EA006658B1 - Method for producing apomictic plant - Google Patents

Abstract

The present invention relates to the induction, stabilization, and control of apomixis (asexual seed formation) in angiosperms for the purpose of producing, improving, and economically using superior-yielding agamic crops (crops possessing a capacity for both sexual and apomictic seed production). "Stabilizing" apomixis refers to processes that either convert a facultative apomict to obligate apomixis or confer mechanisms to a facultative apomict that prevent, during facultative sexual seed formation, the recombination of genes that cause apomixis such that progeny produced sexually from said facultative apomict inherit the allelic combinations required for apomixis. More specifically the present invention relates to (i) the production of two or more sets of genetically divergent sexual lines, either inbred or outcrossed, which sets of parental lines remain homozygous for genes that cause apomixis when hybrids are produced by crossing a member of one of such sets of lines with a member of another set, (ii) the hybridization of sexual inbred or outcrossed parental lines to produce apomicts, (iii) the stabilization of such apomicts through cytogenetic or molecular modifications, (iv) the improvement of apomicts by breeding or genetically engineering parental lines or apomicts, (v) the modification of facultative apomicts to abort female meiosis resulting in obligate apomixis except when a recombinant DNA that aborts meiosis is inhibited, and (vi) the modification of facultative apomicts to remain facultative except during inducible expression of a recombinant DNA that causes obligate apomixis by aborting meiosis. The various facets of this biological operating system may be selectively combined for the efficient production, improvement, and use of agamic crops.

Description

BACKGROUND OF THE INVENTION

The present invention relates to the consolidation of hybrid strength (heterosis) and other traits through apomixis (seed formation not associated with the reproductive process) in flowering plants (angiosperms). More specifically, the invention provides methods for stabilizing apomixis in naturally occurring or artificial facultative apomicts (plants capable of sexual and apomictic reproduction) so that sexually derived offspring, which are sometimes produced optionally from such apomictic plants, tend to become apomictic, like maternal a plant, despite being otherwise genetically recombined, instead of being sexual revertants. The present invention also provides methods for “controlling” apomixis in naturally occurring or artificial apomicts in such a way that such apomicts exhibit obligate apomixis (inability to sexually form seeds), obligate apomixis other than those induced to be optionally apomictic, or facultative apomixis other than those induced to be Obligatory apomictic. This invention uses genetic, cytogenetic and molecular modifications to prevent genetic recombination between loci that is dangerous for the expression of apomixis (stabilization of apomixis), and controls the percentage of seeds that are received apomictically by controlling the frequency of sexually transmitted seeds in naturally occurring or artificial optional apomicts (control of apomixis).

The types of apomixis related to this patent application cause seed formation asexually. Thus, the seeds of apomictic plants contain embryos, which are genetic clones of the mother plant. Such forms of apomixis include adventitious (incremental) embryo and gametophytic apomixis (hereinafter referred to as apomixis), which are usually divided into aposporia and diplosporia. 8.E. Akkeg & b. DSG11pd. Lrosh1h1k ίη P1ap1k (SCS Rgekk 1992) (hereinafter, Questionnaire & 1g1шд).

The developmental signals responsible for apomixis precede megasporogenesis by inducing the formation of a precocious germinal sac either from the mother cell of the megaspore (MMS) (diplosporia), or from somatic nuccellus cells (aposporia). Fertilization is also preceded by fertilization, which often happens before the stigma becomes susceptible to pollen. The fluctuation in the intensity of the signals responsible for apomixis takes into account the optional expression of sexual reproduction among apomictic plants. Consequently, in most apomicts, a certain percentage of seeds produced by a separate apomictic plant will form sexually and this percentage is often influenced by environmental factors. Akkeg & 1eg1shd. With diplomas such as Ayeppap, signals for the formation of a precocious embryonic sac occur very early, completely preventing meiosis. In Taghasit type diplosporia, the signals of the formation of the embryo sac are less early and affect the MMS after meiosis has begun. In case of Hspascality type aposporia, the nucellus cells are affected by early and ectopic (non-specific) signals that induce the embryo sac, and affected somatic cells of the nucellus undergo three cycles of endomitosis to form an 8-nucleus embryo sac. With aposporia of the Raschit type, only two cycles of endomitosis occur, leading to the formation of mature 4-nucleus embryo sacs. In adventitious embryo, embryos are formed from cells, not from an egg, including nucellus cells, integument (integuments), synergides, and antipodal cells. Akkeg & 1eg1shd.

Technologies that induce, stabilize, and control the expression of apomixis in crops have the potential to revolutionize plant breeding and become essential for a competing global agricultural business. With these methods, breeders will “clone” highly desirable plants (showing heterosis, transgenic traits, and the like) through their own plant seed — generation after generation. The increase in yield resulting from the consolidation of heterosis of inbred crops, such as wheat (15%) and rice (35%), will be economically used on a large scale for the first time, which will give Apomixis a huge worldwide commercial significance. Since cloning occurs through seed, apomixis may become the most valuable effective plant mechanism for transferring biotechnological and productive achievements to marginal (small) farm lands in the developed world and poorly endowed natural resources to farms in developing countries. In the 21st century, apomixis may be among the most valuable genetic tools for plant breeders. At a recent Apomixis conference, the following conclusion was reached: “The prospect of introducing Apomixis into sexually propagating cultures provides such revolutionary opportunities to justify the ongoing international scientific effort. If apomixis formed with a sufficiently high degree of flexibility, then its impact on agriculture could be deep in nature and extremely wide in scale. ” Thye Be11adu Arot1x1k SopGhepsse. \ νΐιν ίκ Arot1x1k 1trog1ap1 1o Adpsiig (1998) (L1p: // L1She, yaguagf ebi / arot1x1k / aro1esy.Yt1).

Over the past 100 years, four methods have been proposed for the inheritance of apomixis: chromosomal non-homology (broad hybridization), quantitative inheritance, simple inheritance, and

-1006658 complex inheritance. The hypothesis of chromosomal nonhomology, put forward by A. Ernst, Bacillus hypera ak, Itkasie bet Arodasch 1t ΡΠζαηΐΌίοΙι (P1kieg, 1epa 1918), states that apomixis is a function of chromosomal nonhomology and is one of several cytogenetic widespread anomalies. According to this theory, apomixis is not directly controlled by genes, but is the result of divergence in the structure of the chromosome. This hypothesis is no longer considered fair, mainly because apomixis is found in plants whose chromosomes appear homologous. EU Sagtap, Akupsygopoik Ehrtekkup οί AirNsa1e Sepek ίη Updateukt Mau Saike Arot1h1k, B1krogu, Teiakrotu, apb Ro1uetytuopu, 61 Vu1. 1. bshpeap 8os. 51-94 (1997).

The hypothesis of a quantitative method of inheritance is also considered invalid. In the mid-20th century, this was confirmed by Müntzing, who believed that apomixis occurs as a result of exact balance from several to many recessive genes, and Powers, who believed that recessive genes lead to three main components of apomixis: disruption of meiosis, the formation of an apomictic embryo sac and parthenogenesis. Akkeg & 1ypd.

Over the past 40 years, most apomixis scholars, including Vakyaga, NoDeg. 8au1bap, 8yetgaoob and Nag1ap (Besheu, Nogler, Savidan, Sherwood and Harlan) confirmed the hypothesis of simple inheritance, namely, that one or two dominant genes lead to apomixis. Akkeg & 1ypd. Initially, this conclusion seems convincing that Mendelian analyzes repeatedly produce a simple ratio of inheritance splitting, for example, the 1: 1 ratio of apomictic offspring to sexually derived offspring is often produced by crosses between sexually propagated plants and apomictic plants. Υ. 8au1bap, Arot1h1k: Sepeysk apb Vteebshd, 18 P1ap1 Vgeeb. Wow. 13-86 (2000). However, despite continued efforts, not a single gene responsible for apomixis has been identified or isolated.

In the late 1990s, the hypothesis of asynchrony of double genes or the theory of flower asynchrony resulting from hybridization was proposed for the evolution of apomixis (hereinafter, NRtheory). RS.Sattap, 61 Vyu1. 1. Lpteap 8os, 51-94 (1997). It implies complex inheritance and is based on a synthesis of concepts from various fields of biology. According to this hypothesis, the method of inheriting apomixis is not simple; and it’s not just quantitative, at least not with the standard way of observing quantitative inheritance. On the contrary, it is complex and is most explainable through a series of five principles that are built on top of each other. The first three principles are published, EU. Sagtap, 61 Vyu1. 1. Yppeap 8os. 51-94 (1997), and are summarized below. The last two principles contain unpublished concepts new to the present invention and are presented here.

The first principle, apomixis is a hybrid phenotype divorced during individual development. Apomixis is divorced, not intermediate, from its parental female reproductive phenotypes, which for convenience are designated by the parental phenotypes A and B. Plants showing phenotypes A or B undergo normal sexual reproduction. Phenotypic differences between A and B were detected cytoembryologically using microscopic techniques known in the art. They (phenotypic characters) are simply (easily) not observed and for this reason have not been described previously.

The second principle, the parental phenotypes A and B are clearly different from each other with respect to the time periods during which meiosis, embryonic sac formation and embryo occur, relative to the entire period of development of the ovule.

The third principle, parental phenotypes A and B are themselves quantitatively inherited. Therefore, almost obligate apomixis, in cases where most of the ovules of this plant produce functional apomictic embryo sacs, is expressed due to polygenic heterozygosity. In populations of agamic complexes (populations of interbreeding of sexual and apomictic species) there are multiple alleles for many of the critical loci, i.e., critical loci are polymorphic. The polygenic heterozygosity responsible for the almost obligate apomixis involves, in particular, divergent alleles, which are preserved in natural populations due to natural selection. Conversely, facultative apomixis, in which sexually-formed seeds and apomictic seeds usually develop on the same plant, occurs when some of the more critical loci required for obligate apomixis become homozygous (or acquire alleles that encode similar developmental programs ovules) through genetic segregation.

Effective procedures based on NRA theory are described for creating facultatively apomictic plants from sexually propagated plants. EU Sagtap, MeShobk Roth Rtobisshd Aro1shsys P1a-k, \ νϋ 98/33374 (1998) (here is given by reference). These methods are used to create highly apomictic plants that may or may not be genetically stable as defined above. The solution proposed in νθ 98/33374 is the production of highly apomictic plants, that is, reducing, as far as possible, the case of seed formation by sex in apomictic hybrids, by identifying or producing (through selection) pairs of parental lines that are suitably divergent in their female reproductive programs at

-2006658 provided that facultative sexual development in facultatively apomictic hybrid offspring is minimized. Artificial apomicts produced in this way can be used as apomictic hybrid lines from several to many generations before the grown seed becomes unsuitable for reseeding due to the dangerous contamination of the seeds of sex revertants. Seeds infected with revertants are products of genetic segregation and their presence impairs agronomic value. This situation may be similar to mixing the worst E ₂ and seeds of later generations with the elite Ε ₁ hybrid seeds in the conventional seed production program. The result would be an agronomically worse product. νΟ 98/33374 did not present the subject of stabilization and control of apomixis. Consequently, methods for modifying apomict, if already created, were not provided.

