Production of new plants by sexual reproduction, which produces botanical seeds, is usually not feasible. Asexual reproduction, through the cultivation of somatic or zygotic embryos, has been shown for some species to produce a large number of genetically identical embryos, each with the ability to develop into a normal plant.
    Somatic cloning is a process for producing genetically identical plants based on any plant tissue other than male and female diameters. In a somatic cloning approach, plant tissue is grown in an initiation medium that includes hormones, such as auxins and / or cytokinins, to initiate the formation of embryogenic tissue, such as embryogenase suspender masses, capable of developing into somatic embryos. The embryogenic tissue is then further cultured in a proliferating medium which promotes proliferation and mass production of the embryogenic tissue. The embryogenic tissue is then cultured in a development medium such as promoter development and maturation of cardiac somatic embryos which, for example, are placed on a germination medium for the production of germs and then transferred to soil for further growth, or alternatively placed in seeds and sown in soil where they germinate. seedlings. Manufactured seeds are described, for example, in U.S. Patent Nos. 5,564,224, 5,687,504, 5,701,699 and 6,119,395.
    The typical somatic embryogenesis process is laborious and inefficient. One of the more laborious and subjective steps in the embryogenesis process is the selective harvesting of individual embryos suitable for germination. At the end of the development period, the embryos may occur in a number of stages of maturation and development. Those that are most likely to successfully grow into normal plants are preferably selected using a number of visually evaluated thinning criteria. Usually, one skilled in the art evaluates the morphological properties of each embryo, such as embryo size, shape (eg axial symmetry), leaf development, surface texture , dye and the like, and manually picks out desired 2G embryos using a pinoett and transfers the selected embryos to a germination medium. The linrais process is highly subjective, and the transfer of embryos to the germination medium by hand is still a cumbersome, labor-intensive and ergonomically challenging process. In addition, it results in a significant production bottleneck when the final desired production is on thousands of plants.
    Efforts have been made to use the instrument! image analysis for embryo selection for supplementation or replacement of the visual evaluation performed by technicians. A laborious and complex classification method is described, for example, in U.S. Patent Publication 2OD7 / 026909ö, which describes the classification of plant embryos by applying screening algorithms for digitized images of plant embryos and the absorption, transmittance or reflectance spectra of embryos for determining embryos. likely to develop into germs. U.S. Patent No. 7,610,155 similarly discloses the use of image and spectral data from embryos of known quality to develop a classification model by using a classification algorithm, such as logistic regression (LR) analysis, to classify embryos, such as (i) embryos that are unlikely to germinate; (ii) embryos that can germinate with the help of extra care; and (iii) embryos that will be gromed with minimal care. The classification model is then applied to image or spectral data acquired from a plant embryo of unknown quality, predetermining the probability that the embryo will develop into a germ. Although the determination of the germination potential of embryos using the classification model is a more objective process than the selection of embryos using techniques, such methods involve the use of costly instruments for collecting required images and data for each embryo, as well as extensive studies of embryos of known quality for development. of the model system.
    Thus, there is a need for methods for selecting embryos that are most likely to successfully grow normal plants, which simplifies the process, reduces the need for selection using techniques or uses costly instruments, increases the rate of production to achieve commercial scale.
    The present invention relates to methods for sorting somatic plant embryos according to the germination potential. Summary This summary is provided for the purpose of introducing a variety of concepts in a simplified form, which are further described in the detailed description below. This summary is not intended to identify any characteristics of the patent-pending features, and is not intended to be used as an aid in determining the scope of protection of the patent-pending features.
    Methods for sorting sornatic plant embryos according to germination potential are provided. Each of the methods includes the steps of (a) pacing a newly formed sornatic plant embryo and a liquid in a vessel; (b) agitating the fluid and the somatic plant embryos at a sufficient rate to suspend the somatic plant embryos in the fluid and sorting the somatic plant embryos according to the germination potential, wherein the fluid and the somatic plant embryos are agitated in a vortex arrangement; (c) distributing the somatic plant embryos from the vessel in a liquid flow over a period of time, the somatic plant embryos being digested later in the sub-period having a germination potential greater than the germination potential pre-somatic plant embryos digested earlier in the time period; and (d) depositing the somatic plant embryos on a substrate.
