WO2001032001A1 - Procedes servant a stabiliser et a controler l'apomixie - Google Patents

Procedes servant a stabiliser et a controler l'apomixie Download PDF

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Publication number
WO2001032001A1
WO2001032001A1 PCT/US2000/029905 US0029905W WO0132001A1 WO 2001032001 A1 WO2001032001 A1 WO 2001032001A1 US 0029905 W US0029905 W US 0029905W WO 0132001 A1 WO0132001 A1 WO 0132001A1
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Prior art keywords
apomixis
apomictic
plant
sexual
genetically
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PCT/US2000/029905
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English (en)
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John G. Carman
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Utah State University
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Priority to EA200200507A priority Critical patent/EA006658B1/ru
Application filed by Utah State University filed Critical Utah State University
Priority to IL14933500A priority patent/IL149335A0/xx
Priority to BR0015276-5A priority patent/BR0015276A/pt
Priority to EP00978296A priority patent/EP1227715A4/fr
Priority to CA002389055A priority patent/CA2389055A1/fr
Priority to MXPA02004186A priority patent/MXPA02004186A/es
Priority to JP2001534218A priority patent/JP2003516725A/ja
Priority to NZ519156A priority patent/NZ519156A/en
Priority to AU15772/01A priority patent/AU1577201A/en
Publication of WO2001032001A1 publication Critical patent/WO2001032001A1/fr
Priority to HK03100846.3A priority patent/HK1048741A1/zh
Priority to US10/772,243 priority patent/US7638680B2/en
Priority to US12/416,883 priority patent/US20090217409A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A01H1/022—Genic fertility modification, e.g. apomixis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A01H1/021—Methods of breeding using interspecific crosses, i.e. interspecies crosses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis

Definitions

  • This invention relates to the fixation of hybrid vigor and other traits through apomixis (asexual seed formation) in flowering plants (angiosperms) . More particularly, it provides methods for "stabilizing" apomixis in natural or man-made facultative apomicts (plants capable of sexual and apomictic reproduction) such that sexually-derived progeny, which are occasionally produced facultatively from such apomictic plants, tend to be apomictic like the mother plant, though otherwise genetically recombined, instead of being sexual revertants.
  • It also provides methods for "controlling" apomixis, in natural or synthetic apomicts, such that such apomicts express obligate apomixis (no capacity for sexual seed formation) , obligate apomixis except when induced to be facultatively apomictic, or facultative apomixis except when induced to be obligately apomictic.
  • This invention uses genetic, cytogenetic, and molecular modifications to prevent genetic recombination among loci critical to the expression of apomixis (stabilization of apomixis) and controls the percentage of seeds that are derived apomictically by controlling frequency of sexually-derived seeds in natural or synthetic facultative apomicts (control of apomixis) .
  • apomixis The types of apomixis referred to in the present patent application cause asexual seed formation. Accordingly, seeds of apomictic plants contain embryos that are genetic clones of the mother plant. Such forms of apomixis comprise adventitious embryony and gametophytic apomixis (referred to hereinafter as apomixis) , which is commonly divided into apospory and diplospory. S.E. As er & L. Jerling, Apomixis in Plants (CRC Press 1992) (hereinafter, "Asker & Jerling") .
  • Antennaria-type diplospory signals for precocious embryo sac formation occur very early, completely preventing meiosis.
  • Taraxacum-type diplospory signals for embryo sac formation are less precocious and affect the MMC after meiosis has initiated.
  • tfieracium-type apospory nucellar cells are affected by the precocious and ectopic embryo-sac-inducing signals, and the affected somatic nucellar cells undergo three rounds of endomitosis to produce an 8-nucleate embryo sac.
  • Panicum-type apospory only two rounds of endomitosis occur, resulting in mature 4-nucleate embryo sacs.
  • adventitious embryony embryos form from cells other than the egg, including cells of the nucellus, integument (s) , synergids, and antipodals. Asker & Jerling.
  • Apomixis may become among the most valuable genetic tools for plant breeders in the 21st century. At a recent conference on apomixis, the following conclusion was reached: "The prospect of introducing apomixis into sexual crops presents opportunities so revolutionary as to justify a sustained international scientific effort. If apomixis were generated with a sufficiently high degree of flexibility, the impact on agriculture could be profound in nature and extremely broad in scope.” The Bellagio Apomixis Conference, Why is Apomixis Important to Agriculture (1998) (http://billie.harvard.edu/apomixis/ apotech.html) .
  • HFA theory hybridization-derived floral asynchrony theory
  • apomixis is a developmentally-disjunct hybrid phenotype.
  • Apomixis is disjunct from, not intermediate to, its parental female reproductive phenotypes, which, for convenience, are labeled parental phenotypes A and B. Plants exhibiting phenotypes A or B undergo normal sexual reproduction. Phenotypic differences between A and B are detected cytoembryologically through state-of-the-art microscopy techniques. They are not casually observed, which is why they have not been described previously.
  • parental phenotypes A and B are distinctly different from each other with regard to the time periods in which meiosis, embryo sac formation, and embryony occur relative to gross ovule development .
  • facultative apomixis where sexual and apomictic seeds commonly 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 schedules of ovule development) through genetic segregation.
