CN1267187A

CN1267187A - Method for producing apomictic plants

Info

Publication number: CN1267187A
Application number: CN98803377A
Authority: CN
Inventors: J·G·卡曼
Original assignee: University of Utah
Current assignee: Utah State University USU; University of Utah
Priority date: 1997-02-05
Filing date: 1998-02-05
Publication date: 2000-09-20
Anticipated expiration: 2018-02-05
Also published as: EP1014778A1; AU6055498A; CN1206903C; EP1014778A4; JP2002510964A; WO1998033374A1; IL131196A0; CA2280145A1; AU732743B2; AU732743C; EA004253B1; EA199900712A1; NZ337683A

Abstract

Methods are provided for producing apomictic plants from sexual plants divergent with respect to responses to different photoperiods and schedules of megaspore and gametophyte development. A preferred system is to identify divergent lines from within a species or closely related group of species, accentuate the divergence by breeding where necessary, and produce artificial amphiploids that contain genomes from the apposing divergent lines. Apomixis results from the asynchronous expression of female developmental programs induced by combining the reproductively divergent lines. The procedures for manipulating the expression of apomixis described herein permit the development of true-breeding hybrids of various cultivated crops.

Description

The method for preparing the asexual reproduction plant

The mutual reference of related application

The application requires the right of the U.S. Provisional Application 60/037,211 of submission on February 5th, 1997.

Background of invention

The present invention relates to can be by the hereditary method of cloning the plant of self of the seed (gametophyte agamete) of self from normal amphigenetic plant preparation.More particularly, the method that the present invention relates to comprises the sexual reproduction strain system that (a) selects two or more expression reproduction phenotypes to differ from one another, it needs plant breeding in some cases and selects to obtain the difference of enough degree, (b) breeding phenotype different plant intermolecular hybrid, and (c) carry out amphiploidization (chromosome doubling) before or after the hybridization.

Asexual reproduction seems more can improve than other any plant mechanism the output of food, feed and fiber.Asexual reproduction betides about 0.3% flowering plant species.Present patent application has been described and has not been used two kinds all to be attempted but the not satisfied method of effect, and promptly sexual reproduction plant and wild type asexual reproduction correlative are hybridized and method of mutagenesis, makes the method for sexual reproduction Asexual Reproduction in Plant.Method described herein has been simulated the mode (J.G.Carman that the asexual reproduction plant is evolved naturally, the asynchronous expression of duplicate factor in angiosperm can cause asexual reproduction, two basidiospore formation, tetraspory and polyembryony, and 61 Biol.J.Linnean Soc.51-94 (1997) (are incorporated herein by reference; Hereafter be called " Linnean "), and can make the skilled person in plant breeding and genetics field the inbreeding crops can be comprised that wheat, barley and paddy rice are transformed into the agamic hybrid crops of the inbred variety raising 10-30% of potential production ratio use at present.Hybrid crops such as the corn that uses also can be transformed into agamic at present.The vegetative hybrid of inbreeding or typical hybrid crops is being a hybrid aspect its good output just.The seed of vegetative hybrid is the heredity clone of its maternal hybrid, that is, heredity do not occur and separate.Therefore, year after year, the farmer can also can expect to obtain high yield and homogeneity as seed with the sub-fraction of its harvested product.This will make the very expensive hybrid of common seed be used for poor country first, and another " green revolution " made huge contribution.

Gregor Mendel has carried out gametophyte agametic (plant of asexual generation seed) genetic experiment first under unconscious situation.He has reported the successful hybridization that different hawkweed strains are but has commented on and has prevented autogamous being the devil.What he thought is that high-frequency unexpected selfing (in his facultative apomixis hawkweed strain system) is actually high-frequency asexual reproduction seed formation.Except his setback, he also fails the minority F that manages to produce at him ₁The F of s ₂Observe separation among the s.His F ₂S is actually his F ₁The asexual reproduction of s clone, they express their F separately without any exception ₁Phenotype (S.E.Asker ﹠amp; L.Jerling, the asexual reproduction in the plant (CRC publishing house, 1992) (is incorporated herein by reference; Hereafter be called as " Asker ﹠amp; Jerling ").

When Mendel made member's hybridization of this agamic complex, thousands of kinds of hawkweeds were described.This tangible polymorphism, the result that the facultative apomixis body produces new polymorphism that passes through of observed polymorphism and Mendel makes early stage geneticist infer that the hybridization in the agamic complex is the main mechanism that species form in other agamic complex (Antennaria, Erigeron, Taraxacum, Potentilla).Support the complete evidence of this conclusion, as, (C.S.Campbell ﹠amp in Amelanchier and Crataegus; T.A.Dickinson, asexual reproduction, species concept in the pattern of morphosis and the Maloideae subfamily (Rosaceae), 15 SystematicBot.124-25 (1990) (being incorporated herein by reference), at Antennaria (Bayer etc., Antennaria system generation inference (Asteraceae:Gnaphalieae) according to nuclear rDNA internal transcription interval (ITS), 83 american plants are learned magazine, 516-527 (1996) (being incorporated herein by reference), (E.A.Kellogg in multiple no system of disposition grass group, variation in the agamospermy grass and species restriction, 15 Systematic Bot.112-23 (1990) (being incorporated herein by reference), (Nybom in Rubus, with the experiment of the interspecific cross in the dna fingerprint assessment facultative apomixis black mould (Rubus subgenus Rubus), 122 Hereditas 57-65 (1995) (being incorporated herein by reference), (Richards in Taraxacum, the origin of Taraxacum agamospecies, 66 Biol.J.Linnean Soc.189-211 (1973) (being incorporated herein by reference) etc.In contrast, very fast development the among the geneticist in early days about hybridization two kinds of conflicting ideas of effect in the asexual reproduction origin.Strausburger, Zeitpunkt der Bestimmung desGeschlechtes, Apogamie, Parthenogenesis und Reduktionsteilung, 7 Hist.Beitr.1-124 (1909) (being incorporated herein by reference), Ostenfeld, the experiment of the hawkweed Origin of Species (apogamy and hybridization type), 11 New Phytol.347-54 (1912) (being incorporated herein by reference) and Holmgren, Zytologische Studien uberdie Fortpflanzung bei den Gattungen Erigeron und Eupatorium, 59Kgl.Sven Vetenskapsakad. Ak. Handl. No. 7,1-118 (1919) (being incorporated herein by reference) think that asexual reproduction is controlled by the special inherent cause of asexual reproduction (gene) rather than the result of hybridization.On the contrary, A.Ernst, Bastardierung als Ursache derApogamie im Pflanzenreich, Fischer, Jena (1918) (being incorporated herein by reference) think that being responsible for agamic reproductive cell is the extension of observed genome disorder in distant hybrid unusually.

Ernst has accumulated a lot of evidences and has supported his hybridization hypothesis, comprise the following fact, the asexual reproduction plant has high chromosome number (they are polyploid normally), agamic complex height reproductive cell polymorphic, the asexual reproduction plant is degenerated in observed mode in species hybrid usually.Ernst hypothesis main and to cause its very fast principle of not considered (and causing still formally not considering so far) widely be that hybrid has formed the non-individual body of nourishing and generating to the Zhongdao of asexual reproduction from the sexual propagation that function is arranged fully.The degree that hybrid is suitable for non-individual body depends on the closely-related degree of parent's species, as, if the parent is closely related, hybrid is with sexual propagation, if the parent is moderate relevant, hybrid tends to asexual reproduction, if parent relationship wide apart, hybrid tend to nourish and generate.Therefore, according to Ernst, hybrid only originates from distant hybrid.Ernst does not determine the mechanism of the support vegetative hybrid origin beyond the hybridization popularity.

The hypothesis of Ernst is subjected to supporting (Harrison at twentieth century two, the '30s, the inheritance of the hybrid melanism between continent Tephrosia crepuscularia and Britisht bistortata, reach about what parthenogenesis in the interspecific cross originated from and talk about 9 Genetika 4467 (1927) (being incorporated herein by reference); G.L.Stebbins, the cytology II parthenogenesis species of Antennaria, 94 Bot. Gaz.322-45 (1932) (being incorporated herein by reference)), but when Ake Gustafsson has delivered his comprehensive argumentation, A Gustafsson, the asexual reproduction of higher plant, I. agamic mechanism, 42 Lunds Universitets Arsskrift 1-67 (1946), A Gustafsson, the asexual reproduction of higher plant, the agamic reason of II., 43 Lunds Universitets Arsskrift 69-179, (1947), A Gustafsson, the asexual reproduction of higher plant, III. biotype and species form, 43 Lunds UniversitetsArsskrift 181-370 (1947) (being incorporated herein by reference).In this was discussed, Gustafsson summed up: " can prove that in no instance hybridization itself can produce asexual reproduction.On the contrary, there is no doubt that the asexual reproduction method originates from the species group under many circumstances." be that the fact of autopolyploid has formally been refuted any requirement of recognizing for distant hybridization in some agamete of nineteen forty-six record.Thus, because the hypothesis of Ernst thinks that agamic cytological mechanism is the extension of observed cytological abnormal in the gamete forming process of distant hybrid, it has comprised the discrepant generally genome that stops chromosome pairing in the reduction division process by definition, so it has been disintegrated.Our the present known fact is really not so, that is, chromosome pairing is normal in many agametes.After the argumentation of Gustafsson, seldom there is the geneticist to propose to hybridize effect in agamic complex, this agamic complex has exceeded and has comprised that heredity goes up the species of taxon of the asexual reproduction inducement of decision and form.Attempted analysis this hereditary inducement but prove the task of being difficult to tackle.