In view of the foregoing, it would be advantageous to provide methods that allow the improvement of apomictic lines that are obligate, obligate if not induced to be optional, or optional, if not induced to be obligate. The apomictic line can be improved by inducing facultative apomixis, traditional selection strategies, and then returned to an obligate apomictic state over many years of field cultivation. It should be appreciated that these and other advantages of the present application for invention (discussed below) represent major successes in this field.

SUMMARY OF THE INVENTION

The present invention is the provision of a new breeding method that offers, stabilizes and controls apomixis in order to simplify the production of hybrid seeds, so that all angiosperm cultures can be used as hybrids.

Another objective of the present invention is the provision of specialized methods of plant breeding for the successful improvement of such apomicts.

Another objective of the present invention is the provision of methods that control apomixis by converting an optional apomict, which was previously or was not improved by plant breeding or genetic engineering, into an obligate apomict, thereby preserving its genotypic and phenotypic properties as long as possible. These and other tasks can be solved by providing a method for producing an apomictic plant exhibiting increased genetic stability of apomixis from an angiosperm apomictic parent plant that exhibits apomixis genetic instability, which measures the degree of genetic instability of the apomixis of the parent plant;

if the parent plant shows genetic instability of apomixis, obtaining one or more descendant plants from the parent plant, where each of the descendant plants contains a double chromosome set of the parent plant;

measuring the degree of genetic instability of the apomixis of one or more flowering plants;

comparing the degree of genetic instability of the apomixis of one or more plants of the flow with the degree of genetic instability of the apomixis of the parent plant; and selection of at least one of one or more descendant plants exhibiting increased genetic stability of apomixis compared to the parent plant, where one or more descendant plants are obtained by treating the parent plant with a spindle inhibitor, where the spindle inhibitor is colchicine, where one or several descendant plants are obtained by culturing the parent plant or part thereof in tissue culture, where one or more descendant plants are obtained by self-pollination of the parent th plant or by crossing parent plants with another plant of the same species, genus or family, and form a first progeny; and counting the chromosomes in the first progeny and selecting an amphiploid or partial amphiploid having all the chromosomes from the parent plant in the case of selfing of such a parent plant, or all chromosomes from the parent plant and one or more sets of chromosomes from another plant of the same species, genus or family, or all chromosomes from another plant of the same species, genus or family, and one or more sets of chromosomes from the parent plant if the parent plant is crossed with another plant of the same species, genus or family.

The method further includes backcrossing a partial amphiploid with a parent plant or another plant of the same species, genus or family and obtaining second offspring from them and selecting an amphiploid having an equal (even) level of ploidy, where the partial amphiploid is an euploid or aneuploid, where the amphiploid is an euploid or aneuploid.

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The method further includes increasing the fertility of the selected offspring plants by self-pollination or hybridization with another plant, obtaining seeds from them, sowing said seeds, and selection of sexually-obtained apomictic hybrid plants that detect an increased frequency of non-reduced egg fertility or parthenogenesis.

Another aspect of the present invention is a method for producing an apomictic segmented allopolyploid plant exhibiting increased genetic stability of apomixis from an angiosperms interspecific hybrid plant that exhibits genetic instability of apomixis, comprising measuring the degree of genetic instability of apomixis of an intersortic hybrid plant;

if an intersortic hybrid plant shows genetic instability of apomixis, obtaining one or more descendant plants from an interbreed hybrid plant, where each of the descendant plants contains a double chromosome set of intersortal hybrid plants, leading to the appearance of one or more apomictic segmented allopolyploid plants;

measuring the degree of genetic instability of the apomixis of one or more apomictic segmented allopolyploid plants; and comparing the degree of genetic instability of the apomixis of one or more apomictic segmented allopolyploid plants with the degree of genetic instability of the apomixis of an intersortic hybrid plant to check whether the genetic stability of the apomixis of one or more apomictic segmented allopolyploid plants is increased compared to the interspecific hybrid plant.

The method further includes increasing the fertility of one or more apomictic segmented allopolyploid plants by self-pollination or hybridization with another plant, obtaining seeds from them, sowing said seeds, and selecting sexually obtained apomictic segmented allopolyploid plants that show an increased frequency of non-reduced fertility of egg cells or parthenogen.

Another aspect of the present invention is a method for producing an apomictic allopolyploid plant exhibiting increased genetic stability of apomixis from an angiosperm interspecific hybrid plant exhibiting genetic instability of apomixis, comprising measuring the degree of genetic instability of apomixis of an interspecific hybrid plant; and obtaining one or more apomictic allopolyploid offspring plants from said interspecific hybrid plant and selecting an apomictic allopolyploid plant offspring exhibiting increased genetic stability of apomixis.

The method further includes increasing the fertility of the selected apomictic allopolyploid offspring plant by self-pollination or hybridization with another plant, obtaining seeds, sowing said seeds, and selecting sexually obtained apomictic allopolyploid plants that show an increased frequency of unreduced egg fertility or parthenogenesis. Another aspect of the present invention is a method for producing an apomictic plant exhibiting increased genetic stability of apomixis from an angiosperm, intraspecies, interspecific or interspecific hybrid plant exhibiting genetic instability of apomixis, comprising measuring the degree of genetic instability of apomixis of an intraspecific, interspecific or interspecific hybrid plant; and obtaining apomictic autopoliploid, segmented allopolyploid or allopolyploid offspring plants of an intraspecific, interspecific or interspecific hybrid plant and selection of an apomictic plant exhibiting increased genetic stability of apomixis.

The method further comprises increasing the fertility of the selected apomictic plant by self-pollination or hybridization with another plant, obtaining seeds, sowing said seeds and selecting sexually obtained apomictic descendant plants that show an increased frequency of underestimated egg fertility or parthenogenesis. Another aspect of the present invention is a method for the genetic stabilization of apomixis of an angiosperm, apomictic, intraspecific, interspecific or interspecific hybrid plant exhibiting genetic instability of apomixis, comprising measuring the degree of genetic instability of apomixis of an intraspecific, interspecific or interspecific hybrid plant and obtaining a descendant plant from within the specified apomicis intersortial or characterized by an odd increase in ploidy, pr why female meiosis is usually interrupted, leading to almost 100% formation of apomictic seeds.

The method further includes increasing the fertility of the descendant plant by self-pollination or hybridization with another plant, obtaining seeds from them, sowing seeds and breeding apomictic plants that are sexually transmitted, which show an increased frequency of non-decreased egg fertility or parthenogenesis. Another aspect of the present invention is

-4006658, there is a method for genetic stabilization of apomixis of an angiosperm, apomictic, intraspecific, interspecific or interspecific hybrid plant exhibiting genetic instability of apomixis, comprising measuring the degree of genetic instability of apomixis of an intraspecific or interspecific or interspecific interspecific interspecific interspecific interspecific interspecific interspecific interspecific interspecific interspecific interspecific interspecific interspecies interspecies interspecific interspecies interspecies interspecies interspecies interspecies interspecies interspecies interspecies interspecies interspecies interspecies interspecies interspecies interspecies interspecies interspecies interspecies interspecies interspecies interspecies interspecies interspecies interspecies interspecific a plant of the same species, genus or family, and breeding of offspring, where female meiosis is usually interrupted, leading to almost 100% formation of apomictic seeds.

The method further includes increasing the fertility of the selected offspring by self-pollination or hybridization with another plant, obtaining seeds from them, sowing these seeds, and breeding apomictic offspring plants that are sexually transmitted, which show an increased frequency of unreduced egg fertility or parthenogenesis.

Another aspect of the present invention is a method for producing an apomictic plant exhibiting increased genetic stability of apomixis from an angiosperm apomictic parent plant that exhibits genetic instability of apomixis, comprising measuring the degree of genetic instability of apomixis of the parent plant;

obtaining a descendant plant from the parent plant by doubling the number of chromosomes of the specified parent plant or introducing chromosome aberration in which recombination is suppressed, so that allele segregation during meiosis is inhibited; and measuring the degree of genetic instability of the apomixis of the descendant plant to verify that such a plant exhibits increased genetic stability of the apomixis compared to the parent plant, where said chromosomal aberration involves translocation, where said chromosomal aberration involves inversion.

The method additionally provides for increasing the fertility of a descendant plant by self-pollination or hybridization with another plant, obtaining seeds from them, sowing of said seeds, and selection of sexually obtained apomictic descendant plants that exhibit an increased frequency of unreduced egg fertility or parthenogenesis.

A brief description of several types of graphic materials

FIG. 1 shows the stages in the evolution of agamic complexes. The stages include ecotypic differentiation before apomict formation, the formation of stage I apomicts through hybridization resulting from secondary contacts, the formation of stage II apomicts through structural (karyotypic) stabilization (usually involving polyploidization), and the formation of mature, ecologically diverse agamic complexes (stage III) through optional outcrossing, initially with sexual related forms and secondarily with other related apomicts.

FIG. 2 shows the stages of a megasporocyte (MMS) and dyad in the pistils of the genital diploid Ap1eppap gasecho / a and A. ityshaia. Differences between species in the length of the integument at the stages of MMS and dyad are noted. Arrows = MMC or dyad elements. Lines = magnitude of growth of integument (compare with Fig. 3).

FIG. Figure 3 shows the average values of the lengths of the integument and ovule (actual measurements, the lower left and right figures, and the percentage elongation of the mature integument and ovule, upper left and right figures) at the stages of the dyad, 2-core germinal bag, and mature germinal bag for nine diploid apomictic AI1eiiapa gokea precursor cells. The data in FIG. 8 to 11 depict variability in ovule development programs among plant ecotypes. This natural ecotypically induced variation has never been characterized before, and it is a prerequisite for the occurrence of apomixis in naturally occurring and artificial hybrids.

FIG. 4 shows one of several measurements of the duration of meiosis among 17 ecologically different zoned races and varieties of 8ogd. The duration of meiosis is only one of several types of ecotypically derived interracial / interspecific variability found in ovule development programs preserved by various ecotypes of flowering plants. The columns depict the length of time between the stage of the dyad and the time at which the formation of the embryo sac was initiated (as a function of the growth of the internal integument), i.e., the low and high columns depict lines with a very small and very large delay, respectively, between meiosis and the formation of the embryo sac .