  • HFA theory efficient procedures for synthesizing facultatively apomictic plants from sexual plants have been described. J.G. Carman, Methods for Producing Apomictic Plants, WO 98/33374 (1998) (hereby incorporated by reference). These methods are used to produce highly apomictic plants that may or may not be genetically stable as defined above.
  • the solution offered in WO 98/33374 is to produce highly apomictic plants, i.e.
  • apomictic hybrids by identifying or producing (through breeding) pairs of parent lines that are appropriately divergent in their female reproductive schedules such that facultative sexual development is minimized in the facultatively apomictic hybrid progeny.
  • Synthetic apomicts produced in this manner may be used as apomictic hybrid lines for several to many generations before the harvested seed becomes useless for replanting due to serious contamination from seeds of sexual revertants .
  • the contaminating revertant seeds are products of genetic segregation, and their presence degrades agronomic value. This situation would be analogous to the mixing of inferior F 2 and later generations of seed with elite F- . hybrid seed in a conventional hybrid seed production program.
  • WO 98/33374 did not address the subject of stabilization and control of apomixis. Hence, methods for modifying an apomict once it is synthesized were not provided. In view of the above, it would be advantageous to provide methods that permit development of apomictic lines that are obligate, obligate unless induced to be facultative, or facultative unless induced to be obligate. By inducing facultative apomixis, the apomictic line can be improved, by conventional breeding strategies, and subsequently returned to the obligately apomictic condition for many years of field production. It should -be appreciated that these and other advantages of the present application (discussed below) represent major advancements in the state-of-the-art .
  • high frequency sexual seed formation occurs except in the optional case of an inducible down regulation of a transgenic promoter/gene construct, which gene construct enforces high frequency sexual embryo sac and seed formation when expressed, such that obligate to near obligate apomixis is expressed ( ⁇ 5% sexual seed formation) during which time apomictic hybrid seed may be multiplied; or
  • obligate to near obligate apomixis occurs ( ⁇ 5% sexual seed formation) except during an inducible up regulation of a transgenic promoter/gene construct that when expressed causes high frequency sexual seed formation (>5%) during which time said plant may be further enhanced genetically through plant breeding procedures.
  • FIG. 1 shows stages in the evolution of agamic complexes.
  • the stages include ecotypic differentiation prior to the formation of apomicts, formation of stage I apomicts through secondary contact hybridization, formation of stage II apomicts through structural (karyotypic) stabilization (usually involving polyploidization) , and formation of mature, ecologically-diverse agamic complexes (stage
  • FIG. 3 shows mean integument and ovule lengths (actual measurements, bottom left and right, and as percentages of mature integument and ovule lengths, top left and right) at the dyad, 2- nucleate embryo sac, and mature embryo sac stages for nine diploid progenitors of apomictic Antennaria rosea .
  • the data in figures 8 through 11 depict variation, among plant ecotypes, in schedules of ovule development. This natural ecotypically-derived variation has never before been characterized, and it is a prerequisite for apomixis arising in nature and in synthetic hybrids.
  • FIG. 4 shows one of several measures of duration of meiosis among 17 ecologically diverse Sorghum land races and varieties. Duration of meiosis is only one of several types of ecotypically- derived interracial/interspecific variation observed in the schedules of ovule development maintained by different ecotypes of flowering plants.
  • the bars represent the duration of time between the dyad stage and the time in which embryo sac formation is initiated (as a function of inner integument growth), i.e. short and long bars represent lines with very little and much delay, respectively, between meiosis and embryo sac formation.
  • FIG. 5 shows flower bud maturity at the time of megasporogenesis (female meiosis) as measured by mean inner integument lengths (portrayed as percentages of mature integument lengths) at the dyad stage of meiosis for parent lines of three
  • FIG. 7 shows megasporogenesis and embryo sac development in sexual Sorghum plus apomictic (aposporous) embryo sac development in a synthetic Sorghum hybrid produced from sexual lines. About 5% of pistils in the hybrid exhibit aposporous initials and/or aposporous embryo sac formation. Diplospory is not observed in the parent lines. Note from FIG. 3 that hybrids producing low frequency aposporous embryo sac formation are derived from parent lines that are not strongly divergent in timing of meiosis relative to overall bud development.
  • About 50% of pistils in the hybrid exhibit diplosporous embryo sac formation. Parthenogenic embryo formation from a reduced egg has been observed cytoembryologically (presence of a globular stage embryo with no fertilization of the central cell having yet occurred) .
  • FIG. 9 shows sexual megasporogenesis and sexual and diplosporous embryo sac development in a synthetically stabilized obligately-apomictic trispecific triploid Tripsacum hybrid produced from sexual diploids ( T. laxum / T. pilosum // T. zopilotense) . About 80% of pistils in the hybrid exhibit diplosporous embryo sac formation. The remaining pistils are abortive.
  • apomictic instability of an apomictic plant means the average frequency of sexual seed formation among sexually produced progeny of such plant exceeds that of such apomictic plant.
  • stabilizing means assuring that the average frequency of sexual seed formation among sexually derived progeny of such plant does not exceed that of such apomictic plant.
  • complete apomixis means (1) preemption of megasporogenesis by precocious embryo sac formation, (2) preemption of fertilization by precocious embryony, and (3) formation of endosperm either pseudogamously (through fertilization of the central cell but not the egg) or autonomously (without fertilization of the central cell) .