Before the argumentation of Gustafsson, genetic analysis few in number has been carried out in asexual reproduction, and these experiment shortages draw quantity (the Asker ﹠amp of the required filial generation of specificity conclusion; Jerling).But, their prompting Gustafsson asexual reproduction is that mutual equilibrium system by recessive gene causes.The following by reference example of Gustafsson safeguard this viewpoint (a) wherein embryo's capsule of Parthenium, Poa and Potentilla form and parthenogenesis be under genetic control independently and (b) among Poa, Potentilla and the Rubus between two agametes or agamete and sexual parent between hybrid can be sexual also can be agamic, the result does not have tangible pattern (hinting many recessive genes).In Rubus, sexual F ₁From the asexual reproduction parent, and these F ₁Produce low percentile asexual reproduction F ₂, this points out the separation of multiple recessive factor once more.

Another field that produces the asexual reproduction research of equivocal meaning relates to the effect of manual change agamete ploidy.General tendency is that when agametic ploidy artificially increased, asexual reproduction strengthened.Yet, in Potentilla, Taraxacum, Paspalum, Poa, found exception, wherein artificial induction's ploidy increase causes (a) haploid parthenogenesis, wherein the avette one-tenth of subtrahend and do not have fertilization and grow (b) B _IIIHybridization, the wherein unreduced feritilization of ovum, and (c) recovery fully of sex.Haploidization has recovered the sex of agamic Poa.About so fuzzy meaning, Asker ﹠amp; Jerling reaches a conclusion: " we explain that the difficulty of ' asexual reproduction destruction ' is still relevant for the ignorant of its adjusting with us .... " the so true property of interrogatory promotion Gustafsson overthrown simple asexual reproduction inheritance dominant models.He does not almost observe the evidence of supporting them and is not proved by opinion such among Dryopteris, Hieracium, Hypericum, Potentilla and the Sorbus.Under each situation, Gustafsson provides the rational substitute of the opinion of delivering.

The segregation ratio of distorting has also hindered agamic genetic analysis.Some agamete among Dicanthium and the Themeda tends to sexual when growth under long sunshine, tends to asexual reproduction (Asker ﹠amp when growing under short illumination; Jerling).But, carried out obtaining the research that repeats of consistent segregation ratio at following species: Panicum (Asker; Jerling), Tripsacum (O.Leblanc etc., with the asexual reproduction pattern in corn RFLP mark detection corn-Tripsacum hybrid, 90 theoretical and applied genetics 1198-1203 (1995) (being incorporated herein by reference) and Brachiaria (Valle ﹠amp; Miles, the breeding of asexual reproduction species is at Y.Savidan ﹠amp; J.G.Carman, asexual reproduction progress (FAO, Rome is in the publication) (being incorporated herein by reference), and each research prompting apomeiosis (detected on the cytology) is controlled by single dominant allele.Yet, other nearest research has proposed challenge to this conclusion, as, at O.Leblanc etc., apomeiosis " allelomorph " during 90 Tripsacum theoretical and applied genetics 1198-1203 (1995) research are approaching is a part that is present in the repressed big linkage group of recombinating, similar linkage group be present in asexual reproduction Pennisetum (Grimanelli etc., the molecular genetics in the asexual reproduction research is at Y.Savidan ﹠amp; J.G.Carman, asexual reproduction progress (FAO, Rome, 1998) (in the publication, being incorporated herein by reference)) in.These linkage groups can comprise the required several genes (Grimanelli etc. of apomeiosis, is diploidy sporulation asexual reproduction location among the tetraspory Tripsacum: a gene be a plurality of genes? genetics (1998) (in the publication, being incorporated herein by reference)).

Two seminar are just attempting asexual reproduction gradually is seeped into the corn from Tripsacum at present, and they do not report that it is containing nine expression in the addition line below the Tripsacum chromosome.In a group, two asexual reproduction corn triploids that contain nine Tripsacum chromosomes (3X+9) have been prepared.Cytogenetics shows that with molecular studies perhaps nine Tripsacum chromosomes in each strain system are identical (B.Kindiger etc., asexual reproduction assessment in a series of 39 chromosomes corns-Tripsacum backcross hybrid, 36 crops science 1108-13 (1996) (being incorporated herein by reference)).Same group has prepared the triploid addition line that the 3rd strain contains nine Tripsacum chromosomes (3X+9).Yet nine chromosomes of two strain systems of many nine chromosomes in this strain system and front are different.Maize chromosome is identical in three strain systems.3X+9 plant described later also is agamic, but agamic frequency has only 10-15%, and the frequency that preceding two strains are is 95-100% (Sokolov etc., the prospect of development asexual reproduction corn, genetics preferentially instructs, Russia (1997) (progress report; Be incorporated herein by reference)).These data and other trial make the agamic group of corn do not deliver the result (Grimanelli etc., the molecular genetics of asexual reproduction research is at Y.Savidan ﹠amp; J.G.Carman, asexual reproduction progress (FAO, Rome, 1998) (in the publication)) pointed out asexual reproduction genetic composite mode.In an other research, sexual T.dactyloides dliploid produces aneuploid with highly agamic T.dactyloides triploid hydrid.46 strain F ₁Have only three strains to tend to apomeiosis (cytology is determined) among the s.Yet, the apomeiosis F of these height ₁S comprises seven or more addition chromosome (having surpassed the dliploid level), and all chromosome numbers are near the F of dliploid level ₁S is sexual (Sherman etc., the asexual reproduction in the dliploid X triploid Tripsacum dactyloides hybrid, 34 genome 528-32 (1991) (being incorporated herein by reference)), and this points out compound heredity.At last, asexual reproduction and be divided into from (Blakey etc. in the artificial Tripsacum triploid that produces with five regional collinear Tripsacum linkage groups of three maize chromosomes, Tripsacum fertility and dna marker be divided into from, 42 Maydica 363-69 (1997) (being incorporated herein by reference)), this further makes the agamic simple inheritance mechanism of control fall under suspicion (working as at least in the strain system of attempting asexual reproduction mechanism is transferred to other species or same species).Can suppose that oligogene under recessive condition (regulating or controlling gene) can stop agamic generation, making asexual reproduction look thus is to be under the simple genetic regulation.Yet such gene belongs to that the gene of needs is expressed in many other asexual reproduction and they self can not give other species asexual reproduction (Linnean).Following deduction has been looked back in these researchs: (a) asexual reproduction needs a plurality of genes, (b) the facultative gene of influence can the additivity form work, (c) some Tripsacum chromosome is bigger to facultative influence than other, and (d) can not give homeologous (collinear) counterpart that substitutes their Tripsacum aspect the asexual reproduction from the allelomorph of at least three maize chromosomes.

The meiotic mutants body is the center (Linnean) of simple hereditary hypothesis.Unless meiotic mutants that the prompting of nearest sudden change hypothesis is suitable and the associating of suitable multiple gene inducement, asexual reproduction can not expressed (Mogie, the evolution of Asexual Reproduction in Plant (1992) (being incorporated herein by reference), Grimanelli etc., the molecular genetics of asexual reproduction research is at Y.Savidan ﹠amp; J.G.Carman, asexual reproduction progress (FAO, Rome, 1998) (in the publication)).Yet the evidence that obtains recently shows that the allelomorph that is considered to suddenly change is actually the part of multiple gene inducement and is that marginal habitat sexual propagation is essential.Do not understood in the past but what show at this is uniting of the difference ecotype (racial or plant between) to make asexual reproduction that (also referring to Linnean) take place by secondary contact.

With regard to aforementioned, should be appreciated that it will be progress significant in this area that the method for preparing the asexual reproduction plant is provided.

The invention summary

One of target of the present invention provides not to be used method of mutagenesis or is being to prepare the method for asexual reproduction plant from the sexual reproduction plant under the situation of asexual reproduction plant.

Additional objects and advantages of this invention obtain illustrating in detailed description and can obtain understanding by practice of the present invention.

In order to finish aforesaid target and consistent, the invention provides from two or more identical or relevant sexual reproduction plants and prepare the method for asexual reproduction plant with invention described herein.