FIG. 5 shows the maturation of a flower bud during megasporogenesis (female meiosis) as the measured average lengths of internal integument (depicted as the relative elongation of mature integument as a percentage) at the stage of meiosis dyad for the parent lines of three hybrids. Apospore buds and / or an increase in multinucleated apomictic (apospore) embryo sacs were observed in approximately 5% of the pistils of hybrids 5 and 18, whose parents showed a slight difference in the levels of kidney maturation during megasporogenesis. Conversely, multi-core apo

-5006658 mictic (diplospore) embryo sacs plus apospore rudiments and embryonic sacs form approximately 10% of pestles of hybrid 15, whose parents show a much greater difference in levels of kidney maturation during megasporogenesis. Apomictic embryo sac formation in parental lines occurs only occasionally (<0.1%). The parent lines of hybrids 5, 18, and 15 are Eag1y Kag1o / Kagab Loea1, Uy-5049 / A | 5rip (transformed), and Ue511apb / ADga, respectively.

FIG. Figure 6 demonstrates megasporogenesis and development of the embryo sac in the reproductive Ap1epiapa, plus apomictic (diplospore) development of the embryo sac in the artificial Apepiapodiidae (2n = 2x, reproductive) X Apepiapaea (2n = 2x, genital) interspecific hybrids. Approximately 7% of the pistils in the hybrid show the formation of a diplospore embryonic sac. Diplospora was not observed in parental lines. Note to FIG. 3 such that the two parental lines are not highly divergent with respect to the timing of meiosis.

FIG. 7 demonstrates megasporogenesis and development of the embryo sac in the genital 8og§yish plus apomictic (apospore) development of the embryo sac in the artificial hybrid of 8og§1pcs. produced from genital lines. Approximately 5% of the pistils in the hybrid show apospore rudiments and / or apospore germinal sac formation. Diplospora was not found in parental lines. Note to FIG. 3, hybrids producing a low frequency of formation of an apospore germinal sac are derived from parental lines that are not highly divergent in terms of meiosis relative to the overall development of the kidney.

FIG. Figure 8 shows sexual megasporogenesis and the sexual and diplospore development of the embryo sac in the artificial facultatively apomictic amphiploid Тпр ^ аент (2п = 4х), produced from the hybrid Т.1 achit (2п = 2х, sexual) Х T. Ryokit (2п = 2х, sexual). Approximately 50% of the pistils in the hybrid show diplospore embryonic sac formation. Parthenogenic embryo formation from a reduced egg cell was observed cytoembryologically (the presence of a globular stage of the embryo without fertilization of the already formed central cell).

FIG. Figure 9 demonstrates sexual megasporogenesis and sexual and diplospore development of the embryo sac in the artificially stabilized obligate apomictic trispecific triploid hybrid Tprkaeit derived from genital diploids (T.1 achit / T..Po5it // T. chorPo1ep5e). Approximately 80% of the pestles in the hybrid show diplospore embryonic sac formation. The remaining pestles are abortive.

Detailed description

Prior to the disclosure and description of the present methods for stabilizing and controlling apomixis, it should be understood that the invention is not limited to the particular forms, process steps, and materials disclosed herein, since such forms, process steps, and materials may vary somewhat. It should also be understood that the terminology used here is used solely to describe particular embodiments, and is not intended to be limiting, since the scope of the present invention is limited only by the appended claims and their equivalents.

Publications and other reference materials related to this invention, to describe the premise of the invention and provide further details regarding its use, are hereby incorporated by reference. The links discussed here are provided solely for level disclosure prior to the filing date of this application. Nothing here can be construed as an assumption that the inventor is not entitled to date such disclosure by an earlier invention.

It should be noted that, as used in this description and the attached claims, the singular forms a, an and 1ye include plural reference objects, unless the context clearly dictates otherwise.

The following terminology will be used in the description and claims of the present invention in accordance with the definitions given below.

As used herein, “comprising”, “including”, “comprising”, “characterized” and their grammatical equivalents are inclusive, or incomplete terms that do not exclude additional, not listed elements or steps of the method. “Containing” - should be interpreted as including the more limited terms “consisting of” and “consisting essentially of”.

As used herein, “consisting of” and its grammatical equivalents exclude any element, step or ingredient not specified in the claims.

As used herein, “consisting essentially of” and its grammatical equivalents limit the scope of the claim to the materials or steps described and those that do not substantially affect the main and new features or features of the claimed invention.

As used herein, “genetic instability” of an apomictic plant means that the average frequency of sexually-generated seed formation among sexually-derived offspring of such a plant is greater than the average frequency of sexually-generated seed formation of such an apomictic plant.

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As used herein, “stabilization” of an optionally apomictic plant means it is guaranteed that the average frequency of sexually generated seed among the sexually produced offspring of such a plant does not exceed the average sexually generated seed of such an apomictic plant.

As used herein, “complete apomixis” means (1) the preceding megasporogenesis during the formation of the early embryonic sac, (2) the preceding fertilization during the early embryo, and (3) the formation of the endosperm or pseudogamous (through the fertilization of the central cell, but not the egg), or autonomously ( without fertilization of the central cell).

Principles 4 and 5 of the NEA theory of apomixis are as follows.

Principle 4 states that in the absence of structural or karyotypic heterozygosity, offspring of an almost obligate or facultative apomict obtained sexually are usually reproduced sexually, which means that they are sexual revertants. In the absence of structural heterozygosity, the divergent alleles responsible for parental phenotypes break down during the formation of the sex gamete, which sometimes occurs in apomicts, sometimes during megasporogenesis (female meiosis) and usually often during microsporogenesis (male meiosis). In this regard, the loss of apomixis in the sexual E ₂ generation is similar to the loss of hybrid strength in the E ₂ generation of standard hybrid crop varieties. Both are complex polygenic hybrid phenotypes.

Principle 5 states that in the presence of structural heterozygosity, sexually progeny of an almost obligate or optional apomict usually reproduces either apomictically, imitating a homozygous dominant state, or both sexually and apomictic (with a cleavage factor of almost 1: 1), imitating a heterozygous home state. In true reproductively stabilized apomicts, a high frequency of cleavage to the sexual type is prevented by structural (karyotypic) heterozygosity, which includes, but is not limited to, allopolyploidy, segmented allopolyploidy, sexual sterility or paleopoliploidy. Structural heterozygosity is responsible for apomixis, mimicking simple inheritance.

Intraspecific apomictic diploid hybrids, the sexual offspring of which is usually weakly apomictic or completely sexual due to the recombination of the polygenic heterozygosity necessary for apomixis, are stabilized by inducing triploidy, or another odd polyploid level. This results in an almost obligate apomixis. With an unpaired ploidy level, genetically reduced and recombined functional eggs are rarely formed and are rarely fertilized with genetically reduced and recombined sperm, the production of which is very reduced. Therefore, the intragenomic polygenic heterozygosity responsible for apomixis is rarely impaired in polyploid apomicts with an unpaired ploidy level.

Allopolyploidy (polyploidy involving various species) is generally the most convenient mechanism for limiting recombination. In allopolyploid, recombination mainly occurs only within the genomes, not between the genomes. Therefore, the genes responsible for apomixis are maintained through facultative generations in a homozygous state within the genome, but in a heterozygous state between the genomes.

Other cytogenetic mechanisms can be used to prevent recombination within or between whole genomes, or parts of genomes. This application applies to all such mechanisms, including heterozygous inversion or translocation heterozygosity and mechanisms of genetically controlled meiotic triggering.

Fertility levels of interspecific apomictic diploids exhibiting low fertility are increased by polyploidization of either even (e.g. tetraploid, hexaploid, and the like), or odd (e.g. triploid, pentaploid, and the like) levels. Apomictic polyploids created in this way can produce a certain amount of offspring obtained through sexual contact, that is, they are mainly optional apomicts. Such sexually derived offspring are also optional apomicts since the polygenic heterozygosity necessary for apomixis exists between the genomes and not within the genomes. Allopolyploids record reliable intergenomic heterozygosity so that random intergenomic recombination does not affect the allelic composition of the divergent intergenomic locus. Segment-like allopolyploidization is encouraged by the methods of the present invention to enhance pollen fertility, the formation of an unreduced embryo sac and ovum, viability and unreduced parthenogenesis of the ovum.

The development of NEA theory

By combining the NEA theory (all five principles) with the principles of evolutionary genetics, the present inventor developed a new theory about the origin, stabilization and differentiation of natural “agamic complexes” (interbreeding genital and apomictic plants). The theory is presented here and forms the basis for the production of "agamic cultures."

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During the pre-apomixis phase of the theory (Fig. 1), natural selection occurs along latitudinal and other environmental gradients and sexual ecotypes evolve with divergent wide temporal characteristics of egg development. This is what the hybridization resulting from the secondary contacts follows. In this context, the Pleistocene was the only one in the history of angiosperms in which extensive plant migrations took place, V. Nip! 1eu & T. \ Ulb III, Uede! as a result of secondary contacts, in particular, in the mid-latitudinal heterogeneous protected flora. 1.S. Sagtap, 61 Bu1. I. bshpeap 8os. 51-94 (1997).

New hybrids trying to express two or more specifically divergent, long-term flowering developmental characteristics were reproduced as optional stage I apomicts (Fig. 1). Most of these disappearing apomicts were diploids, and most of them had an optional and short-lived apomixis, that is, their offspring produced sexually were obligate sexual, since the balanced multi-locus heterozygosity necessary for apomixis was disrupted by recombination. Consequently, subsequent generations of these short-lived lines contained several apomicts until all apomicts were replaced by sexual offspring. Such a substitution did not occur in apomicts, either with those existing at the time of their formation, or with rapidly acquired stabilizing mechanisms, such as allopolyploidy. Most of the mechanisms that stabilize apomixis involve polypolydia and all of them are very inhibitory and sometimes eliminate the recombination of various heterozygous genes critical for apomixis and related reproductive anomalies.

Stabilized apomicts step II may be periodically included in the BU and B _w hybridization with related apomicts and environmentally divergent sexual relatives to produce heterogeneous agamic complexes stage III (FIG. 1). Many stage III apomicts continue to assimilate, through optional outcrossing with sexual and apomictic relatives, the genetic capacity continues to migrate to new and ecologically different areas. Cd. Woweg, Euphorius ok Ro1ur1o1b Adat1s Sotr1ekhek \ νί11ι Ehatr1ek kgot Atepapa (Ak! Egaseee), 132 Orega Vo !. 53-65 (1996).

The stabilization mechanisms of the genetic inheritance systems responsible for apomixis complete the NEA model of the origin, stabilization and inheritance of apomixis. As for apomixis, the constituent mechanisms (defined below) are not described in the prior art.