  • Tenets 4 and 5 of the HFA theory of apomixis are as follows:
  • Tenet 4 states that in the absence of structural or karyotypic heterozygosity, sexually produced progeny of a near obligate or facultative apomict generally reproduce sexually, that is, they are sexual revertants.
  • the divergent alleles responsible for parental phenotypes segregate during sexual gamete formation, which in apomicts occurs rarely to frequently during megasporogenesis (female meiosis) and usually frequently during microsporogenesis (male meiosis) .
  • loss of apomixis in the sexual F 2 generation is analogous to loss of hybrid vigor in the F 2 generation of standard hybrid varieties of crops. Both are complex polygenic hybrid phenotypes.
  • Tenet 5 states that in the presence of structural heterozygosity, sexually produced progeny of a near obligate or facultative apomict generally reproduce either apomictically, mimicking a homozygous dominant condition, or both sexually and apomictically (in a near 1:1 segregation ratio), mimicking a heterozygous dominant condition.
  • structural (karyotypic) heterozygosity which includes, but is not limited to, allopolyploidy, segmental allopolyploidy, sexual sterility, or paleopolyploidy .
  • Structural heterozygosity is responsible for apomixis mimicking simple inheritance.
  • Intraspecific apomictic diploid hybrids whose sexual progeny are usually weakly apomictic or totally sexual due to recombination of the polygenic heterozygosity necessary for apomixis, are stabilized by inducing triploidy or other odd polyploid level. This results in near obligate apomixis.
  • genetically-reduced and recombined functional eggs are seldom produced and seldom fertilized by genetically-reduced and recombined functional sperm, the production of which is greatly reduced.
  • the intragenomic polygenic heterozygosity responsible for apomixis is seldom disturbed in odd polyploid apomicts.
  • Allopolyploidy (polyploidy involving different species) is generally the most convenient mechanism for restricting recombination. In an allopolyploid, recombination generally occurs only within genomes, not between genomes. Hence, genes responsible for apomixis are maintained, through facultative sexual generations, in a homozygous condition within genomes but a heterozygous condition between genomes.
  • cytogenetic mechanisms can be used to prevent recombination within or among whole genomes or only portions of genomes. This application extends to all such mechanisms including inversion or translocation heterozygosity and mechanisms of genetically controlled meiotic drive.
  • Fertility levels of interspecific apomictic diploids exhibiting low fertility are increased by polyploidization either at the even (e.g., tetraploid, hexaploid, and the like) or odd (e.g., triploid, pentaploid, and the like) levels.
  • Apomictic polyploids produced in this manner may produce some sexually-derived progeny, i.e. they are generally facultative apomicts.
  • Such sexually-derived progeny are also facultative apomicts because the polygenic heterozygosity required for apomixis exists between genomes not within genomes.
  • Allopolyploidy fixes the responsible intergenomic heterozygosity such that occasional intragenomic recombination does not affect the allelic composition of the divergent intergenomic loci. Segmental allopolyploidization is encouraged by way of the methods of the present invention to enhance pollen fertility, unreduced embryo sac and egg production and viability, and unreduced egg parthenogenesis.
  • stage II apomicts may periodically engage in B-. and B m hybridization with related apomicts and with ecologically-divergent sexual relatives to produce heterogeneous stage III agamic complexes (FIG. 1) .
  • stage III apomicts today continue to assimilate, through facultative outcrossing with sexual and apomictic relatives, the genetic capacity to migrate into new and ecologically diverse habitats.
  • R.J. Bayer Evolution of Polyploid Agamic Complexes with Examples from Antennaria (Asteraceae) , 132 Opera Bot . 53-65 (1996).
  • the parental sexual phenotypes of apomicts are polygenic coadaptations, A.R. Templeton, Coadaptation and Outbreeding Depression, in M.E. Soule, Conservation Biology: The Science of Scarcity and Diversity 105-116 (Sinauer Assocs. Inc., Sunderland, MA 1986) ; B. Wallace, Coadaptation Revisited, 82 J. Hered. 89-95 (1991) , encoded by unique groupings of alleles that function cooperatively to confer fitness to specific ecotypes adapted to specific environments. Any significant recombination between parental genomes, i.e.
  • stage I apomicts are eventually replaced by sexual segregants that generally contribute only sexual progeny to the population.
  • stage II The vast majority of diploid stage I apomicts that successfully progress to stage II (FIG. 1) are stabilized by allopolyploidy or segmental allopolyploidy.
  • the rate at which stabilization occurs depends on the relatedness of the parental lines and on certain conditions in the secondary contact hybridization zone.
  • One parental line, in_such zones, is usually more common than the other.
  • pollen from the predominant parent is more likely to be involved in B I ⁇ hybridizations (fertilization of unreduced eggs) to form triploids with a 2:1 genome ratio.
  • the B ⁇ hybrid may be formed from unreduced pollen of the stage I apomict that affects fertilization of the predominant parent producing the same 2:1 genome ratio.
  • triploids Assuming the triploids also produce unreduced eggs (show tendencies for apomixis) or pollen, a second round of backcrossing involving the same predominant diploid sexual parent results in a 3:1 genome ratio. Such ratios are probably common among apomicts, and they explain simple inheritance segregation ratios and hemizygous apomixis-conferring linkage groups.