A step in the method relates to and obtains two sexual reproduction strains system, its female reproduction phenotype is variant, and female development time table between (illumination length, luminous intensity, temperature regime, etc.) two strain systems has the asynchronism of suitable degree so that under identical environmental condition.Suitably the asynchronism of degree is including, but not limited to following condition, in one of them strain system megaspore the time of beginning takes place is the time that blastular forms in another strain system, with respect to the colored induction time of agametophyte ovule and ovary tissue (megarchidium, integument, pericarp etc.) and other phenology factor such as photoperiod adjusting.Acceleration strain system (the strain system that the experience blastular is grown) will finish to spend to induce with megaspore and take place.

Another step of method relates to makes suitable difference sexual reproduction strain system (with the chromosome doubling technology of standard) become amphiploid, if they can not become polyploid, is to carry out hybridized induction asexual reproduction with these two sexual amphiploid strains.Hybridization also can be carried out before amphiploidization.Under these circumstances, amphiploidization relates to the mitotic spindle inhibitor such as the colchicine of standard, or depends on B _IIIThe formation of hybrid (the not subtrahend feritilization of ovum) is to produce the asexual reproduction triploid.Other method is included among the following embodiment.

The several viewpoint summaries of accompanying drawing

Figure 1A-M has represented that the ideograph that sexual (amphigamy) of typical types and asexual reproduction blastular are grown (sees B.M.Johri etc., angiosperm comparative embryology, the 1st and 2 volumes (Springer-Verlag, 1992); Asker ﹠amp; Jerling).Polygonum-type grow be standard and exclusively more than 99% all angiosperm species in express.The digitized representation stage special gene expression relevant in the horizontal line: (1) ripe predivision interval and meiosis prophase morning (exchange and the filming function of MMCs in callose) with each developmental pattern, (2) reduction division, (3) the megaspore maturation (corpus callosum from the survival megaspore on digestion and the survival megaspore initial vacuolization), (4) blastular is grown, (5) double fertilization and early stage endosperm form, and (6) embryo is taken place by (beginning after endosperm forms usually).The asynchronous expression of the duplicate factor of digitized representation in parallel row supposition, member wherein originates from the genomic fragment of different polyploid genomes or different ancient polyploid mesarcs.Interval representative sudden change (great majority are amorphs) in the numeral and deletion.Should be noted that very general and least general in ancient many spores of polyploid type at interval in polyploid asexual reproduction type.Perhaps, the formation of some amorph can strengthen agamete and set seeds.In growing nonsynchronous polyploid and ancient polyploid, eliminate the main evolutionary mechanism (Linnean) that gene repeats to have seemed to represent new reproduction type (asexual reproduction, polyspory and polyembryony) evolution arbitrarily.

Fig. 2 A-B has represented two can provide the plant of sufficient difference to belong in periodicity of illumination reaction and preparation asexual reproduction plant reproductive timetable: (A) the sexual Antennaria aromatica of dliploid (the little triangle among the Montana), A.Corymbosa (two circles on Idaho Montana border, circle is at the Wyoming of Montana boundary vicinity, a circle is at northern Colorado), A.marginata (at the rectangle of New Mexico and Arizona), A.media/pulchella (star of California), (circle is at Albert for A.microphylla, a circle is at Idaho, the boundary vicinity of Montana and Wyoming, circle is in the central and north of Wyoming, a circle is in the Colorado south), (circle is at BritishColumbia for A.racemosa, two circles are at Idaho Montana boundary vicinity and circle Montana at the Wyoming boundary vicinity), A.rosulata is (at Utah, Colorado, the star of Arizona and New Mexico), A.umbrinella (at the big triangle of Idaho Montana and Wyoming), A.friesiana (starlet of Alaska), A.densifoliaA.E.Porsild. (at the circle of Yukon), A.monocephala DC. is (at Alaska, the big star of Yukon and northwest Territiries) and the agamete A.rosea of polyploid (little rectangle); (B) the sexual Tripsacum bravum of dliploid Gray (large rectangle of Mexico), T.dactyloides (the big star of US), T.dactyloides var hispidum (Hitchc.) De Wet and Harlan (the big star of Mexico), T.latifolium Hitchc. (the little rectangle of Mexico), T.laxumNash (great circle of Mexico), T.maizarHernandez and Randolph (triangle of Mexico), T.manusoroides de Wet and Harlan (near the Mexico starlet in Mexico bay), T.pilosum Scribner and Merrill (small circle of Mexico) and T.zopilotense Hernandez and Randolph (keeping off the Mexico starlet in Mexico bay).Dliploid sexual T.cundinamarce de Wet and Timothy, T.australe var.australe Cutler and Anderson and T.dactyloidesvar.meridionale de Wet and Timothy can obtain (near 15 ° of north latitude, 85 ° in west longitude) from Columbia and not on map.

Fig. 3 has represented the gynoecium developmental stage (before the reduction division or before the apomeiosis, reduction division or apomeiosis, multinuclear blastular) of dliploid (sexual) and tetraploid (diploidy sporulation) T.zopilotense and T.dactyloides with gynoecium length.The length of gynoecium (mm) for＜0.50,0.50-0.75,0.75-1.00,1.00-1.25,1.25-1.50,1.50-1.75,1.75-2.00,2.00-2.25,2.25-2.50,＞2.5 be respectively the 1-10 class.It should be noted that (a) dliploid T.zopilotense than dliploid T.dactyloides the three phases by female sexual development quickly, (b) tetraploid begin apomeiosis than dliploid reduction division begin morning, they than dliploid by three phases faster (tetraploid does not carry out reduction division) and (c) T.dactyloides of diploidy sporulation (be summarized in O.Leblanc ﹠amp by three phases quickly than double sporulation T.zopilotense; Y.Savidan, the opportunity of (Poaceae), 8 Polish Botanical Studies 75-81 (1994) (being incorporated herein by reference) take place in the Tripscaum species megaspore relevant with asexual reproduction and sex).

Fig. 4 has represented to take place according to megaspore the stage of (reduction division or apomeiosis MMCs and monokaryon megaspore) or blastular growth (multinuclear blastular), the gynoecium of 190 sexual Elymus scabrus and 690 agamic Elymus rectisetus distributes, and corresponding pollen development stage (1=PMC meiosis II and tetraspore; The free microspore of 2=; 3=has the pollen grain of end beginning gemma; Tool bubble monokaryon pollen before the 4=Ubish granule forms; The early stage Ubish granule of 5=is formed up to early stage oncus; The 6=oncus; The monokaryon pollen that 7=does not divide again; 8=has the pollen of the monokaryon of engrain; The pollen of the monokaryon that dyes a little less than 9=has; 10=double-core pollen).The accumulative total percentage (the pollen stage before the accumulative total) of the sample gynoecium sum of in the female developmental stage, being adopted given with bar representative the pollen stage separately of pollen on the stage.After the phase III, almost can't see MMCs in the sexual gynoecium.Therefore, the bar after this in this classification almost equates.On the contrary, the bar in the classification of sexual monokaryon megaspore increases from 10 stage of phase III to the length always, and this shows that most of gynoeciums in these stages have developed and has surpassed the MMCs stage.In the 10th stage, two in three sexual gynoeciums comprise the multinuclear blastular.In the asexual reproduction gynoecium, the double-core blastular is at first observed (from C.F.Crane ﹠amp at the 3rd pollen period of the multinuclear blastular (＞2) that occurs in the 6th pollen stage with big percentage; The asexual reproduction mechanism of the ELymus rectisetus of the eastern Australia and New Zealand of J.G.Carman, 74 american plants are learned magazine 477-96 (1987) and are modified) (being incorporated herein by reference)).

Detailed Description Of The Invention

Before this prepares the method disclosure and description of agamic plant, be to be understood that to the invention is not restricted to specific configuration, method step and material disclosed herein because configuration, method step and material can have some change. Be to be understood that equally also it is not to provide constraints because the suitable thing with it of claim that scope of the present invention is only added limits that the term of using only is used for describing specific embodiment herein.

Should be noted that unless clearly explanation in the context, singulative " a ", " an " and " the " comprise plural reference as used in this specification and additional claim.

Describing and requiring in the right of the present invention, following term will use according to following definition.

In history, term asexual reproduction comprises nourishing and generating of various ways when being used for flowering plant (angiosperm). Yet term asexual reproduction herein is confined to not the routine of gamete (single-female generation) associating of the not subtrahend ovum of opposite sex is caused agamic method. These methods usually are described with the agamic form of gametophyte and relate to and destroy the unusual of normal Ovule Development. On the contrary, two Formation of basidiospores and tetracytes state (jointly being called as polyspory herein) also destroy normal Ovule Development, but form normal, the maiotic ovum (Fig. 1 J-M) that needs fertilization. Cytology similitude between asexual reproduction and the many spores is determined (U.S.'s sequence number 60/037,211 recently; Also see Linnean). The general character of these similitude prompting asexual reproduction and polyspory origin.