According to the NEA theory, the parental sex phenotypes of apomicts are polygenic coadaptants, A.K. Tetr1e! Op, Soabar! A! Ju ap apb Oi! 8oy1e, Sopuetkup Vyuodu: Thye 8s1epse ok 8satsyu apb Eguetkyu 105-116 (8th Akkosk. Shs., 8ibet1apb, MA 1986); V. Zayase, Soabar! Ayop R. eu1K1! Eb, 82 I. Negeb. 89-95 (1991), encoded by unique groups of alleles that act cooperatively to confirm compliance with specific ecotypes adapted to specific environments. Any important recombination between parental genomes, i.e. between this critical multilocus heterozygosity in the optional stage I apomicts, there is a consequence of offspring that exhibits dioeciousness or, at best, a very reduced frequency of apomixis, i.e., sexually derived offspring is sexual or mostly sexual (highly optional). Therefore, without stabilization, stage I apomicts are eventually replaced by sexual segregants, which genetically introduce only sexual offspring into the population.

The vast majority of stage I diploid apomicts that successfully advance to stage II (Fig. 1) stabilize with allopolyploidy or segmented allopolyploidy. The speed at which stabilization occurs depends on the interconnectedness of the parental lines and on certain conditions in the hybridization zone resulting from secondary contacts. One parent line in such areas is more numerous than another. In such cases, pollen from the dominant parent is likely to be involved in _W hybridization (fertilization of unreduced eggs) to form triploids with a 2: 1 genome ratio. On the contrary, the hybrid _W can be formed from unreduced pollen of apomict stage I, which affects the fertility of the dominant parent, which forms the same ratio of the genome 2: 1. Assuming that triploids also form unreduced eggs (showing a tendency toward apomixis) or pollen, a second round of backcrossing involving the same dominant diploid sex parent results in a 3: 1 genome ratio. Such ratios are likely to be frequent among apomicts, and they (ratios) explain the simple ratio of inheritance splitting and the hemizygous cohesion groups inherent in apomixis. On the contrary, triploid may be involved in _H hybridization with another parent, in this case, the ratio of the genome is 2: 2. Other forms of polyploidization, involving unreduced pollen and eggs, or somatic doubling, may give similar results.

Most apomictes are outcrossing perennials, i.e. inbreeding of apomicts with annual apomicts is extremely rare (Akkeg & 1yegp§). Mutation based hypotheses do not explain this observation. On the contrary, the hybridization and outcrossing scheme described above,

-8006658 (Fig. 1) depends on outcrossing and the presence of perennials. At the diploid level of the hybrid, BC1 — triploid and BC ₂ — tetraploid levels, the presence of perennials allows us to analyze numerous genetic recombinations (in pollen). Everyone can provide a genetic prerequisite that discusses varying degrees of viability and facultativeness, and genetic prerequisites that discuss maintaining higher viability.

Outcrossing and the presence of perennials are a feature of families with a high degree of natural hybridization, and L51sgassas. Roasee and Kokasee are often located close to the top. N.0. E11kyapb e! A1., ΌίδΙπόιιΙίοη о £ 8rop! apeoic Р1ап! NLpbk, 93 Prgos. ΝηΙ'Ι Asab. 8s1. I8A 5090-5093 (1996). These three families contain 75% of the total apomictic genus. EU Sagtap, 61 Bu1. 1. bshpeap 8os. 51-94 (1997). On the contrary, apomixis is rarely observed in families that are low in the hybridization score, for example, Vgakkuaseeae, Sopasopaea and Arlaseae. Again, although the hypothesis based on mutagenesis does not explain these associations, they completely coincide with the origin of hybridization and backcrossing described above (Fig. 1).

Since most apomicts are allopolyploids, Akkeg & lg1dd, allopolyploidy is probably the most common form of stabilization of apomixis. Recombination in true allopolyploids occurs only within the genomes. Consequently, loci critical for the high frequency of (almost obligate) apomixis are isolated from intergenomic segregation and an independent assortment, i.e., they retain homozygosity within the genomes, but heterozygosity between the genomes. The progeny formed sexually from the optionally apomictic TT T'T 'genomic allopolyploids, where T and T' are divergent and encode divergent features of the development of the egg, remains apomictic, but is phenotypically variable due to intragenomic recombination involving heterozygous apixis not critical.

If TTT T 'apomicts are propagated sexually, then the polygenic capacity of apomixis (from extremely sexual to almost obligate) often segregates according to the simple Mendelian method. This is due to the fact that cosegregation with a non-recombinant T 'univalent (or a large linkage group), which contain most of the many critical divergent alleles, is required for expression of low to high apomixis frequencies. It seems that many genes that are essential for almost obligate apomixis are found on several chromosomes. Consequently, in crosses between TTTT sex lines and TTT T 'apomictic lines, facultativeness will vary from <10% to> 90% among segregants that are usually classified as apomictic. Υ. 8au1bap, Oepeisk apb IYE / ayop o £ Arot1x1k £ og 1Pe 1trgouetep! o £ Oshpeadgakk (Rashsit ottoman Shsts), Prgos XIV 1n1. Ogakk 1. Sopdg., Exchd! Op, ΚΥ, 1981, 182-184 (1983); 8. Riyk e! a1., Ma1e apb Eetae 8godepekk apb Catelogepekk Arotkys VgasyPapa bn / apya, Vasilya pesitiek apb E1 Nypbk \ νι11ι 8exia1 S1cIser than 1pbseb Tebarbiebpb. 78 Airyuys 19-25 (1994). Such apomicts often approach 50% of a segregated population, i.e. a 1: 1 segregation ratio is often approximate, which is erroneous as evidence of simple inheritance. If the chromosome that contains the most loci critical for apomixis is classified as univalent, as expected in the TTT T 'genomic population, then the transmission frequency will not reach 50% due to the formation of microsatellite. This explains the many cleavage factors that describe <50% of apomixis transmission. Such layers on the simple inheritance hypothesis explain this deviation by tetrasome inheritance with an arbitrary chromatid set. Υ. 8au1bap, Arot1h1k: Opeysk apb Vgeebshd, 18 P1ap! Vgeeb. Keu. 13-86 (2000).

The chromosome assortment in the inherent homologous TTT T 'set for apomixis occurs if all four chromosomes are homologous. During meiosis, each of the three homologous T chromosomes has an equal chance of being associated with its corresponding homeologous T 'chromosome. Therefore, if the locus common to all four chromosomes contains alleles that are different from each other, then all six pairwise combinations of four different alleles will occur randomly, i.e. the chromosome set mimics an autopolyploid.

In many apomicts, most polyploid chromosome sets work genomically as autopolyploid sets, but at least one works as an allopolyploid set. This chromosomal behavior is typical for segmented allopolyploidy. O. 81bbk, ν; · vp; · vyop apb EyoYbop w P1ap! K (Somit1a Ishuegku Rgekk, No. \ ν Wogk 1950); I. 8yepda, Syotokot Ratpd Ajpju apb Oyabg | ya1ep1 Rogtayop sh R1ur1o1bk: Eo 8edtep! The alloplastic set (s) between the genomes supports proportional multilocus heterozygosity, which is mandatory for apomixis.

According to the NEA theory, segmented allopolyploid apomicts evolve from the early stage of interracial autopolyploid apomicts or weakly polyploid TTT T 'or TT T'T' apomicts. Apomixis-critical recombination within a homeologous set (s) of chromosomes is often non-adaptive because it usually results in sterile sexual segregants. Therefore, allelic recombinations, chromosomal aberrations, or even mutations that inhibit recombination within the inherent apomixis homeologic set, cause further alloploidization, O. 81bbk, VaPaΐ ^ οη apb EuoYyop w P1ap! K (Somit1a Iyuegkyu Rhekk, No. \ ν Wogk 1950) of this set, which

-9006658 paradise can be highly adaptive, leading to the accumulation of such modifications. Also, recombinations within homeologic sets that are not strongly involved in the discussed apomixis can be highly adaptive. Such recombinations are initially rare, but with each additional recombination, the similarity between the chromosomes within the homeologous sets increases, i.e. these cytogenetic events autopolyploidize chromosome sets by combining segments from divergent homeologous chromosomes into one chromosome. I. Fucking. 39 ce for te 11761184 (1996). Since this recombination does not lead to sexual segregants, apomict viability increases by eliminating the maladaptive allelic combinations existing in the original hybrid and by the formation of new and adaptive allelic combinations. This mechanism can explain why apomicts in Tpkasit, E. Spaptapeu! A1., Marrtd E | p1okrogopk Λροιηίχίδ ίη Te1gar1o1b Tpkasit: Opera Sepe og 8susga1 Sepek ?, 80 Negebyu 33-39 (1998), Ό. Spaptape11u e! a1., No. p-Meppbyt Tgkppkküp oG Arot1h1k sh Ma1 / e-Tpr5asit Nüpbk Saikeb Lew a Tgkppkkup Kabo EShoyyup, 80 Negebyu 40-47 (1998), Repykeut, R. O / 1a8-Lkt A1., 95 Pr. Νπ1'1 Asab. 8sk I8A 51275132 (1998), at Sepskgik, Ό. Koske, Ap Arokrogu-kres Schs Sepisp Keszczop Сопκ Sopkegueb Be1 ее п Вп Г Г Г е е д (((С (Sepskgik пkapk..) At RepYke! e! A1 ,. 130 Negebkak 1-11 (1999), behave genomically as autopolyploids, do not even undergo recombination in the homologous chromosome set or linkage characteristic of apomixis.

1. 8yepda, 39 Sepot 1176-1184 (1996), convincingly proved that segmental allopolyploidy is eliminated by autopolyploidization at the beginning of the evolution of polyploids that appear as weak allopolyploids or as interracial autopolyploids. Segmented allopolyploid apomict appears to be an exception. Moreover, the optional apomixis associated with segmented allopolyploidy is an interconnected and highly adaptive feature, that is, they act synergistically in the evolution and stabilization of mature extremely successful agamic complexes (Fig. 1).

In nature, there are several seemingly stable, diploid apomicts and some of them are probably stabilized by almost obligate sexual sterility, which prevents segregation. These apomicts can be formed either by intraspecific hybridization of the sexual diploids, or from allopolyploid apomicts by parthenogenesis of reduced eggs. Examples include diploid apomicts at Po1ep1111a, MiXxcd & Miiptd, Tke Mob oG Kergobisop oG Nupbk Be1 \ uep 8xia1 apb Arotuys Ro! Ep! 111a agdep! Ea, 1945 Vo !. No. 1. 49-71 (1945), at H1egoskoe, S. ^ e1tagsk, Arot1x18 apb 8ehia1ku w H1egoskoy aikyakk apb w 3 \ ueb1kk N. Obog! And op EShegep! Ро1ур1о1б Леуе1к, 120 Wok No. 1. 209-235 (1967), at 8bik, A. 1apkip & M. Kouapba, Arot 1x18 a! е ke о 1 1 у б у у е 1 8 8 ог ик ик ик ((((((к (к ощ ощ 1 8 8 8!!!! Kou, Tke Vgeebshd 8uk! Etc. OG 81x 8res1ek OG AgaYk (Vgakkaseee), 82 Integ. 1. In !. 869-877 (1995). In each case, the diploid genomes are divergent, and sex gametes rarely form.