  • the triploid may be involved in B m hybridization with the other parent, in which case a 2:2 genome ratio occurs.
  • Other forms of polyploidization, involving unreduced pollen and eggs or somatic doubling, may produce similar results.
  • allopolyploidy is probably the most common form of apomixis stabilization. Recombination in true allopolyploids occurs within genomes only. Hence, loci critical to high frequency (near obligate) apomixis are isolated from intergenomic segregation and independent assortment, i.e. they remain homozygous within genomes but heterozygous across genomes.
  • T and T 1 are divergent and encode divergent patterns of ovule development, remain apomictic but are phenotypically variable because of within-genome recombination involving heterozygous loci not critical to apomixis.
  • TTT T' apomicts reproduce sexually, the polygenic capacity for apomixis (from mostly sexual to nearly obligate) often segregates in a simple Mendelian manner. This occurs because it cosegregates with a nonrecombinant T' univalent (or large linkage group) that contains most of the more critical divergent alleles required for expression of a low to high frequency apomixis. It is likely that the many genes essential to a near obligate apomixis occur on several chromosomes. Hence, in crosses between TTTT sexual lines and TTT T' apomictic lines, facultativeness will vary from
  • Chromosome assortment in an apomixis-conferring homeologous TTT T' set occurs as if all four chromosomes are homologous.
  • each of the three homologous T chromosomes has an equal chance of associating with its respective homeologous T' chromosome.
  • the chromosome set mimics an autopolyploid.
  • segmental allopolyploid apomicts evolve from early stage interracial autopolyploid or weakly allopolyploid TTT T' or TT T'T' apomicts.
  • Recombination within the homeologous set(s) of chromosomes critical to apomixis is often nonadaptive because it usually results in sterile sexual segregants.
  • allelic recombinations, chromosomal aberrations, or even mutations that inhibit recombination within the apomixis-conferring homeologous set cause a further allopolyploidization, G.L.
  • segmental allopolyploidy is eliminated by autopolyploidization early in the evolution of polyploids that originate as weak allopolyploids or interracial autopolyploids.
  • the segmental allopolyploid apomict appears to be an exception.
  • facultative apomixis coupled with segmental allopolyploidy are interdependent and highly adaptive traits, i.e. they function synergistically in the evolution and stabilization of mature highly successful agamic complexes (FIG. 1) .
  • diploid apomicts exist in nature, and some of these are probably stabilized by near obligate sexual sterility, which prevents segregation. These may form either by interspecific hybridization of sexual diploids or from allopolyploid apomicts by parthenogenesis of reduced eggs. Examples include diploid apomicts in Potentilla, Muntzing & Muntzing, The Mode of Reproduction of Hybrids Between sexual and Apomictic Potentilla argentea , 1945 Bot. Not. 49-71 (1945), Hierochloe, G. Weimarck, Apomixis and Sexuality in Hierochloe australis and in Swedish H. odorata on Different Polyploid Levels, 120 Bot. Not.
  • homeology is sufficient to restrict facultative recombination to like genomes. This homeology mechanism maintains the cross-genome heterozygosity that often causes apomixis to appear to be simply inherited when apomicts are used as male parents in crosses between sexuals and apomicts.
  • sexual sterility provides added stabilization to polyploid and aneuploid apomicts.
  • a few examples include (i) triploid apomicts in
  • bispory, tetraspory and polyembryony are also polygenically-determined, anomalous, and developmentally-intermediate (hybrid) phenotypes.
  • many bisporic, tetrasporic and polyembryonic species are diploids, and nearly all bisporic and tetrasporic species are completely sexual. J.G. Carman, 61 Biol. J. Linnean Soc.
  • Possible mechanisms of formation include ascending or descending aneuploidy and structural reorganizations of parental genomes.
  • such mechanisms may also arise as polygenic hybrid phenotypes that are stabilized by normal or segmental allopolyploidy, sexual sterility, diploidization, or other cytogenetic mechanisms that prevent recombination of the multilocus heterozygosity critical to their maintenance.
  • the type of stabilization mechanism differentially affects heterosis and gene flow.
  • allopolyploidy of the form TT T'T' instantaneously stabilizes apomixis, but, barring mutations and infrequent outcrossing, few mechanisms exist for improving the fertility of such apomicts by modifying the original coadapted ovule-development programs.
  • potentially effective mechanisms for genome modification exist among segmental allopolyploid apomicts. In such apomicts, recombinational mixing occurs within those homeologous chromosome sets not directly involved in conferring apomixis, which probably includes the majority.
  • Recombinations within these sets may enhance sexual pollen development, asexual egg development, parthenogenesis of unreduced eggs, and heterosis.
  • apomicts originating as inter-racial autopolyploids or weakly interspecific hybrids may rapidly acquire, through natural selection and autopolyploidization of nonapomixis-conferring chromosome sets, recombinations that confer high seed sets and high pollen fertility.
  • Intergenomic recombinations deleterious to female sexuality reinforce selection against sexual revertants. Some intergenomic recombinations may cause the duplication or deletion of certain ovule development steps as seen in bispory and tetraspory.
  • apomixis may serve as an evolutionary springboard in the evolution of reproductively novel sexual species and genera including some that are bisporic, tetrasporic, or polyembryonic. J.G. Carman, 61 Biol. J. Linnean Soc. 51-94 (1997) .