Megaspore generation (female meiosis) in the angiosperm occurs under megasporocyte (MMCs) and the normal condition grows and next female gamete (blastular) from one of four meiosis products (megaspore). Most of angiosperm blastulars are eight nuclears (Figure 1A) and comprise an ovum (fertilization forms embryo), two synergidaes, central cell (fertilization forms endosperm) and three antipodeos (Polygonum-type blastular) that contain two polar cores. Each nuclear is monoploid at first, from the nuclear (Figure 1A) of the megaspore that survives through the interior mitosis of three orders. This process has been broken in asexual reproduction, has caused diploidy sporogenesis (Fig. 1 C-F) or apospory (Fig. 1 G-H).

In the asexual reproduction of known diploidy sporulation form, that the MMC that unreduced blastular is damaged from reduction division forms and formed by precocious blastular and to replace (Fig. 1 C-F).Finishing that blastular forms also is precocious, through the prefecundation of being everlasting unreduced ovum carry out parthenogenesis form proembryo (as, Linnean).The asexual reproduction that is called as the apospory form is similar to the diploidy sporulation, (Fig. 1 G-H) that to be unreduced blastular form from the somatic cell near the integument wall of MMC.In apospory, maiotic MMC or young sexual blastular abortion and replaced (Asker ﹠amp by one or more no spore blastulars; Jerling), this also tends to precocity (Linnean; Asker﹠amp; Jerling).Therefore, these are that blastular forms the result that the precocity of required gene is expressed unusually, and if not all, most asexual reproduction is facultative sexual, this means that sex is retained in its evolutionary process and the accidental (Linnean of expression; Asker ﹠amp; Jerling).

Haploid nuclei division (nuclear division) takes place but tetraspory (Fig. 1 I-K that cell division (division of cytoplasm) does not take place; M.T.M.Willemse ﹠amp; J.L.van Went, the female gamete body, at B.M.Johri, embryology of angiospermis 159-96 (Springer-Verlag, 1984) is incorporated herein by reference; B.M.Johri etc., the angiosperm comparative embryology, the 1st and 2 volume (Springer-Verlag, 1992) (be incorporated herein by reference)), also owing to " precocious gametophyteization " (E.Battaglia of MMC, the evolution of angiosperm female gamete body: the key of explanation, 47 Annali di Botanica 7-144 (1989) are incorporated herein by reference)).The MMCs of tetraspore and diploidy spore has experienced vacuolation, normally takes place in the sexual megaspore of the survival in the beginning process that blastular forms.Such vacuolation also betides aposporous initial cell (Linnean).In diploidy sporulation and tetraspory, the formation of blastular is unremitting to be carried out.In normal species, megaspore is taken place and blastular is grown and separated in time, during this period, on the wall of the megaspore that survives callose (β, 1-3 glucan) (Linnean) is decomposed.

The MMC wall of diploidy spore and tetraspore species lack normal condition early stage parcel form spore and dispore species MMC (Figure 1A-B, L-M) and in the megaspore generating process, be deposited on callose (Linnean) on the opposite walls.These sedimentss are decomposed at postmeiotic, make the beginning Rapid Expansion that megaspore forms at blastular.Lack callose in diploidy spore and the tetraspore species and make that (Fig. 1 C-F I-K) is the further evidence (Linnean of MMC gametophyteization to the precocious expansion of MMCs; E.Battaglia, 47 Annali di Botanica 7-144 (1989); M.D.Peel etc., megasporocyte callose among asexual reproduction Buffelgrass, Kentuky Bluegrass, Pennisetumsquamulatum Fresen, Tripsacum L. and the Weeping Lovegrass, 37 crops science 724-32 (1997)).Myriospored form, the same with agamic form, from unusual (Linnean) of multiple origin widely.

The polyembriony of various ways is meant in each ovule and forms more than one embryo (synergidae and antipode embryo, cleavage polyembryony tire phenomenon, adventitious embryony) (Linnean).The same with polyspory with asexual reproduction, the polyembriony of various ways is polytopic and relates to the time and the development program of spatial offset.

Multi-source takes place and cytology research has been illustrated agamic origin.Cytology comparison shows that some asexual reproduction type more is similar to unusual sexual type than other asexual reproduction type, as, Adoxa-type tetraspore (sexual) is identical with Antennaria-type diploidy spore, and just causing the nuclear divisions of four nuclear blastulars is reduction division and be mitosis (Fig. 1 E ﹠amp in Antennaria-type diploidy spore in tetraspore; 1I).Equally, Ixeris-type diploidy spore (Fig. 1 D) and sexual couple of basidiospore (Fig. 1 L ﹠amp; 1M) identical, just there is not reduction division among the former.Ixeris-type diploidy spore and two basidiospore form (and apospory and tetraspory) and all occur in Erigeron belongs to.Analyzed multi-source generation data and found that asexual reproduction and many spores belong to through being everlasting and section's level (highly significantly correlation) takes place.Equally, preleptonema chromosome condensation and the no function megasporocyte that is substituted by the function megasporocyte subsequently are formed in the species that are closely related with asexual reproduction and many spores species and take place, and this has also pointed out the similar mechanism of evolving.

The high basic number of chromosome (X 〉=10) shows ancient polyploidy usually, this means have or do not have rise or the diplodization of decline aneuploidy after polyploidy.Many radixes during each belongs to have also reflected ancient polyploidy.Have been found that the basic number of chromosome 80% of all genus determined to comprise the species of agamic, myriospored or polyembriony.Statistical analysis shows myriospored and the normally ancient polyploid of species polyembriony, and normally the agamete of polyploid comprises the protogene group usually.And (2.7 ± 0.4SE) than asexual reproduction but not the X value (1.7 ± 0.1) of myriospored species is many in the X of each genus value for the genus of many spores but not agamic species.This means agamete to tend to have the duplicate factor of balance (protogene group) and the species of many spores and polyembriony have unbalanced gene (ancient polyploid genome, that is, because the part that causes of aneuploidy repeats or be triple) usually.Therefore, show the growth order that the interaction between (a) balance genome (comprise and breed gene completely) is taken place at development later stage under the normal condition for some female sexual development order in the polyploid of being made up of the ecotype of breeding difference in the present invention, as replacing fully of in asexual reproduction, taking place be essential, as, megaspore take place to wait non-equilibrium " ancient polyploid genome " that (b) comprise breeding genes incomplete repetition or triple only female sexual development order by asynchronous replacement or by the other parts of growth order as at many spores, polyembriony, it is essential that preleptonema aggegation and MMC repeat when replacing, and (c) asexual reproduction, the breeding long-term with it stability may be normal and grow springboard (rather than clearing end) in novel sexual species of ancient polyploid and the genus evolutionary process when by ancient polyploid process influence.

The type that can cause the developmental mechanism of asexual reproduction, polyspory and polyembriony of having illustrated to the research of yeast heterocaryon.When the yeast cells that is in the G1 phase when being in the cytomixis of S phase, whole cell cycle phase is skipped, that is, G1 phase chromosome is duplicated in advance.The frequency of replication initiation depends on S:G1 nuclear ratio in the heterocaryon.With the mitotic yeast cells of interkinesis cytomixis in, energic nucleus (all stages) enters mitosis (B.Lewin, gene V, (Oxford University Press, 1994) (being incorporated herein by reference)) in advance.

Asexual reproduction may take place in a similar fashion.If a genomic blastular is grown signal and another genomic megaspore generation signal overlap, reduction division may be omitted (diploidy sporulation) or the blastular growth may dystopy (apospory).Correspondingly, only take place and the very big-difference on the opportunity (with respect to the growth of agametophyte ovule and ovary tissue) that blastular is grown when being present between the prototype ecotype or the species megaspore, the asexual reproduction sample is inclined to and comes across polyploid (table 1).Perhaps, such natural variation body only just is found in all over the species of giving birth at height usually,, has latitude and the ecological species that distribute widely that is.

The duplicate factor that table 1 has been described asynchronous expression how to cause comprising two in female development time table the time express the diploidy sporulation and the apospory of the genomic polyploid of (floral induction, megaspore form and gametophyte is grown) difference.Because expressing in advance, the checkpoint gene of genome II is omitted mutually when growing by the italic of genome I coding.