On the contrary, a complete reversal to dioeciousness within one to several sexual generations occurs in dioecious fertile diploid (or weak dihaploid) apomicts. Such unstable apomicts are formed either by interracial hybridization of sex diploids, or from segmented allopolyploid apomicts by parthenogenesis of reduced eggs. Note to the latter case, such stabilized polyploid apomicts of stage I and III can be parental for unstable non-diploid apomicts of stage I (Fig. 1). Unstable apomictic diploids were found in RaikeUit, Ό.υ. Segk! E1 & XV. M. Mkkapes, Op! Ke [pkegkaps oG Arot1kh1k t RaikeUit agdep1a! Um, 115 Vo !. Caζ. 96-106 (1950), in Kapipsi1ik, S.A. №deg, 94 Vo !. Not 1. 411-422 (1984) and possibly by Tketeb, L.T. Euapk & K.V. Kpoh, Epuyotep1a1 Sop! Go1 oG Kergobisyop t Tketeba aikyakk, 17 Hayk !. I. In !. 375-89 (1969), by Vgasyap, T.K. K-stow e! A1., Arot1h1k apb 8ekhia1ku e Elp1o1b apb Te! P1ap! Kergob. 43-52 (1999), and 8gdkit, S.U. Thap e! A1., Arot1x1k t 8gdkit Lshek apb Tkey E1 Rgodeyek, 141 Vo !. Caζ. 294-299 (1980); I.K. Miyu, Arrga1ka1 op! Ke Rgekep! 8! A! Ik oK Kekeagsk op Arot1h1k t 8ogdkit, 64 Sig. 8sk 315-316 (1993), the last of which appears to arise through the hybridization of sexual diploids.

If apomixis was controlled by a simple dominant gene, then approximately 75% (if heterozygous) or 100% (if homozygous) of all sexually transmitted offspring, optional diploid apomicts, should be apomictic. However, such a segregation coefficient has never been observed. Instead, the sexually-derived offspring of facultative diploid apomicts is completely sexual, or only slightly apomictic. These observations are incompatible with simple inheritance, but they are fully consistent with the NEA theory, that is, the recombination of balanced multilocus heterozygosity, critical for apomixis, usually results in sexual offspring. In short, at the diploid level, with optional recombination of weakly homeologous genomes, apomixis is lost. At the polyploid level, homeology is sufficient to limit optional recombination to identical genomes. This mechanism of homeology supports cross-genomic heterozygosity, which often causes apomixis to look like simple inheritance when apomicts are used as male parents in crosses between genders and apomicts.

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As discussed above, these hypotheses, which endorse simple inheritance, explain the 100% reversal of dioecious sexually reproducing offspring of optionally apomictic diploids by stating that the dominant apomixis allele behaves like a recessive lethal in haploid gametes. Therefore, according to this explanation, apomixis cannot be inherited from haploid gametes of diploid apomicts.

In addition to stabilizing certain diploid apomicts, sexual sterility provides additional stabilization to polyploid and aneuploid apomicts. A few examples include (ί) triploid apomicts in Tagahasit, Akkeg & DsgNpd. Epdegoi, Ό.Ά. 8! Ha! 1. In !. 723-728 (1991), in Eir! Ogsht, M.8. Veg! Akko-Vogdek & 1.K. Come on, here is one Sui! Ode! Yuk oG Eira! Ogsht rapyPogpt aiy E. 1i! Eigteisht (Sotrokyaye), 21 Seeie !. Mo1. Vu1. 507-514 (1998), Tprkasit, B1akeu e! A1., Co-Gedged! Yui OG ΌΝΑ Magkegk \ νί! 1ι Tkrkasit EegShyu, 42 Mauyua 363-369 (1997), at Rakra1it, V.L. Vigkoy & M.A. Nikkeu, Su! O1od OG Rakra1it taasasoryu11pt aiyk Ke1a! Yikyr! O R. Schegdeiki ai R. Y11a! A! Um, 159 1i !. 1. R1ai! 8SK 153-159 (1998), and in S1k! Aisye, V. Rahu, P1p1o1y1 / a! Yui Yaige aiy Arot1x1k sh OgOaisyaseee, 128 Vo !. 1. Yii. 8os. 99-103 (1998), (and) in aneuploid apomicts E1utik, EB. Ours, 8bkekhia1 Kergoyisyoi sh Adgorugoi, 10 Negeyyu 129-160 (1956), Ytoshit, 1.A. Kokke11o e! a1., Ishit sagua11yu1 (P1itbadshaseeae), and No. \ ν Eiyetu 8 reeks Ggot! 1e Ba1eaps 1k1aiyk, 56 Ai1ek Ee1 1aishu Vaashso Ee Maipy 23-31 (1998), at Tpkasii ai! Sagtai, unpublished, and (ίίί) in unequal tetraploid (three homologous x = 5 genomes plus one homeologous x = 4 genome) apomicts (nucellular embryo) in No.! Yoksogyit, K. 1oyek, Koyekoshai Yikyui ai Sei! Ps E1kyuyu! ure Euo1i! yui oG N1deyg P1ai! k, 64 Vo !. Ket. 273-289 (1998).

According to the NEA theory, bisporia, tetrasporia, and polyembryony are also polygenically determined, abnormal, and evolutionarily intermediate (hybrid) phenotypes. 1.S. Sagtai, 61 Vyu1. 1. Yesheai 8os. 51-94 (1997). Like apomixis, they occur due to the intragenomic heterozygosity of genes involved in the synchronization of megasporogenesis, development of the embryo sac, and / or embryo. However, unlike apomicts, many bispore, tetraspore and polyembryonic species are diploid and almost all bispore and tetraspore species are completely dioecious. 1.S. Sagtai, 61 Vyu1. 1. Etpeai 8os. 51-94 (1997). Therefore, multilocus heterozygosity, critical for these anomalies, is not stabilized by normal polyploidy or sexual sterility in the case of bispore, tetraspore or optionally polyembial diploids. This raises questions about how such heterozygosity arises and how it stabilizes.

Bispora and tetraspore species and many polyembryonic species are paleopoliploids, which, apparently, should have formed from evolutionarily asynchronous genital or apomictic polyploids. 1.S. Sagtai, 61 Vyu1. 1. Yesheai 8os. 51-94 (1997). Possible mechanisms of formation include increasing or decreasing aneuploidy and structural reorganizations of the parental genomes. E.E. 8o1! 1k & P.8. 8o1! 1k, Ro1ur1o1yu: Kesshtei! Yogtayoi aiy Seyote Euo1i! Yui, 14 Tgeiyyk Eso. Euo1. 348-352 (1999), both of which can be stabilized by diploidization. Without diploidization, segregation to the formation of a normal monospore embryo sac of the type Ploudoyit should occur. Diploidization converts polyploid sets of homeologous chromosomes in which recombination occurs randomly into recombinantly distinct (diploid) chromosomes in which recombination between newly marked and potentially reorganized diploid pairs never occurs. Therefore, hereditary multilocus intragenomic heterozygosity, critical for bisporia, tetrasporia and polyembryony, is constantly stabilized by diploidization. Apomixis in undoubted diploid apomicts Agaik and, possibly, in several other apomicts (diploid or polyploid), can also be constantly stabilized by diploidization.

The presence of extensive aneuploidy or large unbalanced chromosomal rearrangements before diploidization could make monosporia (norm) impossible for some bispore and tetraspore species and make sexual development of the embryonic sac in some apomicts impossible. However, cases of complete obligate bisporia, tetrasporia, and apomixis in plants are likely to be rare, if at all. N. H) e1tdu1k !, Wapa! Yuik sh Ebpo 8as Eeee1ortei !, 14 P11y! Rewarded with code 186-196 (1964); Akkeg &1eg1shd; V.M. 1st e! A1., Sotragayue Etbodu oG Ayidyukregtk, Uo1. 1 ai 2 (Νον Wogk: 8rgshdeg-Ueg1ad 1992). On the contrary, many unusual dioecious and asexual reproductive systems of insects, amphibians and reptiles are obligate. In the case of plants, most of the abnormal reproductive pathways are clearly associated with hybridization, polyploidy, diploidization, and other cytogenetic mechanisms. E. 8 and our e! A1., Su! o1oduyi Eyu1i! yui sh Rag! yeyodeeyek1k (SKS Rgekk, Vasa Ka! oi, E 1987). Therefore, such mechanisms can also arise in the form of polygenic hybrid phenotypes that stabilize by normal or segmented allopolyploidy, sexual sterility, diploidization, or other cytogenetic mechanisms that prevent the recombination of multilocus heterozygosity, which is critical for their maintenance.

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A typical stabilization mechanism differentially affects heterosis and gene drift. For example, allopolyploidy such as TT T'T 'instantly stabilizes apomixis, but there are several mechanisms that fix mutations and rare outcrossing to improve the fertility of such apomicts by modifying the initial co-adaptation egg development programs. Conversely, among segmented allopolyploid apomicts, there are potentially effective mechanisms for genomic modification. In such apomicts, recombination mixing occurs within such sets of homeologous chromosomes that are not directly involved in the discussed apomixis, which probably contains the majority. Recombinations within these kits can enhance pollen sexual development, asexual egg development, parthenogenesis of unreduced eggs, and heterosis. In this sense, apomicts arising as intraspecific autopolyploids or weakly intraspecific hybrids can quickly acquire, by natural selection and autopolyploidization of neapomixis chromosome sets, recombinations that produce large batches of seeds and high pollen fertility. Intragenomic recombination, harmful to the manifestation of the gender of females, enhance selection against sexual revertants. Some intragenomic recombinations can cause duplication or deletion of certain stages of egg development, as seen in bisporia and tetrasporia. In this regard, apomixis can serve as an evolutionary trigger in the evolution of reproductive new sexual species and genera, including those that are bispore, tetraspore or polyembryonic. TS. Sagtap, 61 ΒίοΙ. 1. bshpeap 8os. 51-94 (1997). On the contrary, apomicts arising strictly in the form of genomic allopolyploids, or TT T'T 'or TTT T', can retain an unlimited number of intragenomic heterozygosities that are not fully adapted to apomixis.