  • apomicts originating strictly as genomic allopolyploids either TT T'T' or TTT T', may retain indefinitely many intergenomic heterozygosities not well adapted to apomixis.
  • the present invention is directed to processes for producing genetically stabilized apomictic plants and genetically stabilizing natural or synthetically produced apomictic plants that exhibit genetic instability. It is also directed toward processes for controlling the expression of apomixis (facultativeness) for purposes of plant improvement, seed production, and crop production. It is convenient to separate the processes of the present invention into four categories: (a) assessing genome homeology, facultativeness, and apomixis stability, (b) plant breeding, amphiploidization, and mutagenesis processes, (c) gene mapping and cloning processes, and (d) genetic engineering processes.
  • a feature of the present invention is the stabilization of apomixis in natural or synthetic apomicts by creating karyotypic (structural) heterozygosity. This is readily accomplished when apomicts are synthesized from sexual plants by choosing interspecific or interracial parental lines that also conform to the requirements of divergence in reproductive schedules of ovule development as taught in WO 98/33374.
  • a preferred method of assessing the degree of karyotypic homeology of two sexual lines involves conventional genome analyses where hybrids are produced and the extent of chromosome pairing is evaluated at metaphase I in pollen mother cells (PMC). D.R.
  • Facultativeness is a measure of the percentage of viable seeds that are formed sexually from an apomictic plant. A preferred method for determining this percentage is to conduct progeny tests in which the progeny are compared with the mother plant. Modern molecular fingerprinting techniques are preferred because of their reliability and ease of use once the systems are optimized. 0. Leblanc & A. Mazzucato, Screening Procedures to Identity and Quantify Apomixis, in Y. Savidan & J. Carman, Advances in Apomixis Research (FAO, CIMMYT, ORSTOM, in press) .
  • Degree of stability is assessed by conducting progeny tests on the off types identified in the first generation progeny tests.
  • Progeny families whose members are apomictic like the mother plant come from a genetically stable (karyotypically heterozygous) apomictic mother.
  • Progeny families whose members are represented by high percentages of sexual revertants come from genetically unstable apomicts.
  • synthetic or natural diploid apomicts or natural dihaploid apomicts are unstable.
  • Synthetic or natural polyploid apomicts may or may not be stable.
  • a preferred method is to increase genetic diversity and combining ability of sexual parental lines known to produce apomictic diploids or polyploids.
  • Plant breeding or genetic engineering are used to genetically modify two sets of delineated parent lines of a plant species or closely related group of plant species that are differentiated in their reproductive phenotype such that hybridizing any plant from one of the two sets of delineated lines with any plant from the other set of delineated lines produces an apomictic plant or a plant that becomes apomictic through amphiploidization or further hybridization.
  • Combining ability of parent lines is improved by standard crossing and inbreeding procedures or by single cross, double cross, or multi cross (outcrossing) procedures that are conducted within each set of delineated lines.
  • a feature of the present invention is the delineation of a new hybrid breeding system by which synthetically-derived hybrid apomicts are obtained.
  • the system involves not only the identification of sexual inbred parent lines, which express good combining ability, but the identification of hybrid or multiply- outcrossed parental lines within the two sets of delineated lines such that good combining ability is expressed when a plant from one of the two sets of delineated lines is hybridized with a plant from the other set of delineated lines.
  • this new operational system produces single or multicross hybrids that are either apomictic or become apomictic through amphiploidization or further hybridization. By this means, many apomictic hybrid genotypes can be produced (from each cross).
  • each individual genotype may be increased through apomictic seed formation for field testing and/or cultivar release. Consequently, an unlimited number of new apomictic genotypes is rapidly produced.
  • This technique will greatly increase the genetic diversity of plants used for agriculture and greatly increase the ability of breeders to provide apomictic hybrid varieties specifically adapted to highly, moderately or marginally productive agricultural regions.
  • a feature of the present invention extends the standard definition of combining ability to include development of divergent but highly heterozygous sexual parent lines that when hybridized (or hybridized and amphiploidized) result in apomictic plants with superior hybrid vigor.
  • the genetically heterogeneous apomictic progeny obtained from crosses involving heterozygous (outcrossed) parental lines (sexual or apomictic) are individually evaluated for agronomic desirability and selected for cultivar development.
  • a preferred method is to cross a facultatively apomictic plant with genetically divergent sexual or apomictic lines to produce derived lines with enhanced agronomic traits.
  • the chromosome numbers of hybrids are doubled using standard colchicine techniques, e.g., J. Torabinejad et al . , Morphology and Genome Analyses of Interspecific Hybrids of Elymus scabrus, 29 Genome 150-55 (1987) .
  • standard colchicine techniques e.g., J. Torabinejad et al . , Morphology and Genome Analyses of Interspecific Hybrids of Elymus scabrus, 29 Genome 150-55 (1987) .
  • recently developed tissue culture techniques may be used.
  • a preferred method for doubling chromosomes of intraspecific and interspecific hybrids is to use one or more of the colchicine (or other known spindle inhibitor chemical) treatment methods listed above.
  • a preferred method for doubling chromosomes of interspecific hybrids involves backcrossing to one of the sexual parents and counting chromosomes in root tips to determine partial amphiploidy (usually triploidy) . This is followed by backcrossing to the other parent to obtain a full amphiploid, or to the same parent to obtain a partial amphiploid (three genomes from one parent and one genome from the other) . Amphiploidization may precede or follow hybridization.