    DESCRIPTION OF THE DRAWINGS The aspects described above and many of the accompanying advantages of the present invention will become more apparent by reference to the following detailed description when taken in conjunction with the accompanying drawings. Figure 1 schematically shows a system which can be used in the practice of the methods according to the present description. Figure 2 shows a diagram of the relative percentages at specific times of the totaiantaiet digested embryos developed for seedlings in category t. 2G. Figure 3 shows a diagram of the reiative proceritandeien at least squares method at specific times of the totaiantaion fordeide Figure 4 shows a diagram of the relative percentages with the time of the totaiantaiet distributed embryos developed for the germs according to category 1. Figure 5 shows a diagram of the relative percentages at specific times of the totaiantai distributed at the embryo. seed substances in the same category t + 2. Figure 1 shows a graph of the relative percentages of the least squares method at specific times of the total pre-embryonic embryos developed into seedlings of category 'i-i-Z'. Figure 7 shows a diagram of the relative prooentnaites with the time of the total dispensed embryos developed into seedlings in category 14-2.
    DETAILED DESCRIPTION Unless otherwise specifically stated, the terms used herein are the same as those skilled in the art of the present specification.
    The term "embryogenic suspensor mass" when used (ESiii / i) refers to embryos at an early stage of the process of proliferation by budding and cleavage. The term "plant embryo" when used refers to a somatic plant embryo. Somatic plant embryos can be prepared by debridement of embryogenic tissue using standard laboratory conditions where the tissues comprising the tissue are separated from each other and induced to develop into very small independent embryos. The term "plant embryo", when used, includes embryos at different stages of development.
    The term "seed" when used refers to an immature pianta which is a highly developed root substance with a narcissistic structure with a single-growing epicoty, both of which can be perceived by the biotic eye, and which are ready for pianizing in soil. The amphibian, for example, usually has an epicoty of about two mm more. The term "singulating" or "singulating" as used herein refers to the method of distributing embryos in the form of individual discrete embryos in a substrate.
    The Somali embryogenesis process is a process for the development of plant embryos in vitro. Methods for preparing somatic plant embryos are known in the art and have been previously described (see, e.g., U.S. Patent Nos. 4,956,666, 5,634,326, 5,636,667, 5,641,382, 5,236,841, 5,264,549, 5,462,657, 5 563 661 and 5 621 126. In general, the somatic embryogenesis process includes the steps of (1) initiating or inducing to initiate imaging of embryogenic tissue, such as an embryogenic dispenser mass (ESM), which is a white mucous mass which includes embryos at an early stage and which has a long, thin-walled suspender connected to a narrow head with dense oytoplasm and large nuclei; (2) proliferation, sometimes called maintenance, for the proliferation and mass production of embryogenic tissue; (3) development for the purpose of developing and forming mature heart-leaf-somatic embryos; , such as separation, slngularlser-ing, stratiflerlng, germination, placement in manufactured seeds and transfer to soil further growth and development.
    At the end of the proliferation step, the embryogenic tissue in the form of embryogenic suspender mass can be transferred to a development medium over a period of time for development into a plurality of heart-leafed embryos. At the end of the developmental period, most of the heart-leafed embryos are of varying degrees attached to and embedded in suspensor tissue and remaining underdevelopedSlvl together with incompletely developed embryos, abnormally shaped embryos, under- or over-human embryos and other parts of non-embryos, as well as other non-embryo embryos. It is important for subsequent normal germination to separate each embryo from the suspender mass and other embryos to obtain a plurality of individual embryos.
    Most individual embryos can be separated from ESh / l by using an aqueous fluid, such as water or an isotonic nutrient solution, to facilitate removal and washing of any unwanted material, such as undersized embryos, tissues and residual ES1V1. After the embryos have been separated from the ESM, the embryos can be further separated 1G apart !! individueiia discrete embryos, which is referred to as "singu! arization", in order to inform the seykttonen of embryos which with the greatest probability germinate successfully tii! normaia piantor.Embryos can be singutarized by inhaling the embryos in a liquid in a single vessel, dissolving the embryos and the fluid from the vessel, and depositing the embryos in the form of individual discrete embryos on a substrate.
    The procedures in the foregoing description relate to !! sorting avornatic plant embryos attt after germination potentialia. Groningspotentiaien ärreiaterad ti !! the somatic plant embryos are separated from a loved one. Each of the methods includes the steps of (a) filling a posterior plantar bridge and liquid into a vessel; (b) agitating the fluid and the somatic plant embryos at a sufficient rate to suspend the somatic plant embryos in the fluid and sorting the somatic plant embryos according to the germination potential; (c) distributing the desomatic plant eyebrows from the vessel in a liquid flow over a period of time, the somatic plant embryos during the time period being distributed in order according to the germination potential; and (d) depositing the sornatic plant myoriones on a substrate. In some embodiments, the somatic plant embryos that have been pre-dried later in the time period have a germination potential that is greater than the germination potential of somatic plant embryos that were pre-dried during the time period.