Table 1

	The critical stage * that grows
	The critical stage * that grows				Genome	????1	????2	????3	????4
Genome I (not modified)	Archesporium	Reduction division	Blastular	Double fertilization/early embryo	Genome	????1	????2	????3	????4
Genome I (not modified)	Archesporium	Reduction division	Blastular	Double fertilization/early embryo	Genome I (modification)	Reduction division	Blastular	Double fertilization/early embryo	Just centrocyte fertilization
Genome II	Blastular	Double fertilization/early embryo	Just centrocyte fertilization		Genome I (modification)		Blastular	Double fertilization/early embryo	Just centrocyte fertilization

* by (low latitudes) evolves in short daytime and long season among the genome I growth signal and the genome II that evolves in long daytime and short season (high latitude) the initial ovary development of compromising.Therefore, the initial sum speed of ovary development is supposed a kind of " intermediate phenotype " in the mode similar with other observed intermediate phenotype in the parent different from form (leaf length and width, plant height etc.) amphiploid.1. in the beginning of phase I, genome II produces maiotic signal in advance, because the archesporium mother cell does not also form, so failed, that is, it is to be grown by the midrange speed of middle phase phenotype control.2. in the beginning of second stage, the reduction division of genome II stops closing card signal and reduction division is stopped and makes genome I and genome II synchronous in the mode similar to observed mode in asynchronous yeast heterocaryon (summarizing at this).If reduction division is successfully stopped, one of several diploidy sporulations (Fig. 1) then appear, that is, directly form blastular from megasporocyte (sporulation of Antennaria-type diploidy) or young female gonotocont (Taraxacum or the sporulation of Ixeris-type diploidy).If reduction division not by the termination of success, apospory (Fig. 1) may take place, that is, form one or more blastulars from the nucellar cell who closes on.This mainly comes across in the species that comprise a plurality of or indefinite neck egg cells.In apospory and diploidy sporulation, grown the hereditary unreduced blastular of.The tissue of agametophyte ovule or ovary continues to take place according to centre-phenotype (delay) timetable.On the contrary, the growth of gametophyte (blastular) continues in advance because the blastular development gene of genome I (only in blastular) is synchronous with the blastular development gene of genome II.3. from two avette one-tenth of synchronous genomic signal induction and parthenogenesis, for the growth of the agametophyte tissue of ovule and ovary, they take place in most of agametes in advance.4. pollination takes place according to the intermediate phenotype timetable, but ovum no longer be accept but in most of the cases divide.Central cell then is fertilized if not autonomous, and endosperm and parthenogenesis embryo are grown.

This duplicate factor asynchronism hypothesis is being explained many genomic uniqueness and the many inconsistencies in the asexual reproduction record that show the unusual species of reproduction on the primary level.For example, asexual reproduction, polyspory and polyembriony be rare but tend to occur simultaneously in all over the section of giving birth to because there are enough ecotype variations etc. the time of breeding beginning, and be rare but often see in all over the section of giving birth in most of sections.The facultative sexual propagation that single spore Polysporic type occurs in the asexual reproduction of agamete and many spores species (Linnean), because except deletion and sudden change, each parental gene group comprises the required gene of normal breeding, and growth conditions may be more conducive to cause the outer genomic expression of genome that has sexual development to take place sometimes, as taking place in Dicanthium, Themeda and countless other agamete.The facultative influence that is subjected to following two factors: (a) the genome difference silence that causes by genetic background difference, or, cause sexual development to take place like this (b) by accelerating or the genome (photoperiod or thermotonus) that slows down reduces the environmental factor of asynchronous degree with respect to gene expression in another genome.(incomplete breeding gene series) polyspory or the polyembriony that causes expressed in whole uneven genomic competition by some outpost of the tax office system disappearance.On the contrary, the competitive expression of the genome of the breeding gene series that comprises balance usually in the asexual reproduction stopped by checkpoint gene, makes like this and excessively arrives asexual reproduction (table 1) reposefully.In two parental gene groups in the agamete at least one must have sufficient DNA and breed the duration (reduction division duration etc.) of growth so that express abundant asynchronism with extension.Therefore, asexual reproduction seldom appears containing seldom to measure in DNA and the quick maiotic therophytes.Equally, polynary polyploidy can be eliminated the asynchronism that causes reversing back sex.Asexual reproduction in the outbreeding species than general many in the inbreeding species, because they are easier to form between the ecotype or polyploid between planting when secondary contact takes place, as, at the higher weather drift (L.A.Frakes etc. relevant of frequency with Pleistocene eight big glacial actions or numerous glacieret effect, phaneroxoic climatic model (Cambrige University Press, 1992) (being incorporated herein by reference)).Equally, more agamete is allopolyploid rather than autopolyploid, because pass through B _IIIHybrid forms usually the vital polyploidization of asynchronous expression (Linnean) for the different genes group of female sexual development, and occurrence frequency is higher in species hybrid than in ethnic species hybrid.Equally, therophytes F1 hybrid between most of sterile ecotypes or between planting B takes place _IIIThe probability that hybrid forms is lower more for years than the perennial plant that blooms.This factor has further limited therophytes to be become agamic probability and has explained that further probability takes place abiogenous agamic therophytes low.At last, when agamete and sexual reproduction plant corpus or the hybridization of another agamete,, can expect that offspring's sex is equivocal no matter hybridization is inbreeding or outbreeding.How the modes of reproduction that shows among the offspring influences asynchronism with depending on the genome that adds or remove, if do not know the female development time table by the genome encoding that participates in advance, this point is difficult to prediction (Linnean).Duplicate factor asynchronism hypothesis can explain fully that these abilities that are considered to inconsistent phenomenon main in the asexual reproduction record are strong evidences of its validity.

Many agametic distribution patterns show Pleistocene origin (G.L.Stebbins, the chromosome evolution of higher plant (Addison-Wesley, 1971) (being incorporated herein by reference); Asker﹠amp; Jerling), that is, many agametic geographical distributions and center of diversity are the center with the edge in Pleistocene glacier, but their scope usually covers the ecological edge of the sexual ancestors of the supposition that is positioned at periglacial the north and south.Eight big coverings the glacier of 20% earth surface take place in the Pleistocene.They are continued thousands of year to periods between the warm glacier in 100000 years separately at every turn.Equally, most glacier incident is formed (L.A.Frakes etc., phaneroxoic climatic model (Cambrige UniversityPress, 1992) by the glacier that interrupt period between the little glacier that is continued thousands of years.Therefore, in the Pleistocene, the northern latitude of North America and Eurasia repeatedly removes glacierization, can be adapted to cold weather, long illumination and short season of growth all over the natural disposition taxon replant by.

It is to the high latitude area adaptation in short summer that the reduction division of the precocity in young ovule and blastular are grown.The glacier development of following period between the glacier turns cold low latitudes to allow the flora of high latitude to enter the flora of low latitudes.This ecotype of giving the precocious female sexual development with supposition is (from high latitude, promptly, temperate climate is to arctic climate) provide many chances formation to have the polyploid ecotype (or different species) of the female sexual development of the delay of supposition (from low latitudes, that is, tropical climate is to temperate climate).Such polyploid shows the female sexual development of asynchronism, that is, and and asexual reproduction.

The another kind of adaptation of high and low latitudes is included in the seasonal variation reaction of blooming is made in specific photoperiod variation.Many plants are classified to photoperiodic reaction according to them, as, " long-day plant " is adapted to high latitude, spring and early summer bloom when sunshine is long, " short-day plant " is present in low latitudes (torrid zone) usually and blooms in the weak point " winter " sunshine in the torrid zone, " dual day long plant " requires induce reproductive bud to form sunshine short or length, be respectively long or short sunshine then, so that the bud maturation that forms becomes flower, " day intermediate plant " can not bloom under the oversize or too short situation at sunshine, " day-neutral plant " is meant and adapts to the duration of day hardly, in the flower induction that takes place under the duration of day sunshine widely.Also there are several specific other classification (Salisbury ﹠amp; Ross, plant physiology (1992) (being incorporated herein by reference)).

Salisbury and Ross have selected 85 from research so far the different photoperiods are bloomed about 300 plant species that react.These 85 species have clearly been represented above-mentioned specific photoperiodic reaction classification, and these 85 species have clearly been represented 67 genus.Should be noted that at this 33% (in 67 22) of (first) these genus comprise and have the unusual species of female reproduction (gametophyte asexual reproduction, many spores or polyembriony; The appendix of Salisbury and Ross table 23-1 and Linnean is compared).If breeding does not rely on adaptation to different latitudes unusually, can expect that the number that belongs to has increased by 9 times, that is, it is unusual to have only 3.8% genus to express breeding.Therefore, breed unusual not relying on different adaptation of photoperiod, it is unusual that the species that so only have the genus of 3.8% (rather than 33%) to be defined as comprising to have the different photoperiods to adapt to also should show breeding.33% is divided into: 12% comprises gametophyte asexual reproduction (with 1% comparing that all angiosperm belongs to, promptly, 12 times have been increased), 13% comprises myriospored species (with 1.6% comparing that all angiosperm belongs to, that is, increased by 8 times), 7.5% species that comprise polyembriony are (with 1.7% comparing that all angiosperm belongs to, that is, 4.4 times have been increased).Comprise the genus of expressing the unusual plant of breeding and comprising the additional evidence that the tangible correlation of height between the genus of the different adaptive plants of photoperiod (different latitudes) expression is used as the duplicate factor asynchronism.It is believed that the genomic amphiploid of the multiple ecotype that comprises these features (visibly different photoperiodic reaction and visibly different female development time and speed) difference is agamic, it is former because nonsynchronous female sexual development causes not formation of subtrahend blastular and parthenogenesis (table 1) subsequently.