  • Hybrids are produced between sexual varieties or lines that display appropriate degrees of divergence in photoperiod responses and female developmental schedules. Intraspecific hybrids are made using standard techniques as taught in plant breeding texts, e.g.
  • a feature of the present invention involves controlling facultativeness by modifying expression of quantitative trait loci (QTLs) important to facultative expression using antisense technology.
  • QTLs quantitative trait loci
  • a preferred method begins with QTL mapping of the divergent sexual parental reproductive phenotypes responsible for apomixis occurring in hybrids produced by crossing said phenotypes.
  • the method involves producing an F 2 mapping population, consisting of sexually derived F 2 progeny of a facultative synthetic F- . apomict produced by hybridizing the original reproductively-divergent parent lines, and identifying molecular markers that associate with each phenotype, e.g. A.W. Heusden et al . , Three QTLs from Lycopersicon peruvianum Confer a High Level of Resistance to Clavibacter michiganensis ssp. Michiganensis, 99 Theor. Appl. Genet. 1068-1074
  • Important QTL(s) are then fine-mapped to a given chromosome using a large segregating population and yeast artificial chromosomes (YACs) encompassing the chromosomal region are isolated by using flanking markers.
  • YACs yeast artificial chromosomes
  • a cosmid clone is then produced containing the QTL and complementing cosmids are identified by transformation into the mutant.
  • the QTL transcript is then identified by cDNA isolation using the complementing cosmids, e.g. H.Q. Ling et al . , Map-based Cloning of Chloronerva, a Gene Involved in Iron Uptake of Higher Plants Encoding Nicotianamine Synthase, 96 Proc. Nat'l Acad. Sci.
  • BACs bacterial artificial chromosomes
  • S.S. Woo et al. Construction and Characterization of a Bacterial Artificial Chromosome Library of Sorghum bicolor, 22 Nucleic Acids. Res. 4922-4931 (1994).
  • a feature of the present invention is to control degree of facultativeness by controlling the expression of a QTL important to facultative expression.
  • Another feature of the present invention is to permanently (or reversibly) convert facultative apomicts to obligate apomicts by controlling the expression of meiosis-specific genes .
  • the preferred method for accomplishing obligate apomixis is to breed or transform a facultatively apomictic plant such that it contains a genetic material that causes female meiosis to abort resulting in essentially 100% apomictic seed formation.
  • the genetic material may be a meiotic mutant, introduced through breeding, or a transgenic promoter/gene construct that when expressed disrupts female meiosis.
  • transgenic promoter/gene construct which gene construct causes meiotic abortion when expressed, allows for facultative apomixis to occur.
  • facultative apomixis may occur except during an inducible up regulation of the transgenic promoter/gene construct thus causing meiotic abortion and essentially 100% apomictic seed formation.
  • 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 that when expressed fatally disrupts meiosis, e.g., V.I. Klimyuk & J.D.G. Jones, AtDMCl, the Arabidopsis homologue of the yeast DMC1 gene: characterization, transposon-induced allelic variation and meiosis-associated expression, 11 Plant J. 1-14 (1997); PCT/GB97/03546.
  • the transgenic material which is normally cytotoxic to female meiocyte development, may be controlled by a suppressor molecule encoded by a gene that is controlled by a chemically inducible promoter, which may be a female-meiocyte-specific promoter, such that female fertility (facultativeness) is inducible in such apomict.
  • the transgenic material may contain a gene from the group of sense or antisense genes that when expressed during meiosis fatally disrupts meiosis or is otherwise cytotoxic to the female meiocyte.
  • the method for restoration of a low level of female sexuality in a transgenically-derived obligate apomict may involve expression of a suppressor by induction of the inducible promoter.
  • Introduction of the transgenic material into the host plant may employ any available technique well known to those skilled in the art.
  • the presently preferred procedure of selecting appropriate sexual parent lines is to (a) identify, from the literature or field studies, natural ecotypes and unimproved land races of a given crop species and its closely related species that differ with regard to shade tolerance, latitude, photoperiod requirements for flowering, altitude, and moisture preferences, (b) cytoembryologically characterize physiologically and ecologically divergent lines by relating stages of megasporogenesis and embryo sac development to stages of integument and gross ovary development, (c) characterize and statistically analyze the cytoembryological differences among lines, and (d) choose lines that are divergent physiologically (e.g. photoperiodism) , cytoembryologically, and taxonomically .
  • plants classified as different species i.e. pairs of plants whose hybrids are sterile, should possess sufficient genome homeology to assure karyotypic heterozygosity once the hybrid produced between them is amphiploidized.
  • Example 2 Synthesizing Genetically-stable Facultative and Obligate Apomicts
  • the techniques in Example 1 are used as guidelines to obtain three or more sexual lines with an early meiosis/early gametophyte development relative to development of the integument (s) .
  • the same techniques are used as guidelines to obtain three or more sexual lines of a closely related species with a late meiosis/late gametophyte development relative to development of the integument (s) .
  • the several lines of each category are selected such that embryo sac formation in one set of lines occurs at about the same time as prophase to early meiosis in the other set of lines relative to development of the integument (s) .