    In some embodiments, the fluid and somatic plant emiorias are agitated in a vortex arrangement. Without committing to !! any specific theory of the vortex arrangement sort the somatic plant tempyrons attt according to the defystatic properties, e.g., density, shape, buoyancy, and surface stresses according to other physical properties that are related to it! germination potentials.
    In some embodiments, the somatic plant embryos are distributed from the vessel for a period of less than about 10 minutes. about 65 min. In certain embodiments, the time period extends from about 5 minutes to 10 !! about 10 min. In some embodiments, the time period is about 30 minutes. In some embodiments, the time period is about 6G min.
    Somatic plant embryos suitable for use in the methods of the invention may be derived from various plant species such as heist, such as 2G single-celled plants, gyro nosoforms, etc. Conifer embryos are suitable for use in the methods of the invention and may be related to includes, but is not limited to, species within the genera Pinus, Picea, Tsuga, Pseudotsuga, Thuia, Juniperis, Larix, and Sequoia.
    In some embodiments, the diversity of somatic plant embryos is coniferous embryos. In some embodiments, the diverse desomatic coniferous embryos are somatic embryos of the family Pinaoeae. In certain embodiments, the diversity of somatic coniferous embryos is somatic embryos of the southern state stai ("iobioiiy" -taii) .In certain embodiments, the multiforme of somatic coniferous embryos is somatic embryos of Dougias spruce.
    In some embodiments, the methods according to the present description further include the steps of transferring the diversity of somatic plant embryos to a germination medium and culturing the sornatic plant bridges to produce germs.
    Examples The following examples are intended to provide, but are not limited to, the present description. The examples of this example describe an example of a system which may be used in the procedures of the foregoing description, which is shown in Figure 1. Referring to Figure 1, the system incorporates a a programmable yoghurt styrene (PLC) 20, a mechanism 22 used for depositing individual embryos on a substrate 24 and an embryo digestion tube 26. The embryo distribution assembly 10 includes a vessel 12 and a stirring pawl 14. An embryo distribution tube 22 extends into the embryo.
    In operation, the embryos are received in the embryo distribution assembly 1G and deposited in the vessel 12. A suitable liquid, for example pure water, is also fed to the vessel12.
    The vessel 12 communicates with a agitator plate 14 for agitating the liquid in the vessel 12 to a suitable extent for maintaining the embryos in 2G suspension in the fluid in a vortex arrangement. The stirring plate 14 is connected to the above-mentioned PLC 20 for automatic adjustment of the degree of stirring that takes place. The PLC is programmed in such a way that the agitation speed is reduced inwardly, but to a different degree, when the liquid level in the vessel 12 drops as the embryos are distributed from the vessel 12 through the embryo digestion tube 26.
    The embryo distribution tube 25 extends along the vessel 12 and the mechanism 22. Embryos are transported from the vessel 12 to the mechanism 22 by means of a fluid flow through the embryo distribution tube tube 25. The velocity of the fluid flowing through the tube tube 26 may be from 250-550 rpm / min. The feed rate of the embryos through the tube 26 may be 250-450 embryos / min. The embryos abut the mechanism 22 and are deposited on a substrate 24. The substrate 24 may be mounted in a frame.
    Example 2 In this example, the germination potential of somatic embryos is observed as a function of the order in which the embryos are distributed from a core and deposited on a substrate.
    Using an embodiment of the system described in Figure 1 in the description system, embryos of gui southern state were suspended in about 6.5 liters of water in a vessel and stirred at 22 Hz. Liquid was removed from the vessel at a rate of about 250-300 ml / min. The embryos were digested from the vessel at a rate of 250-450 embryonicimine and deposited on a number of substrates mounted in frames for a period of time. Three time periods were studied, as shown in Tabeii 1.