The methods of the present invention

The present invention is directed to methods for creating genetically stabilized apomictic plants and genetically stabilized naturally occurring or artificially created apomictic plants that exhibit genetic instability. The present invention is also directed to methods for controlling the expression of apomixis (facultativeness) in order to improve plants, seed production and crop production.

It is convenient to divide the methods of the present invention into 4 categories: a) determination (assessment) of genomic homeology, facultativeness and apomixis stability, b) plant breeding, amphiploidization and mutagenesis processes, c) methods of gene mapping and cloning, and i) methods of genetic engineering.

Assessment of genomic homeology, facultativeness and stability of apomixis

A feature of this invention is the stabilization of apomixis in naturally occurring or artificial apomicts by creating karyotypic (structural) heterozygosity. This is easily accomplished if apomicts are obtained from sexually propagating plants by selecting intraspecific or interracial parent lines that also meet the divergence requirements in reproductive egg development programs, as described in XVO 98/33374. The preferred method for assessing the degree of karyotypic homeology of two genital lines (considered as hybridization pairs) includes conventional genomic analysis, where the produced hybrids and degree of chromosome pairing are evaluated in metaphase I in pollen mother cells (PMCs). Ό.Κ .. Uhhhh. Sepot 1s apy P1y1odepeis Re1a1yup51pr5 atopd ΝοΠίι Ashepsap Repepsha TTFseae, ίη TE. E51e5 e1 a1., Sgakkek apy StakNapyk: 8u51eta11S5 apy Esoodu (Ishuegayu οί Ok1a1yuta Rgekk 1982). At the diploid hybrid level, homeologous chromosomal pairing in PMSC often far exceeds that observed during amphiploidization of the diploid hybrid. R.R-S. ‘No apd, ApkkkteSh OG depot apa1u§18 Bakey pkotoguyte ratpd sh Nupyk oG regepsha1 TtShseae, 32 Sepot 179-189 (1989). Therefore, diploid hybrids even with a limited degree of reduced chromosome conjugation in PMCs may be suitable for creating karyotypic heterozygosity by producing amphiploid.

Facultativeness is measured as the percentage of viable seeds that are formed sexually from an apomictic plant. The preferred method for determining this percentage is to analyze the offspring, in which the offspring are compared to the mother plant. Modern molecular fingerprinting methods are preferred because of their reliability and ease of use when systems are optimized. O. Ehl1aps & A ^ akh / isaYu, 8stespd Rgoseyigek 1o 1yep (Well apy OnapiGu Arot1hs, t.

The degree of stability is assessed by analyzing the offspring in selected samples identified in the first generation of offspring. The totality of the offspring whose members are apomictic as a mother plant is obtained from a genetically stable (karyotypically heterogeneous) apomictic mother plant. The totality of offspring, whose members are represented by a high percentage of sexual revertants, comes from genetically unstable apomicts. Typically, artificial, or naturally occurring diploid apomicts, or I meet

-12006658 naturally occurring digaploid apomicts are unstable. Artificial or naturally occurring polyploid apomicts may or may not be stable.

Plant breeding, amphiploidization and mutagenesis

For several purposes of the present invention, generally accepted methods of plant propagation were used, as taught in standard texts on plant breeding, for example, RoyEtai, Vteeyshd G1e1y Sgork (Wap No. K1gapy Vesh1yuy 1987). A preferred method is to increase the genetic diversity and combining ability of the genital parental lines to produce apomictic diploids, or polyploids. Plant breeding, or genetic engineering, was used to genetically modify two sets of transdifferentiated parental lines of plant species, or a closely related group of plant species that are differentiated by their reproductive phenotype, so that any plant hybridizes one of two sets of transdifferentiated lines with any of the plants from another set of transdifferentiated lines produces an apomictic plant or plant that becomes apomictic through amphiploidy hybridization or further hybridization. The combining ability of parental lines was improved by standard crossbreeding and selection, or by simple crossbreeding, double crossbreeding or multicrossing (outcrossing) that accompany each set of transdifferentiated lines.

A feature of the present invention is the transdifferentiation of a new hybrid selection system by which artificially derived hybrid apomicts are obtained. The system (method) involves not only the identification of sexually inbred parental lines that express good combining ability, but also the identification of hybrid or outcrossed parental lines within two sets of transdifferentiated lines, so that this good combining ability is expressed when a plant is hybridized from one of two sets of transdifferentiated lines with a plant from another set of transdifferentiated lines. Thus, this new operating system produces hybrids from crossing two inbred lines or multiple crosses, which are either apomictic or become apomictic through amphiploidization or further hybridization. Thus, many apomictic hybrid genotypes can be produced (from each cross). In addition, each individual genotype can be obtained through apomictic seed formation for field testing and / or release of a cultivated plant variety. Consequently, an unlimited number of new apomictic genotypes are produced rapidly. This method greatly increases the genetic diversity of plants used in agriculture, and greatly increases the ability of breeders to provide varieties of apomictic hybrids that are specifically adapted to high-medium or low-productive agricultural regions.

A feature of the present invention extends to the determination of combinational ability to enable the development of divergent, but highly heterozygous reproductive parental lines which, when hybridized (or hybridized and amphiploidized) result in apomictic plants with higher hybrid strength. Genetically heterogeneous apomictic offspring obtained from crosses involving heterogeneous parental lines (genital or apomictic) (outcrossing) is individually evaluated for its agronomically desired qualities and selected for propagation as a cultivated plant variety. Also, an advantage of the method is the crossing of an optionally apomictic plant with genetically divergent genital or apomictic lines to produce derived lines with enhanced agronomic traits.

For amphiploidization, the chromosome sets of hybrids were doubled using standard colchicine methods, for example, 1. Totally) a e1 a1., Mogr1oodu apy Sepot Apaluyek! 1p! Ekkresyyu Nypyk oh! E1utik xagik, 29 Sepot 150-55 (1987). Alternatively, newly developed tissue culture techniques can be used. O. LeBeops e1 a1., Sytotokot EoIypd ίη Takes part: Do not Rhoisyop oh! AySch1a1, 8ekhia1 Te (gar1o1y P1ai1k, 114 P1ai1 Vgeey, 226-30 (1995); Soyey & Uao, 1i Uygo Sytotokot EoIypd! S1a1ak & Edaue, Ohuhayp HUNDRED \ νίΐ1ι AyuepShunk VedepegaPyup! Og ap Yaep!

Partially amfiploidnye 2n + n (B _w) hybrids are often formed at a low frequency (0.5 to 3%) at interspecific backcross F _{1 to} 8 for example, Ζ. \ Ν. Bsh e! A1., 89 Tieog. Arr1. Sepe !. 599-605 (1994), and this frequency can be much higher if hybrids have a tendency to apomixis (the formation of an unreduced egg), as taught in O. Lebens! A1., Vergoyisyue Weaouyou w Ma1 / e- Tkrkasit Ro1uyar1o1y P1ap! k: 1shr1yuyopk! ogue 1 TgapkGeg about! Arot1h1k 1p1o Msh / e, 87 1. Numb. 108111 (1996). Thus, the preferred method for doubling the chromosomes of intraspecific and interspecific hybrids is the use of one or many methods of treatment with colchicine (or another known chemical spindle inhibitor) listed above. Also, a preferred method for doubling the chromosomes of intraspecific hybrids involves crossbreeding with one of the reproductive parents and counting the chromosomes at the root ends to determine partial amphiploidy (usually triploidy). This is the one followed by backbreeding with the other parent for

-13006658 obtaining a complete amphiploid, or with the same parent to obtain a partial amphiploid (three genomes from one parent and one genome from another). Amphiploidization may precede hybridization, or follow hybridization.

Traditional methods of mutational selection, as taught in the open press, for example, RoyN1tap, Vteyashd Pe1y Stork (Uap No. k! Gapy ResNo1y 1987), are used to induce chromosomal inversions or translocations that isolate from recombination portions of chromosomes that contain the genes required for apomixis . Preferred methods include regeneration of plants with chromosomal rearrangement from plant tissue cultures, 8. 1 our e! a1., 8otay1oyia1 Uapayop apy 1pyisey Mi1ayopk ίη Sgor 1trouuetep1, Siggep! P1ap! 8c1epse apy Vyu! EsNpododu ίη AdpsiNige 32, (K1i \\ 'e Asayetu PuNkjetk 1998), the obtained plants with rearranged chromosomes are further treated with ionizing radiation, R.K. Sir! A, Mi1aiop Vteeyshd sh Segea1k apy Editek, w 8.M. .Yes! A1., Sittep! P1ap! 8ssssse apy vyu! EsNpododu w Adpsiite 32 (K1i \ uye Asayetu Rybnkyetk 1998).

Use of male sterile lines or castration procedures is desirable if the plants are not dioecious or self-incompatible. Hybrids are produced between genital species or lines that show appropriate degrees of divergence in photoperiodic responses and in female organ development programs. Intraspecific hybrids are created using standard techniques, as taught in plant breeding texts, for example, RoyEtap, Vteeyshd G1e1y Stork (1987). Successful production of interspecific or intergeneric hybrids may require rescue procedures to treat hormones of flowers of inflorescences and embryos with hormones, as taught in recent references relating to broad hybridization, for example, ΖΑΥ. Bsh e! A1., Nypyk apy Vasksgokk Ртодешек БеАтееп unea! (Tpsit aekyuit b.) Apy Arotyuns AikIaPap \ Una! Dgakk [E1utik ges11ke1yk (No. kk 1n bait.) A. Bouy & Sopog]: Kapoaryu apy Sepotu Apaoukek, 89 Tneog. Arr1. Sepe !. 599-605 (1994). Hybrids are checked by checking their intermediate phenotype.

Gene Mapping and Cloning

A feature of this invention is the involvement of the control of facultativeness by modifying the expression of quantitative trait loci (fTk) required for optional expression using anti-sense technology. The preferred method begins with mapping fTk divergent sexual parental reproductive phenotypes responsible for the apomixis found in hybrids produced by crossing these phenotypes. The method involves the production of a G ₂ mapped population consisting of G ₂ sexually progeny, optional artificial E ₁ apomict produced by hybridization of the original reproductive divergent parent lines, and identification of molecular markers that are associated with each phenotype, for example, A.U. Neikyep e! A1., T1ee FGK, GGGT Husoretkyop regiu1apit SopGega N | dN booooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooooo! McDapeksch 99 Tneog. Arr1. Sepe !. 1068-1074 (1999). Important fTbk were then accurately mapped for a given chromosome using a large segregated population and artificial yeast chromosomes (UASC), and the surrounding chromosome was isolated using flanking markers. Then, a cosmid clone containing ftB was produced, complementary cosmids were identified by transformation into a mutant. The PTF transcript was identified by isolation of cDNA using complementary cosmids, e.g. N.f. Bshd e! a1., Mar-bakey ош ош С отоп отоп отоп ету ету а а,, а а а 1 1!! Н Н Н Н Н Н Н Н Р Р Р Р Р Р Р Р Р Р с с ой ой ой ой ой 96 96 96 96 96 96 96 96 96. No. G1 Asai. 8cG I8A 7098-7103 (1999); E.8. Badian e! a1., Mar-bakey ош п а еп е е 8 8 8 8 8 Sep. ! Not SrEZ no! Oye Rek1k! Apse bosik oG HUNean 40 Sepot 659-665 (1997). Alternatively, bacterial artificial chromosomes (BACs), which are much easier to work with, for mapping-based cloning can be used. YOU libraries were obtained for many types of cereals, for example, 8.8AUoo e! A1., Sopkyysyop apy SNagas! epha! yup oG a bac! epa1 AgiPs1a1 SNGotokote b1bagu oG 8ogdNit b1co1og, 22 # c1ek As1yk. Century. 4922-4931 (1994).