As mentioned above, the discovery among the present invention makes trial that asexual reproduction is changed into sexual species or induces the important hypothesis of its scientist who from the beginning expresses dependence invalid by sudden change to a great extent.Gradually (carrying out distant hybridization with the asexual reproduction correlative) important hypothesis of infiltration method is that asexual reproduction is can suitably bring into play function at other species by one or more " asexual reproduction gene " control and these genes.Duplicate factor asynchronism hypothesis think asexual reproduction be by between the race (between the ecotype) or plant between genome repeat and the control ovary in asynchronous (overlapping) of gene series of whole female sexual development order (MMC differentiation, reduction division, megagametophyte are grown, embryo take place) cause.Therefore, as long as specific, the interaction of the gene series of differential gene group will cause the asynchronism of suitable degree and induce asexual reproduction.Such specificity is different between species, that is, induce agamic box gene perhaps can not bring into play function rightly in another species in species and induce asexual reproduction.And, in for opportunity being the specific species of the box gene of two differences of average (may be most species) and the asynchronism that appropriate degree the is provided of no use female reproduction box gene that substitutes average photoperiodic reaction of (by plant breeding and genetic engineering) its existing scramble time and female sexual development order, a photoperiodic reaction and female sexual development order can not induce asexual reproduction.Perhaps the someone thinks that one or more key-genes control the opportunity of these orders, has only the key-gene that needs to be transferred, that is, and and another genomic box gene of genomic key-gene controlled.Yet, as Wilson, form and the breeding of physiology shape, K.J.Frey (editor), plant breeding II (Iowa State University Press, 1981) (being incorporated herein by reference) points out that photoperiodic reaction is a quantitative character.And system's generation data show that asexual reproduction is lost when box gene is broken into fragment by ancient polyploidization method.Therefore, although can and backcross by distant hybridization asexual reproduction is transferred to the crops species from edge species far away, the difficulty of not expecting before can expecting very few with understanding comprises and need shift many genes from more than one chromosome.

The main hypothesis of mutational breeding method is that asexual reproduction is single-gene or approaching monogenic proterties.When lacking between genome asynchronism, can expect to have only imitation to be responsible for the asynchronous interaction of many asexual reproduction (all pass through gynecogenic form cell effect) sudden change partly from subtrahend blastular not.Be difficult to the imagination, induce the accumulation of asexual reproduction sample reproductive behavior sudden change can not give other cell, breed or the whole plants process bring injurious effects.Asynchronism hypothesis prediction mutation method will run into great difficulty.

Sexual reproduction plant hybridization method (A.Ernst, Bastardierung als Ursache derApogamie im Pflanzenreich, Fischer, Jena (1918)) main hypothesis is the fertility Restoration Mechanism after asexual reproduction originates from distant hybridization and amphiploidization.People have prepared thousands of kinds of distant hybrid and amphiploid, but have only observed asexual reproduction (Asker in individual other offspring; Jerling; Linnean).The asynchronism hypothesis explains that the reason of this observed result is to induce the required asynchronism significantly of asexual reproduction to be difficult to observe in nature.The example of this rare event is new agametic evolution and the radiation in Rosaceae, Asteraceae, Poaceae and other section in the nature, these sections are in Pleistocene glaciation, the species environmental or that be closely related different when total same species body are repeatedly taken to a time-out by the weather drift, and they have experienced hybridization and have formed between the race subsequently or asexual reproduction polyploid between planting.Therefore, asexual reproduction is just with very low percentage occurring in artificial distant hybrid or amphiploid at random.On the contrary, the amphiploid that comes from parent ecology type appropriate selection or that cultivate or strain system (based on the breeding and the selection of observed photoperiodic reaction on the cytology and female sexual development time difference on opportunity) development of asynchronism hypothesis prediction will be agamic.

The present invention described herein depends on the associating of specific technology, and this ground of deducing of uniting is determined can cause gametophyte asexual reproduction expression when idioplasm during by hybridization or amphiploid associating clearly.When uniting for polyploid, the deduction ground of giving the specific associating of agamic sexual reproduction matter is determined that (asynchronism by giving photoperiodic reaction and the female reproduction opportunity of other form) makes with the present invention and is different from prior art.

The present invention does not rely on the existence with appropriate asexual reproduction gene of expressing in different taxon (genome).In fact, the present invention depends on the non-existent idea of this gene.The present invention does not rely on when sudden change will induce the allelic existence of asexual reproduction.Moreover the idea that the present invention relies on is that such allelomorph can be expressed harmful pleiotropy and can not produce the asexual reproduction form that needs when sudden change.The present invention do not rely at random or previously generated may the expression also may not express asexual reproduction source far away hybrid.On the contrary, the present invention has determined to produce agamic family or box gene with deducing when hybridization or associating.Listed herein basic idea and the practice agametic method of generation that expect in the past with other or that use in the world at present has fundamental difference.

The asexual reproduction gene that other people proposition utilizes molecular labeling to help gradually oozes from the wild type agamete relevant with the crops species and prepares the asexual reproduction plant.The such tag application of Zong Shu data suggest is limited herein, gives asexual reproduction because the gene of their location will have only when other a lot of genes are transferred Shi Caihui simultaneously, and this will lower the desirable property of agronomy of crops widely.These asexual reproduction branching programs have to be determined with general " asexual reproduction gene " or general " asexual reproduction box gene " and becomes the same long-term target of patent.As what thought herein, being responsible for giving gene in the agamic genome in comprising two genomic polyploid species with different female development time tables, to have only the genome that comprises when receptor species be that this gene just can given other species or environmental asexual reproduction in the appropriate difference of donor gene group of the female development time table of coding.Genomicly generally give agamic box gene and perhaps do not exist from one.

Target of the present invention is the method for preparing gametophyte agamete (expressing the plant of apospory or amphidiploidy sporulation) at the plant from the normal sexual propagation of typical experience.

The present invention is particularly at preparing the gametophyte agamete by hybridization with the two or more sexual reproduction plants identical or tight akin species of amphiploid associating, the difference of these sexual reproduction plants is with respect to the environmental condition of (a) plant growing (photoperiodic reaction etc.), and (b) total growth incident such as ovule is initial, endocarp g and D and gynoecium length and width in the plant, the time that megaspore is taken place and or duration, megagametophyte (blastular formations), pollination, fertilization and embryo's generation take place.

The present invention is also in the amount that is used for preparing the required female development time table asynchronism of agametic amphiploid two or more sexual species.

The present invention is also at the selection pressure and the breeding plan that are used for producing in the sexual reproduction strain system of the female development time table of not expressing needs at first female development time table (breeding asynchronism) difference of wishing.

The very convenient method that the present invention is divided into is divided into four classes: (a) select or prepare to be applicable to the sexual reproduction matter that produces the asexual reproduction plant from the sexual reproduction plant (b) cross method, (c) amphiploid method, and the program that (d) confirms the asexual reproduction expression.

Selection and cultivation are used to produce agametic sexual reproduction matter

A feature of the present invention is that quickening to stimulate responsible in the nature is agamic process from the sexual reproduction plant evolution.Natural process needs tens thousand of years, that is, and and the progress in (and being adapted to different weathers and photoperiod) glacier that the plant community that it need cause separating thousands of years is united again.In the present invention, the duration of whole process reduced to the several years from tens thousand of years.

From the sexual reproduction plant select to be applicable to the preferable methods of the idioplasm that produces the asexual reproduction plant relate to determine same species or tight akin species, comprise the photoperiod and be adapted to extensive difference latitude (long-day plant, short-day plant, day-neutral plant etc., Salisbury ﹠amp; Ross, plant physiology (1992)) ecotypic plant.Many such " all over giving birth to " taxons are present in Poaceae, Asteraceae and Rosaceae (Linnean; See embodiment, hereinafter).Idioplasm group's selection makes latitude of the environmental origin of their representatives extreme (a), (b) there is the reaction of blooming the different photoperiods, and (c) with respect to the megaspore generation of agametophyte ovule and ovary tissue, megagametophyte generation and embryo opportunity.

When if there is no the female development time difference of appropriate level is different in present available mutation or strain system, then plant breeding is the essential part of method for optimizing that obtains preparing the idioplasm of asexual reproduction plant from the sexual reproduction plant.Preferable methods comprises: (a) determine several or more be adapted to the mutation of high latitude or environmental and several or more be adapted to the mutation identical or that be closely related or the ecotype of low latitudes, (b) two latitudes are adapted to intervarieted cross in each of classifications, (c) according to different photoperiod being handled the response difference degree and megaspore is taken place, megagametophyte takes place and embryo opportunity of blooming, from F ₁Select idioplasm among the s, and (d) use conventional breeding system (as, mix selection, single seed progeny), continue each proterties is selected.For those skilled in the art, these methods are well-known, as, Poehlman, Breedingfield Crops (Van Nostrand Reinhold, 1987) (being incorporated herein by reference).