    Tabeii 1 WW WTidsperiod Ram Totait Kärt Omrörings- Kariramnummer voiym mi hastighet Gmrörings-i (Hz) ííííííí W biandningstid1 1}}}}} W 1 6590 22 ö: 04: 521 2 2 61:86 22 Ozüöïöö1 3 3 5858 22 Wö; O6: 5 1 4 4 5559 22 011381101 5 5 5238 _ 22 0109: 061 6 6 4963 22 W 9710: 032 1 7 36307 ¿22 0: 25: 4ö2 2 8 5954 v22 61: 26: 502 3 9 5560 22 0127 : 552 4 1QW 5290 22 O: 28: 522 5 11 5018 j 22 0: 29: 5OWW2 6 12 4757 22 _ 0131: 383 1 te WWWWWWW Wszsz 22 osams3 2 14 5934 22 O: 5ö: 4O3 3 15 5636 22 (2571453 4 16 5333 22 O: 58: 473 íííííí W 5 17 5950 22 0591593 6 18 j 4766 22 1111: 01 Siffror i fetstii betaktnar vandpäfyiiningar.
    For time period 1, the embryos were digested from the vessel for a period of time beginning after about 5 minutes and continuing for another island minute. For time period 2, the embryos were digested from the vessel for a period of time beginning after about 25 minutes and continuing for another 5 minutes. For time period 3, the embryos were digested from the vessel for a period of time beginning after about 55 minutes and continuing for another 5 minutes. The embryos were deposited on the substrates ("frames") with a density of about 200-409 embryonic substrates. The embryos were conditioned overwater and then transferred in large quantities to a germination medium. efforts were made to screen embryos based on some criteria. fi) The embryos were cultured for a period of time sufficient for the production of seedlings. Results The seedlings were graded according to certain criteria: category t - bipolar and an epicotyl of 5 mm; category 2 - bipolar; category 3 - root only; category 4 - epicotyl only and category 5 - dead or unchanged. The data analysis was performed as follows: the proportional responses were modeled using a generalized linear mixed model with binomial distribution and logit link. The log transform was used to obtain homogeneous variance. credit values and confidence intervals were transformed back to the natural scale.
    The results are shown in Tables 2 and 3 and in Figures 2-7.
    It was found that the average percentage of the total number of distributed embryos that developed into germs according to category 1 (when represented by "P-1") increased linearly with time, ie P-'l during time period 1 <Pf during time period 2 < Pl during time period 3 (values not shown). the values in Table 2 summarize test results where the combined mean value for germs according to category t is compared.
    Table 2: .Comparison of estimated averages at germination (category t) Time period N Test at d = 0.16'i C2 W * B3 A The column "test at oi = 0.10" summarizes test results where combined averages are compared. Averages with different symbols are statistically different at oi = 0.19.
    As shown in Table 2, the differences in the average percentage of the total number of distributed embryos developed into the category t germs during each time period were statistically significant. The relative percentage shares and the relative percentage share values according to the least squares method are also shown in Figure 2 and Figure 3, respectively. In Figure 3, the error bars represent 96% confidence limits. Figure 4G shows a diagram of the relative percentages of the total number of distributed embryos with time that developed to the germs according to category t. Figure 4 shows that the percentage of total number of distributed embryos that developed into germs according to category in increasing line with time .
    The percentage of embryos that developed into a frog-like category in as a function of the order in which the embryos were distributed from a male and deposited on a substrate was found to be statistically significant (a p-value of QOGÛS).
    It was also found that the average percentage of the total number of distributed embryos developed into the germs according to category ll + 2 (represented by "P-1 + 2") increased linearly with time, ie P-'1 + 2 during the time period 'in <P-'+ 2 during time period 2 <P-'l-Z during time period 3 (values not shown). the values in Table 3 summarize test results where combined mean values for germs according to category '1 + 2 are compared.
    Table 3: Estimated average care for germination (category H2) oon comparisons Time period Test at oi = 9.10 gggg Wi g H C2 B3 A The column "test at oi = 0.10" summarizes test results where combined average values are compared. Mean values with different symbols' are statistically different at d = 0.10.
    As shown in Table 3, the differences in the average percentage of the total number of distributed embryos that developed into Category 1 + 2 seedlings at each time period were statistically significant. The relative percentages and the relative percentages of co-care according to the least squares method are also shown in Figure 5 and Figure 6, respectively. Figure 1 represents the error bars 90% confidence limits. Figure 7 shows a diagram of the relative percentage data with time for the total number of distributed embryos that developed into germs according to category i + 2. It can be seen from Figure 7 that the prooentuelia proportion of the total- 1G 2G 12 the number of distributed embryos developed into seedlings according to category'-i-Z increased linearly with time.