  • Pairs of parent plants are hybridized and amphiploids are produced using standard procedures described above. It will be appreciated that the genetic background in which the lines are derived may influence the expression of apomixis. Thus, selection or production of additional lines incorporating different genetic backgrounds and more or less divergence in timing of meiosis may be necessary.
  • Facultative apomicts which are unstable, meaning they produce sexual segregants as a result of facultative sexual reproduction, are synthesized as a result of hybridization-derived floral asynchrony by producing synthetic diploid Antennaria corymbosa (2x sexual) X A. racemosa (2x sexual) hybrids (FIG. 6) and synthetic diploid Sorghum (2x sexual) ssp. hybrids (FIG. 7) .
  • Aposporous embryo sacs form in Sorghum hybrids 5-1 X 4-1 and 9-1 X 1-2 at about a 5% frequency, and diplosporous embryo sacs, similar to those in
  • Tripsacum (FIG. 8) , form in Sorghum hybrids 5-2 X 9-2 at about a 10% frequency. Note that the divergence in timing of meiosis relative to integument development is substantial (FIG. 5) in the parental pairs whose progeny form diplosporous embryo sacs. Structurally heterozygous (stable) facultative apomicts may be produced from the interspecific Antennaria and Sorghum F x hybrids by doubling their chromosome number using techniques discussed above. Stabilization of the intraspecific Sorghum hybrids (referred to above) requires a genetic modification that causes female meiosis or its immediate cell produces to abort, which not only stabilizes apomicts but makes them obligate.
  • a meiotic mutant into the line through standard hybridization procedures, by inducing triploidy through B ⁇ hybridization or amphiploidization followed by hybridization with a diploid, or by transforming the diploid with a promoter/gene construct that is cytotoxic to the female meiocyte using the methods discussed above.
  • inducible promoters as discussed above, genetically-stable apomicts with induced obligate or facultative expression may be produced.
  • T. pilosum (2x sexual) is a stable facultative apomict with 50% diplosporous embryo sac formation (FIG. 8) .
  • Crossing this plant with T. zopilotense (2x sexual) or r. Jbravum (2x sexual) produces stable obligate apomicts with about 80% diplosporous embryo sac formation and 20% abortive meiocyte or sexual embryo sac formation (FIG. 9) .
  • Brachiaria brizantha Brachiaria decumbes and F- . hybrids with sexual colchicine induced tetraploid Brachiaria ruziziensis, 78 Euphytica 19-25 (1994); C.B. Do Valle & J.W. Miles, Breeding of apomictic species, in Y. Savidan et al . , Advances in Apomixis Research (2000); P. Ozias-Akins et al., 95 Proc. Nat'l Acad. Sci. USA 5127-5132 (1998), lumped plants into the apomixis category in which percentages of ovules developing apomictically were as low as 12, 28, 17, and 7 %, respectively.
  • QTL mapping is conducted for the divergent sexual parental reproductive phenotypes responsible for apomixis occurring in hybrids (FIGS. 2-5).
  • Examples 1 through 3 are used as guidelines to synthesize genetically-stable highly-facultative apomicts with inducible obligate expression or genetically-stable obligate apomicts with inducible highly-facultative expression.
  • apomixis is analogous to a computer operating system.
  • biological operating system include the following: (i) in farmers' fields, true-to-type "cloning" of hybrids from the hybrids' own seed - generation after generation, (ii) in plant breeders' nurseries, partial sexuality for plant improvement followed by reversion to strict apomixis, (iii) large numbers of rapidly-produced and genetically-diverse cultivars tailored to diverse agricultural niches, (iv) an increase in overall genetic diversity for protecting against widespread crop devastation by pests, and (v) a win-win reduction in expenses, i.e. farmers pay less for seed, and seed companies pay less to develop superior crop varieties .

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Abstract

L'invention concerne l'induction, la stabilisation et le contrôle de l'apomixie (formation de semences asexuées) dans des angiospermes dans le but de produire, d'améliorer et d'utiliser de façon économique des récoltes agamiques de productivité supérieure (récolte possédant une capacité de production de semences à la fois sexuées et apomictiques). La stabilisation de l'apomixie concerne des procédés qui soit convertissent une plante apomictique facultative en apomixie obligatoire, soit confèrent des mécanismes à une plante apomictique facultative qui empêchent, pendant la formation facultative de semences sexuées, la recombinaison de gènes provoquant l'apomixie, de sorte que la descendance produite sexuellement à partir de ladite plante apomictique facultative hérite des combinaisons alléliques nécessaires à l'apomixie. Plus particulièrement, l'invention concerne: (i) la production de deux ou plusieurs ensembles de lignées sexuées génétiquement divergentes, fécondées par la même espèce ou par une variété différente de cette espèce, ces ensembles de lignées parentales restant homozygotes pour des gènes provoquant l'apomixie quand on obtient des hybrides par croisement d'un membre d'un de ces ensembles de lignées avec un membre d'un autre ensemble; (ii) l'hybridation de lignées parentales sexuées de la même espèce ou obtenues par croisement avec des variétés différentes afin de produire des plantes apomictiques; (iii) la stabilisation de ces plantes apomictiques par modifications cytogéniques ou moléculaires; (iv) l'amélioration de plantes apomictiques au moyen de la production naturelle ou par génie génétique de lignées parentales ou de plantes apomictiques; (v) la modification de plantes apomictiques facultatives afin de faire échouer la méiose femelle provoquant une apomixie obligatoire sauf quand un ADN de recombinaison faisant échouer la méiose est inhibé et (vi) la modification de plante apomictique facultative afin de conserver le caractère facultatif sauf pendant l'expression pouvant être induite d'un ADN de recombinaison provoquant une apomixie obligatoire par échec de la méiose. On peut combiner de façon sélective les différentes facettes de ce système opérationnel biologique afin d'améliorer l'efficacité de la production et de l'utilisation de récoltes agamiques.