Cross method

If selected species be dioecious or self-compatible, have the short brephic and be easy to carry out the cytogenetics and the embryology analysis of the tip of a root and ovule respectively, hybridization and the amphiploid process will accelerate.If plant is not dioecious or self-compatible, must uses male sterile plant system or carry out the castration process.When the difference of performance photoperiodic reaction difference of appropriate level and female development time table, the hybrid of sexual mutation or strain system has just produced.Prepared species hybrid according to the standard method of being taught in the plant breeding textbook, as, Poehlman, Breeding Field Crops (1987).Described in the nearest reference that relates to distant hybridization, as, Z.W.Liu etc., wheat (Triticum aestivum L.) and asexual reproduction Australian Wheatgrass (Elymusrectisetus (Nees in Lehm.) A.Love ﹠amp; Connor]: caryogram and genome analysis, 89 theoretical and applied genetics 599-605 (1994) (being incorporated herein by reference) is hybridized and the backcross progeny kind between or the successful preparation of bigenering may need HORMONE TREATMENT Xiao Hua and embryo rescue program.Confirm hybrid by phenotype in the middle of it.

The amphiploid method

The colchicine technology of standard can make chromosome number double, as, J.Torabinejad etc., the form and the genome analysis of Elymus scabrus species hybrid, 29 genome 150-55 (1987) (being incorporated herein by reference).Optionally, can adopt the tissue culture technique of latest developments, as, O.Leblanc etc., the chromosome doubling among the Tripsacum: the preparation of artificial, sexual tetraploid plant, 114 plant breeding 226-230 (1995) (being incorporated herein by reference); Salon ﹠amp; Earle, Eastern Gamegrass synthesize the definite of tetraploid modes of reproduction, Agron.Abs.114 page or leaf (1994) (being incorporated herein by reference); Cohen ﹠amp; Yao, the external chromosome doubling of nine strain Zantedeschia cultivated speciess, 47 plant cells, tissue, organ culture 43-49 (1996) (being incorporated herein by reference); And Chalak ﹠amp; Legave, Oryzalin and indefinite regeneration are united and are used for the effective chromosome doubling of triploid Kiwifruit, 16 plant cells breeding 97-100 (1996) (being incorporated herein by reference).F between planting ₁When s backcrosses, part amphiploid 2n+nB _IIIHybrid often produces (0.5%--3%) with low probability, as, Z.W.Liu etc., 89 theoretical and applied genetics 599-605 (1994), if the tendency of asexual reproduction (formation of ameiosis ovum) exists, what this probability may be high is many, as, O.Leblanc etc., the reproductive behavior of corn-Tripsacum polyhaploid plant: relate to asexual reproduction and transfer in the corn, 87 genetics magazine 108-111 (1996) (being incorporated herein by reference).Therefore, planting interior preferable methods with the species hybrid chromosome doubling is colchicine (or other known spindle poison) processing method listed above the one or many.The preferable methods of species hybrid chromosome doubling equally, comprise with one of sexual parent with the counting tip of a root in chromosomal number with determining section amphiploid (being generally triploid).Backcross to obtain completely amphiploid or to backcross to obtain partial diploid (three genomes are from a parent, and a genome is from another parent) with another parent then with same parent.Amphiploidization can carried out before the hybridization or after hybridization.

Confirm the program that asexual reproduction is expressed

The expression of asexual reproduction in artificial amphiploid passed through according to J.G.Carman ﹠amp; S.L.Hatch, aposporous asexual reproduction among the Schizachyrium Poaceae:Andropogoneae, 22 crops science 1252-55 (1982) (being incorporated herein by reference) analyze the megaspore generation and blastular is grown to analyze aposporous asexual reproduction, according to C.F.Crane ﹠amp; J.G.Carman, 74 american plants learn magazine 477-96 (1987) (being incorporated herein by reference) and analyze diploidy sporulation asexual reproduction.Progeny analysis also is useful, according to Asker ﹠amp; The carrying out that Jerling tells about.

Some feature of the present invention can be by being understood with reference to certain embodiments better.Be to be understood that the following examples are indicative rather than restrictive, they are to be used to illustrate basic religious doctrine of the present invention and idea rather than restriction the present invention.Expectation those skilled in the art can use embodiment to neutralize other information that comprises herein to use under the situation that the present invention not have herein special description.

Embodiment 1

Select and collect idioplasm---dicotyledon

A feature of the present invention provided from species or one by selecting in the colony of the species composition that is closely related and collecting only strain system to produce the method for asexual reproduction plant.These methods are considered to simulate and quickened greatly initial natural process from the sexual reproduction plant to the asexual reproduction plant evolution, that is, those cause taking place the secondary natural process that contacts in the weather that has adapted to big big-difference and photoperiodic taxon.

In this embodiment, explained the preferable methods that is used for Dicotyledonae in detail.In embodiment 2, explained the preferable methods that is used for monocotyledon subclass in detail.Dicotyledon embodiment has comprised the sexual species that belong to (X=14) from Antennaria.Expectation those of ordinary skill in the art can successfully apply to the method many species, comprises various dicotyledonous crops, as, strawberry, Raphanobrassica, potato, cherry, apple and beet.

Selecting at present the preferable methods of suitable given species or the system of the strain in the tight akin species group to begin is that (a) determines geographical distribution from record and/or from the research of field, collects with (b) and all relevantly spends biological information (photoperiodic reaction, embryology etc.) and various ecotypic any adaptation especially in the extreme ecotype of latitude.For example, dicotyledon Antennaria belongs to the polyploid agamic complex (asexual reproduction) that comprises 33 sexual species (great majority are dliploid, and some are arranged is tetraploids) and 5 big height polymorphisms.They are dicots, draft, perennial and common tool stolon.Antennaria asexual reproduction takes place naturally in one of six regions usually, and Catepes comprises sexual dliploid (2X) and from the sexual and agamic polyploid of 4X to 12X.All members of this population all have on stolon and the sex be dimorphism (Bayer, the kind system of Antennaria gaertner (Asteraceae:Inuleae) rebuilds 68 Canad.J.Bot 1389-97 (1990) (being incorporated herein by reference); Bayer is the evolution of example polyploid agamic complex with Antennaria (Asteraceae), 132 OperaBotanica 53-65 (1996) (being incorporated herein by reference)).

Ecotypic widely, form and genetic research show 5 agamic complexs of Catepes, and A.alpina (L.) Gaertn, A.howellii E.L.Greene, A.parlinii Fern., A.parvifolia Nutt. and A.rosea are from sexual spore.For example, the multifarious center of A.rosea agamic complex is the Rocky Mountain (near periglacial) of Western North America, its scope is from New Mexico and California south, to Alaska and NorthwestTerritories, Alberta, Saskatchewan, Great Lakes the north are passed in the east, at Atlantic Canada discrete colony are arranged.These sexual species tools are thought the initial evolution that has participated in A.rosea, its expansion subsequently comprises A.aromatica Evert, A.corymbosaE.Nelson, A.pulchella E.Greene, A.marginata E.Greene, A.microphylla Rydb. (Bayer, 132 Opera Botanica 53-65 (1996)).

The agamic sexual correlative of A.rosea meets and listed prepares geography and the reproduction criteria of asexual reproduction plant from the sexual reproduction plant herein.They have Latitude Distribution and variant on flowering time very widely, have been noted that as, some asexual reproduction form is bloomed than near late three week of sexual form (G.L.Stebbins, 94 Bot.Gaz.322-45 (1932)).At last, the Antennaria species are easy to cultivating and growing, are easy to carry out interspecific cross (owing to lacking inner breeding separation mechanism) and when by vernalization, can produce every year two generation seed.

The ramet of Antennaria is to represent (Fig. 2 A) that latitude is extreme and collect in latitude intermediate point, site ecology and the multifarious vegetable material of form from providing.Collect at the florescence (for low latitudes, is June; For high latitude, be July), at this moment species can be proved and hybridize required male and female and can be identified.The preparation and the execution of collecting stroke comprise study wax leaf voucher specimen and obtain collecting permission.Clone's velamen is cleaned and is enclosed in then in the moist bog moss.Cutting is carried out getting wet in potted plant and the process of taking root in the potting mixtures of standard.Be to be understood that and collect with take root may some difference for different dicotyledons.

Embodiment 2

Select and collect idioplasm---monocotyledon

In this embodiment, explained the method for selecting and collecting idioplasm from monocotyledon subclass in detail.This monocotyledon embodiment has comprised the sexual species that belong to (X=18) from Tripsacum.Expectation those of ordinary skill in the art can successfully apply to the method many species, comprises various unifacial leaf crops, as, Chinese sorghum, wheat, barley, paddy rice and corn.