    The percentage of embryos that developed into a seed-like category ^ i + 2 as a function of the order in which embryos were distributed from a vessel and deposited on a substrate was found to be statistically significant (p-value <QOOG1). In this Example 2, embryos from the vessel were distributed over time periods of up to one hour. It was expected that the percentage of embryos distributed over time periods exceeding one hour ooh which developed into germs of category t or category '1 + 2 would be higher than the percentage of embryos distributed previously developed into germs of category 1 or category' l + 2r It is also expected that the linear nature of the increase would continue for time periods exceeding one hour.
    The results described above were surprising. It was believed that the agitation process itself, or the length of time spent embryonic in the fluid in the vessels, would have a detrimental effect on the embryos and ultimately on germination. Therefore, it was expected that embryos that were previously distributed would have a higher germination potential than embryos that were distributed later. However, it was unexpectedly discovered that embryos that were distributed later had a higher germination potential than embryos that were distributed earlier and that the increase in germination potential was linear as a function of time.
    Although illustrative embodiments have been shown and described, it is to be understood that various changes may be made therein without departing from the spirit and scope of the invention.
    权利要求:
    Claims (10)
    [1]
    A method of sorting somatic plant myorvon at the aftergrowth potential, comprising the steps of: (a) pacing a diverse somatic plant embryo and monovalent in a vessel (t2); (b) introducing the liquid and the somatic plant tumors at a sufficient rate to suspend the sornatic plant embryos in the liquid and sorting the somatic plant prionates according to the germination potential, the liquid and the somatic plant embryos being stirred in a vortex; (c) distributing the somatic plant embryos from the vessel in a liquid period over a period of time, wherein the somatic plant embryos digested later in the time period have a germination potential greater than the germination potential of somatic plant embryos digested earlier in the time period; and (d) depositing the somatic plant embryos on a substrate (24).
    [2]
    A method according to claim t, wherein the time period extends from less than 1G min to about 65 min.
    [3]
    A method according to claim 2, wherein the time period ranges from about 5 minutes to about ten minutes.
    [4]
    A method according to claim 2, wherein the time period is about 3G min.
    [5]
    A method according to claim 2, wherein the time period is about 6G min.
    [6]
    A method according to claim 1, wherein the sornatic plant embryos are distributed from the vessel (12) at a liquid period rate of about 259-450 embryos / min. 14
    [7]
    The method according to claim 1, wherein the plurality of sematic plant embryos are sematic conifer embryos.
    [8]
    The procedure according to claim 7, wherein the plurality of sematic needle embryos are semantic embryos of the family Pinaceae.
    [9]
    The present claim 8, wherein the multiplicity of Sernatian conifer embryos is somatic embryos of the southern state ("iobioiiy" -tat).
    [10]
    The present claim 8, wherein the diversity of somatic coniferous embryos is somatic embryos from Deugiasgran. A method according to claim 1, further comprising the step of transferring the diversity of the sematic plant embryos to a germination medium. The method of claim 11, further comprising odiation of the sernatic plant embryos on the branching medium for beading crops.
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    同族专利:
    公开号 | 公开日
    SE1351518A1|2014-06-29|
    BR102013033765A2|2016-08-02|
    US9320209B2|2016-04-26|
    US20140186956A1|2014-07-03|
    CA2836615C|2016-11-01|
    CA2836615A1|2014-06-28|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US20040072143A1|1998-06-01|2004-04-15|Weyerhaeuser Company|Methods for classification of somatic embryos|
    US9040301B2|2009-10-09|2015-05-26|Georgia Tech Research Corporation|Separator device, deposition device and system for handling of somatic plant embryos|
    AR089280A1|2011-12-29|2014-08-13|Weyerhaeuser Nr Co|AUTOMATIC SYSTEM AND METHODS TO SEPARATE AND ISOLATE EMBRYOS FROM PLANTS|US9138750B2|2011-12-29|2015-09-22|Weyerhaeuser Nr Company|Spray apparatus and method for separating plant embryos|
    EP2898547A4|2012-09-18|2016-04-27|Glo Ab|Nanopyramid sized opto-electronic structure and method for manufacturing of same|
    US10883081B2|2016-12-20|2021-01-05|J. D. Irving, Limited|Systems, methods and apparatuses for processing plant embryos|
    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    US201261632433P| true| 2012-12-28|2012-12-28|
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