Genetic engineering

A feature of the present invention is to control the degree of facultativeness by controlling the expression of PTB, which is important for optional expression. Another feature of the present invention is the conversion of permanently (or reversibly) optional apomicts into obligate apomicts by controlling the expression of meiosis-specific genes.

The preferred method for perfect obligate apomixis is to breed or transform an optionally apomictic plant so that it contains genetic material that causes female meiosis to interrupt, resulting in essentially 100% apomictic seed formation. The genetic material may be a meiotic mutant introduced through selection, or a transgenic promoter / gene construct, the expression of which interrupts female meiosis. Inducible down regulation of transgenic, promoter / gene constructs, the gene construct of which upon expression causes meiotic abortion, which allows facultative apomixis to exist. Alternatively, facultative apomixis can occur except during the inducible up-regulation of the transgenic promoter / gene construct, thus causing meiotic abortion and essentially 100% apomictic seed formation.

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The promoter / gene construct may contain a promoter from the group of promoters that are expressed immediately before or during female meiosis and a gene construct whose expression fatally interrupts meiosis, for example, ν.Ι. KNwick & 6.Ό.Ο. Bopeh, AYUMS1. Р1е ЛгаЬ1бор818 1Тё1е о £ Не аз Э Р Р Э 1 EMC1:: с: syagas Rephiop, Prinzrosop-tbiseb a11e11s uapaRup apb teu818-a88os1aReb expgeziup, 11 P1apr b. 1-14 (1997); PCT / OV97 / 03546. A transgenic material that is normally cytotoxic for the development of a female meiocyte can be controlled by a suppressor molecule encoded by a gene that is controlled by a chemically inducible promoter, which can be a specific promoter to a female meiocyte, so that female fertility (optional) is inducible in such apomict. The transgenic material may contain a gene from a group of sense or antisense genes whose expression during meiosis fatally disrupts meiosis or is otherwise cytoxic to a female meiocyte. In addition, a method for restoring a low level of female dioecious in a transgenic-derived obligate apomict may involve suppressor expression by induction of an inducible promoter. For the introduction of transgenic material into the host plant, any available method well known in the art can be used.

Examples

Some of the features of the present invention can be better appreciated by reference to specific examples. It should be understood that the following examples are illustrative in nature rather than limiting in nature, and are intended to demonstrate the basic teachings and concepts of the present invention and not to limit the invention. It is expected that a person skilled in the art will be able to use the information contained in the examples and elsewhere here to apply the present invention to situations not specifically described herein.

Example 1. Selection of lines suitable for the synthesis of stable apomictic plants.

A feature of the present invention is the provision of procedures for selecting sex lines within the primary, secondary or tertiary germplasm pools of a given crop to synthesize stable optionally apomictic plants (those that do not easily form sexual segregants) from plants with sexual characteristics or unstable optionally apomictic plants. This example illustrates the preferred procedures for use on plants from the subclass O1coPu1ebopae, namely, the genus species from the genus Lpreppapa and from the subclass Moposoupheba, namely, the sex species from the genera Trzasit and Zogdkit. It is expected that a person skilled in the art will be able to successfully apply these procedures to many other crops, such as rice, sugar beets, apple trees, cherries, tomatoes, soybeans and lettuce.

Now, the preferred procedure for selecting suitable genital parental lines is (a) identification, from literature, or from areas of study, natural ecotypes and non-reclaimed land, the race of these crop species and their closely related species, which differ in relation to shade resistance, latitude, photoperiodic requirements for flowering, altitude and preferred moisture, (b) the cytoembryological characteristic of physiologically and ecologically divergent lines by the ratio Nia megasporogenesis stages and development of the embryo sac with stages of development of the integument and ovary weight, (c) the characteristic cytoembryological and statistical analysis of differences between the lines and (b) selecting lines which are divergent physiologically (e.g., photoperiodism) cytoembryological and taxonomically. In general, plants classified as different species, for example, pairs of plants whose hybrids are sterile, must have sufficient genomic homeology to ensure karyotypic heterozygosity if the hybrid produced between them is amphiploidized.

Sexual diploids and polyploids in Lpnepapa, Trzasit and Zogdkit correspond to the geographical, physiological, environmental, cytoembryological and taxonomic criteria listed here to create genetically stable optional apomictic plants from plants with sexual characteristics. Within the entire Rocky Mountain in Cordillera, from the Arctic Circle of northwestern Canada to the U.S. border with Mexico, there are numerous varieties of reproductive animals that collectively occupy a wide range of habitats, but are often individually narrowed to specific habitats. Waueg, 132 Orega WoRasha 53-65 (1996). Significant differences in the synchronization of meiosis relative to the development of integument were observed among these species (Figs. 2 and 3). Within America, from 42 ° north to 24 ° south latitude, there are numerous species of genital Tprzasit, which also collectively occupy a wide range of habitats, but are often individually narrowed to specific habitats. Ee VeR eP a1., ZuzRetaysz o £ Takes up bassRu1o1 without (Ogateae), 69 Lt. b. Thief. 1251-57 (1982). Significant differences in the synchronization of meiosis with respect to the development of integument, similar to those observed among the subspecies of Lpreppap, were also observed among these species (data not shown). Systematization throughout most of the African continent, Australia, and South Asia shows that sexual diploid and polyploid ecotypes, races, and species are always numerous. Significant differences in the synchronization of meiosis relative to the development of integument were also observed among these species (Figs. 4 and 5). The selection of suitable genital lines for the creation of facultatively apomictic plants can be made from these data.

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They suggest that the collection, characterization and selection procedures for the partial change of any monocotyledonous and dicotyledonous will be appreciated.

Example 2. The creation of genetically stable facultative and obligate apomicts.

As instructions, the methods of Example 1 were used to obtain three or more genital lines with early meiosis and early gametophyte development relative to the development of integument (s). The same techniques were used as instructions for obtaining three or more genital lines of closely related species with late meiosis and late gametophyte development regarding the development of integument (s). Several lines were selected from each category, so that the formation of the embryo sac in one set of lines occurred at about the same time as prophase in early meiosis in another set of lines relative to the development of integument (s). Pairs of parental lines (one plant from each of the two groups) were hybridized and amphiploids were produced using standard procedures described above. It will be appreciated that the genetic background from which the lines were derived can affect the expression of apomixis. Thus, the selection or creation of additional lines that combine different genetic backgrounds and a greater or lesser difference in the timing of meiosis may be necessary.

Optional apomicts, which are unstable, meaning that they produce sexual segregants as a result of facultative sexual reproduction, were synthesized as a result of flowering asynchrony resulting from hybridization, by creating artificial diploid hybrids of varieties L1spspapa sogutoza (2x sexual) x A. gasetosis (2x ) (Fig. 6) and artificial diploid hybrids of 8ogdNit varieties (2x genital) (Fig. 7). Apospore embryo-sacs are formed in hybrids 8-1 5-1 X 4-1 and 9-1 X 1-2 with a frequency of about 5% and diplospore embryonic sacs similar to those in Trzasit (Fig. 8) are formed in hybrids 8ogdNit 5-2 X 9-2 with a frequency of about 10%. It is noted that divergence in terms of meiosis relative to the development of integument is significant (Fig. 5) in parental couples whose offspring form diplospore embryonic sacs.

Structurally heterozygous (stable) facultative apomicts can be produced from the intraspecific hybrids L1eppap and 8ogd1st by doubling their sets of chromosomes using the methods described above. Stabilization of the intraspecific hybrids of 8ogd1sh (indicated above) requires a genetic modification that causes abortion of female meiosis, or the formation of a meiotic cell, which not only stabilizes apomicts, but also makes them obligate. This is achieved by introducing a meiotic mutant in a line using standard hybridization methods, inducing triploidy via _W hybridization, or amphiploidization followed by hybridization with a diploid, or transformation of a diploid with a promoter / gene construct that is cytotoxic for a female meiocyte using the methods described above. Using inducible promoters, as discussed above, genetically stable apomicts with induced obligate or facultative expression can be produced.

The artificial diploid amphiploid Trzasit 1chit (2x genital) X T. rhyositis (2x genital) is a stable optional apomict with 50% formation of a diplospore embryonic sac (Fig. 8). Crossing of this plant with T. chorapoje (2 genital) or with T. lauwait (2 genital) produces stable obligate apomicts with approximately 80% formation of a diplospore embryonic sac and with 20% formation of an abortive meiocyte or the formation of an embryonic sac through sexual contact (Fig. 9) .

Example 3. Mapping and cloning of genes responsible for optionality.

Genetic analyzes of apomixis were carried out by pollinating genital plants with pollen from apomictic plants and counting eggs in the offspring during sexual or apomictic development. Usually in these studies, if any apomictically developing eggs were found, the offspring were considered apomictic. For example, Υ. 8au | yap, ya! Yee e! Neegeye ye 1 = arot1x1e skeh Rapuyit tachypit 1ass. |., RYu (Nemu Ishuegye Rapzi XI, Bgayse (1982); 8. Lijes e! A1., Mälé apy £ eta1e 8rögépépé8e apSa, vGasYapa YesTiEs apy B ₁ NuGuyk χνίΐΐι Зехия1 с1СЫСТЕ tyisei 1е! Gar1О1й ВгасЫапа ги7171п818, 78 Ерюуйса 19-25 (1994); S.V. e! a1., Ayuapsez sh Arot1x18 Vezeagsy (2000); R. O / 1a5-Lkt8 e! a1., 95 Pr. developing apomictically were lower than 12, 28, 17 and 7% What happened in these studies (whether it was intentional or not) was the identification of the minimum number of linked groups required to encode at least some degree of functional apomixis. In some cases, gaps were observed among the offspring as a percentage of eggs expressing Apomixis, M. II) agit & Nappa, Arotuis apy zechia1 react1 ty1e! X REVIEW 8ciati1a1it Nuib1y8, 74 I. Negeu. 277-279 (1983), but in other cases the expression range was somewhat prolonged in some places, 8. L e e! A1., 78 Airyuys 19-25 (1994). Consequently, it is believed that several major and, possibly, minor genes with quantitative effects (а. 8au1yap, аЫЫге! !Егегегей й й 1 1 1 1 = отот111х скехехехехехЫЫЫЫЫЫЫ т т т т т 1ассассассасс 1ассасс 1 1 1асс 1 1 1 | | | | |,, Р Р Р Р Р 1 1ййййй8881818. ИЫЫапапапапапапапапапапапапапапапапапапап ап,, 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 1982 Leys e! A1., 78 ErNuysa 19-25 (1994)) affect the facultativeness (degree of expression) of apomixis.