PCT/US2000/029905 1997-02-05 2000-10-30 Procedes servant a stabiliser et a controler l'apomixie WO2001032001A1 (fr)

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MXPA02004186A MXPA02004186A (es) 1999-10-29 2000-10-30 Metodo para estabilizar y controlar apomixis.
IL14933500A IL149335A0 (en) 1999-10-29 2000-10-30 Methods for stabilizing and controlling apomixis
BR0015276-5A BR0015276A (pt) 1999-10-29 2000-10-30 Métodos para estabilizar e controlar apomixia
EP00978296A EP1227715A4 (fr) 1999-10-29 2000-10-30 Procedes servant a stabiliser et a controler l'apomixie
CA002389055A CA2389055A1 (fr) 1999-10-29 2000-10-30 Procedes servant a stabiliser et a controler l'apomixie
EA200200507A EA006658B1 (ru) 1999-10-29 2000-10-30 Способ получения апомиктичного растения
JP2001534218A JP2003516725A (ja) 1999-10-29 2000-10-30 アポミクシスを安定化および調節する方法
NZ519156A NZ519156A (en) 1999-10-29 2000-10-30 Methods for stabilizing and controlling apomixis
AU15772/01A AU1577201A (en) 1999-10-29 2000-10-30 Methods for stabilizing and controlling apomixis
HK03100846.3A HK1048741A1 (zh) 1999-10-29 2003-02-06 穩定和控制無融合生殖的方法
US10/772,243 US7638680B2 (en) 1997-02-05 2004-02-06 Methods for stabilizing and controlling apomixis
US12/416,883 US20090217409A1 (en) 1997-02-05 2009-04-01 Methods for increasing the frequency of apomixis expression in angiosperms

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US7638680B2 (en) 1997-02-05 2009-12-29 Utah State University Methods for stabilizing and controlling apomixis
CN102960242A (zh) * 2012-12-17 2013-03-13 沈阳金色谷特种玉米有限公司 一种用炔苯酰草胺诱导玉米孤雌生殖的育种方法
CN103081804A (zh) * 2012-12-17 2013-05-08 沈阳农业大学 一种用甲基胺草磷诱导玉米孤雌生殖的育种方法
US8878002B2 (en) 2005-12-09 2014-11-04 Council Of Scientific And Industrial Research Nucleic acids and methods for producing seeds with a full diploid complement of the maternal genome in the embryo
CN104145805A (zh) * 2014-07-14 2014-11-19 西北农林科技大学 使有性玉米产生显性和隐性无融合生殖杂种的方法
US9326463B2 (en) 2005-03-03 2016-05-03 Rijk Zw Aan Zaadteelt En Zaadhandel B.V. Near reverse breeding
US10131919B2 (en) 2011-05-30 2018-11-20 Leibniz-Institut Fur Pflanzengenetik Und Kul Turpflanzenforschung Gatersleben (Ipk) Means and methods to induce apomixis in plants

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ES2554471T3 (es) * 2005-03-03 2015-12-21 Rijk Zwaan Zaadteelt En Zaadhandel B.V. Mapeado de progenie inverso
CN115119744A (zh) * 2022-07-26 2022-09-30 山东农业大学 一种苹果无融合一系四群体育种方法

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7638680B2 (en) 1997-02-05 2009-12-29 Utah State University Methods for stabilizing and controlling apomixis
US9326463B2 (en) 2005-03-03 2016-05-03 Rijk Zw Aan Zaadteelt En Zaadhandel B.V. Near reverse breeding
US9332697B2 (en) 2005-03-03 2016-05-10 Rijk Zwaan Zaadteelt En Zaadhandel B.V. Near reverse breeding
US8878002B2 (en) 2005-12-09 2014-11-04 Council Of Scientific And Industrial Research Nucleic acids and methods for producing seeds with a full diploid complement of the maternal genome in the embryo
US10131919B2 (en) 2011-05-30 2018-11-20 Leibniz-Institut Fur Pflanzengenetik Und Kul Turpflanzenforschung Gatersleben (Ipk) Means and methods to induce apomixis in plants
US10907174B2 (en) 2011-05-30 2021-02-02 Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung Gatersleben (IPK) Means and methods to induce apomixis in plants
CN102960242A (zh) * 2012-12-17 2013-03-13 沈阳金色谷特种玉米有限公司 一种用炔苯酰草胺诱导玉米孤雌生殖的育种方法
CN103081804A (zh) * 2012-12-17 2013-05-08 沈阳农业大学 一种用甲基胺草磷诱导玉米孤雌生殖的育种方法
CN104145805A (zh) * 2014-07-14 2014-11-19 西北农林科技大学 使有性玉米产生显性和隐性无融合生殖杂种的方法

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