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A feature of this invention is the up-regulation or down-regulation of facultativeness by modifying the expression of OTBb using antisense technology. Using the methods described above, OTB mapping is performed for divergent sexual parental reproductive phenotypes responsible for the apomixis that occurs in hybrids (Fig. 2-5). Important OTbs were then finely mapped for a given chromosome and identified as described above.

Example 4. The creation of genetically stable highly facultative apomicts with iducible obligate expression or genetically stable obligate apomicts with inducible highly facultative expression.

The methods of Examples 1 to 3 were used as guidelines for creating genetically stable highly optional apomicts with inducible obligate expression or genetically stable obligate apomicts with highly optional expression.

In the present invention, apomixis is similar to a computer operating system. The features of this “biological operating system” include the following: (1) on farmers' fields, clear cloning of hybrids from the actual hybrid seeds — generation after generation, (and) in plant breeding nurseries, partial dichotomy for plant improvement, with subsequent reversal to strict apomixis, (w ) a large number of rapidly produced and genetically diverse cultivated plant varieties specially adapted to a variety of agricultural niches, ()ν) an increase in the overall genetic diversity varieties for protection against tax extermination by pests, and (ν) winning costs, namely, farmers pay less for seeds and seed companies pay less for the development of excellent varieties of crops.

It should be noted that the present invention can be embodied in other specific forms without deviating from its spirit or essential characteristics, which are in the disclosure of the five principles of theory. The described stages and materials should be considered in all respects only as illustrative and not restrictive, and the scope of the invention is indicated in the attached claims, and not in the description. All changes, within the meaning and equivalence of the claims, should be included in its scope.

Claims (23)

CLAIM 1. A method of obtaining an apomictic plant exhibiting increased genetic stability of apomixis from an angiosperms apomictic parent plant that exhibits apomixis genetic instability, comprising: (a) measuring the degree of genetic instability of the apomixis of the parent plant; (b) if the parent plant exhibits genetic instability of apomixis, obtaining one or more descendant plants from the parent plant, where each of the descendant plants contains a double chromosome set of the parent plant; (c) measuring the degree of genetic instability of the apomixis of one or more flowering plants; (i) comparing the degree of genetic instability of the apomixis of one or more flowering plants with the degree of genetic instability of the apomixis of the parent plant; and (f) selecting at least one of one or more descendant plants exhibiting increased genetic stability of the apomixis compared to the parent plant. 2. The method according to claim 1, where one or more descendant plants are obtained by treating the parent plant with a spindle inhibitor. 3. The method according to claim 2, where the spindle inhibitor is colchicine. 4. The method according to claim 1, where one or more descendant plants are obtained by culturing the parent plant or part thereof in tissue culture. 5. The method according to claim 1, where one or more descendant plants receive: (a) by self-pollination of the parent plant or by crossing the parent plant with another plant of the same species, genus or family and obtaining the first offspring; and (b) counting the chromosomes in the first offspring and selecting an amphiploid or partial amphiploid that has all the chromosomes from the parent plant in case of self-pollination of such the parent plant or all chromosomes from the parent plant and one or more sets of chromosomes from another plant of the same species, genus or family, or all chromosomes from another plant of the same species, or as a family, and one or more sets of chromosomes from the parent plant in the case of crossing a parent plant with another plant of the same species, genus or family. 6. The method according to claim 5, further providing for the reverse crossing of a partial amphiploid with a parent plant or with another plant of the same species, genus or family and obtaining second offspring from them and selection of an amphiploid having an equal (even) ploidy level. 7. The method according to claim 5, where the partial amphiploid is an euploid or aneuploid. -17006658 8. The method according to claim 5 or 6, where the amphiploid is an euploid or aneuploid. 9. The method according to any one of claims 1 to 8, further comprising increasing the fertility of the selected offspring plants by self-pollination or hybridization with another plant, obtaining seeds from them, sowing said seeds, and selection of sexually-obtained apomictic hybrid plants that exhibit an increased frequency not reduced egg fertility or parthenogenesis. 10. A method of obtaining an apomictic segmented allopolyploid plant exhibiting increased genetic stability of apomixis from an angiosperms intersortic hybrid plant that exhibits genetic instability of apomixis, comprising: (a) measuring the degree of genetic instability of the apomixis of an intervarietal hybrid plant; (b) if an intervarietal hybrid plant exhibits apomixis genetic instability, obtaining one or more offspring plants from an intervarietal hybrid plant, where each of the offspring plants contains a double chromosome set of an intervarietal hybrid plant, resulting in the appearance of one or more apomictic segmented allopolyploid plants; (c) measuring the degree of genetic instability of the apomixis of one or more apomictic segmented allopolyploid plants; and (ά) comparing the degree of genetic instability of the apomixis of one or more apomictic segmented allopolyploid plants with the degree of genetic instability of the apomixis of an intersortic hybrid plant to check whether the genetic stability is increased or several apomictic segmented allopolyploid plants compared to intersortion hybrid Asten. 11. The method of claim 10, further comprising increasing the fertility of one or more apomictic segmented allopolyploid plants by self-pollination or hybridization with another plant, obtaining seeds from them, sowing said seeds, and selecting sexually obtained apomictic segmented allopolyploid plants that show an increased frequency not reduced egg fertility or parthenogenesis. 12. A method of obtaining an apomictic allopolyploid plant exhibiting increased genetic stability of apomixis from an angiosperms interspecific hybrid plant exhibiting genetic instability of apomixis, comprising: (a) measuring the degree of genetic instability of the apomixis of the interspecific hybrid plant; and (b) obtaining one or more apomictic allopolyploid offspring plants from the indicated interspecific hybrid plant and selecting an apomictic allopolyploid plant flow showing increased genetic stability of apomixis. 13. The method of claim 12, further comprising increasing the fertility of the selected apomictic allopolyploid offspring plant by self-pollination or hybridization with another plant, obtaining seeds, sowing said seeds, and selecting sexually obtained apomictic allopolyploid plants that show an increased frequency of non-decreased egg fertility or parthenogenesis. 14. A method of obtaining an apomictic plant exhibiting increased genetic stability of apomixis from an angiosperm, intraspecific, interspecific or interspecific hybrid plant exhibiting genetic instability of apomixis, comprising: (a) measuring the degree of genetic instability of the apomixis of an intraspecific, interspecific or interspecific hybrid plant; and (b) obtaining apomictic autopolyploid, segmented allopolyploid or allopolyploid offspring plants of an intraspecific, interspecific or interspecific hybrid plant and selection of an apomictic genetic plant exhibiting increased apomictic stability. 15. The method of claim 14, further comprising increasing the fertility of the selected apomictic plant by self-pollination or hybridization with another plant, obtaining seeds, sowing said seeds, and selecting sexually obtained apomictic descendant plants that show an increased frequency of non-reduced egg fertility or parthenogenesis. 16. A method for the genetic stabilization of apomixis of an angiosperm, apomictic, intraspecific, interspecific, or interspecific hybrid plant exhibiting genetic instability of apomixis, which measures the degree of genetic instability of apomixis of an intraspecific, interspecific or interspecific hybrid plant and produces a descendant plant from the specified apomictic non-subspecies or interspecific non-species or an increase in ploidy, moreover, female meiosis is usually interrupted, leading to almost 100% formation of apomictic seeds. 17. The method according to clause 16, further comprising increasing the fertility of the plant by flowing by selfing or hybridization with another plant, obtaining seeds from them, sowing -18006658 seeds and selection of sexually-obtained apomictic plants that exhibit an increased frequency of unreduced egg fertility or parthenogenesis. 18. A method for the genetic stabilization of apomixis of an angiosperm, apomictic, intraspecific, interspecific, or interspecific hybrid plant exhibiting genetic instability of apomixis, comprising measuring the degree of genetic instability of apomixis of an interspecific, interspecific, or interspecific hybrid plant, and crossing this intraspecific, interspecific hybrid plant or interspecific marsanti or interspecific hybrid plant of the same species, genus or family and the selection of offspring, where feminine meiosis usually interrupted, resulting in almost 100% the formation of apomictic seeds. 19. The method according to p. 18, additionally providing for increased fertility of the selected offspring by self-pollination or hybridization with another plant, obtaining seeds from them, sowing of said seeds and selection of sexually-obtained apomictic descendant plants that show an increased frequency of unreduced egg fertility or parthenogenesis . 20. A method of obtaining an apomictic plant exhibiting increased genetic stability of apomixis from an angiosperm apomictic parent plant that exhibits apomixis genetic instability, comprising: (a) measuring the degree of genetic instability of the apomixis of the parent plant; (b) obtaining a descendant plant from the parent plant by doubling the number of chromosomes of said parent plant or introducing a chromosome aberration in which recombination is suppressed, so that allele segregation during meiosis is inhibited; and (c) measuring the degree of genetic instability of the apomixis of the progeny plant to verify that such a plant exhibits increased genetic stability of apomixis compared to the parent plant. 21. The method according to claim 20, where the specified chromosomal aberration involves translocation. 22. The method according to claim 20, where the specified chromosomal aberration involves inversion. 23. The method according to any one of claims 18 to 22, further comprising increasing the fertility of the offspring plant by self-pollination or hybridization with another plant, obtaining seeds from them, sowing said seeds, and breeding sexually obtained apomictic descendant plants that show an increased frequency of not decreased egg fertility or parthenogenesis. Free plants Preapomixis Differentiation of sexual ecotypes and species according to ecological gradients Stage I Stage II a. Climate degradation a. Stabilization b. Hybridization, occurring apomixis, resulting from secondary b. Improved contact, expressed in a climate of flower asynchrony (apomixis) Stage III a. Periodic outcrossing of apomicts with sexual forms b. Differentiation agamic complex Stabilized agamic complex (mainly polyploid) Genetically stabilized apomicts (usually polyploids)