Present embodiment relates to monocotyledon Tripsacum and belongs to, it is that New World is distinctive, survive in 42 ° N-24 ° N latitude (de Wet etc., the phylogeny of Tripsacum dactyloides (Gramineae), 69 american plants are learned magazine 1251-57 (1982) (being incorporated herein by reference), from 0-2600 rice height above sea level (Berthaud etc., Tripsacum: diversity and conservative are at Taba, maize genetic resource (CIMMYT, 1995) (being incorporated herein by reference)).Hermaphrodite herbaceos perennial, male flower and female flower are separated from each other along spike.Be typically in many agamic complexs, the variation of key feature has overlapping in 16 species, and this is difficult to they are divided into definite individuality.The T.dactyloides agamic complex is the most various during this belongs to.Its scope has covered the scope of whole genus, and other dliploid of two individual characteies and asexual reproduction polyploid all have distribution (de Wet etc., 69 american plants magazine 1251-57 (1982) everywhere; Berthaud etc., Tripsacum: diversity and conservative be at Taba, maize genetic resource (CIMMYT, 1995)).

The Tripsacum dliploid is sexual, and all studied on embryology abiogenous are agamete (Burson etc., the asexual reproduction of Eastern Gamagrass and sex, 30 crops science 86-89 (1990) (being incorporated herein by reference); O.Leblanc etc., the megaspore of several Tripsacum (Poaceae) species is taken place and megagametophyte takes place, 82 american plants are learned magazine 57-63 (1995) (being incorporated herein by reference)).These are different with Antennaria, the latter's autotetraploid abiogenesis.Recently, make by colchicine and be adapted to that North America, temperate zone weather is diplontic to have doubled to prepare sexual Tripsacum amphiploid (Salon ﹠amp; Earle, Eastern Gamegrass synthesize the definite of tetraploid modes of reproduction, Agron.Abs.114 page or leaf (1994); Salon ﹠amp; Pardee, the registration of Eastern Gamegrass SG4X-1 idioplasm, 36 crops science 1426 (1996) (being incorporated herein by reference)), by with the diploid hybrid that is adapted to tropical Mesoamerican weather, carry out colchicine then and double (O.Leblanc etc., chromosome doubling among the Tripsacum: the preparation of artificial, sexual tetraploid plant, 114 plant breeding 226-230 (1995) (being incorporated herein by reference)).

Sexual Tripsacum dliploid (with artificial sexual amphiploid) meets and listed prepares geography and the reproduction criteria of asexual reproduction plant from the sexual reproduction plant herein.They have geographical distribution very widely and the sexual ecotype spread all over the world (Fig. 2 B).These ecotypes are easy to cultivating and growing, are easy to hybridize.Their ovule by disturb the difference and fluorescence microscopy is examined and have ecotype variation (Fig. 3) in female development time table.At last, the torrid zone keeps green and (10-12 hour/day) late fall the or early winter blooms under the short-day condition the environmental whole year.On the contrary, the temperate zone ecotype needs vernalization, follows by the long-day (14-16 hour).The torrid zone and the temperate zone ecotype can not rebloom before specific stimulation repeats.

The ramet of Tripsacum is to represent the extreme and latitude intermediate point of latitude, site ecological and the multifarious vegetable material of form collection from providing, as, Fig. 2 B.Collect at the florescence (for high latitude, is June and July; For low latitudes, be September And October), at this moment species can be proved and obtain (CIMMYT) from the nursery of the U.S. and Mexico's work.Collect the preparation and identical (embodiment 1) of execution of Tripsacum idioplasm with Antennaria.Be to be understood that and collect with take root may some difference for different monocotyledons.

Embodiment 3

The different photoperiods are quantitative to the influence of blooming

One of feature of the present invention provides carries out quantitative methods to being chosen as the only sexual reproduction strain system that is used to prepare asexual reproduction plant (embodiment 1 and 2) to the influence of the growth of different photoperiodic flowers.Be to be understood that and announced many methods of determining the quantity of this influence in nearest document, those skilled in the art can find that the method beyond the method described herein more is applicable to some species.

Present preferably to wait at the paramount mountain mists of temperate climate (medium latitude) (high latitude) be the photochemical and thermal reaction quantitative methods of natural Antennaria species be to Oxyria digyna traditional research modify (Mooney and Billings, the comparison Physiological Ecology of the Oxyria digyna arctic and high mountain population, 31 Eco Monog.1-29 (1961) (being incorporated herein by reference)).Selected a series of ecotypes (each contains 12 clones) from being confirmed as the only ecotype that is used to prepare asexual reproduction plant (embodiment 1), that is, they represent the extreme and latitude intermediate point of latitude.Bloom the back during the 6th week, and each ecotypic four clone is placed on temperature and is made as in 4 ℃ of (constant) vernalization growth rooms and 6 hours photoperiod (low light level).All around, each ecotypic other four clone is placed in the vernalization growth room, when the 8th week, carries out the experiment of the 3rd series then.

When the 12nd week, all clones are taken out from the vernalization growth room, three groups of different vernalization open-assembly times (4,8 and 12 week) are provided.Each is ecotypic, each open-assembly time group four clones assign at random two flowers induce the photoperiod (12 and 14 hours) each be set to 22/12 ℃ sunshine/dark temperature scenario.Measure all clones from vernalization finish to bud form, archesporium forms, megaspore is taken place, megagametophyte takes place and the time interval of bloom (dependent variable).Each dependent variable being carried out the factorial experiment (the ecotypic number of 3X2X) of variance analysis and conduct repetition (each environmental analysis twice) analyzes.Cluster analysis (Sneath ﹠amp; Sokal, numerical taxonomy: skyline diagram principle with put into practice (1973) (being incorporated herein by reference)) ecotype that is used to show similar female sexual development (with phenology, cytological and photo-thermal data) is divided into one group, the result be used to predict when artificial amphiploid respectively at the environmental of breeding difference or breed when forming between the similar ecotype, be asynchronous (asexual reproduction) or synchronous (normally) female sexual development (table 1).

Embodiment 4

The different photoperiods are quantitative to the influence of blooming

Present preferably in tropical climate (low latitudes) to temperate climate (medium latitude) being the photochemical and thermal reaction quantitative methods and different (embodiment 3) that are used for Antennaria of natural Tripsacum species.The torrid zone keeps green and (10-12 hour/day) late fall the or early winter blooms under the short-day condition the environmental whole year.On the contrary, the temperate zone ecotype needs vernalization, follows by the long-day (14-16 hour).The torrid zone and the temperate zone ecotype can not rebloom before specific stimulation repeats.Preferable methods of the present invention has been utilized these differences.Selected a series of ecotypes (each contains 16 clones) latitude be extreme to represent, latitude intermediate point and other tangible female sexual development difference form.Induce each ecotype to bloom, bloom subsequently stop after, under non-inductive condition (13 hour photoperiod, 30/25 ℃ sunshine/dark temperature) be held at least 6 weeks.Then, each ecotypic four clone is placed on that temperature is made as in 6 ℃ of (constant) vernalization growth rooms and photoperiod (low light level) of 13 hours.All around, each ecotypic other four clone is placed in the vernalization growth room, when the 8th week, carries out the experiment of the 3rd series then.When the 12nd week, all clones are taken out from the vernalization growth room, three groups of different vernalization open-assembly times (0,4,8 and 12 week) are provided.

After vernalization finishes, each is ecotypic, each open-assembly time group four clones assign to two flowers at random and induce in the treatment combination and (two clones are arranged) in each, each combination by the photoperiod determine (10 and 16 hours) each be set to 30/25 ℃ sunshine/dark temperature scenario.Measure all clones from vernalization finish to bud formation, bolting, archesporium form, megaspore is taken place, megagametophyte takes place and the time interval of bloom (dependent variable).Each dependent variable being carried out the factorial experiment (the ecotypic number of 4X2X) of variance analysis and conduct repetition (each environmental analysis twice) analyzes.Cluster analysis be used to show similar phenology, the ecotype cytological and the photo-thermal feature is divided into one group, as used in Antennaria embodiment, the result is used for prediction when being asexual reproduction sample or normal development (table 1) during amphiploid between the artificial preparation ecotype.

Embodiment 5

Difference in the female development time table is carried out quantitatively

The difference that a feature of the present invention has provided between ecological property in the female development time table is carried out quantitative methods.The method that preferably can be used for dicotyledon (as Antennaria) and monocotyledon (as Tripsacum) is at present united the utilization noninvasive measurement and is destroyed sampling, and bud forms to measure, archesporium forms, megaspore is taken place, megagametophyte takes place, bloom, fertilization and early embryonic development (2-16 cell stage) time interval.These information can be in the embodiment 1 and the 2 selected ecotypes, that is, those Dai Xuan latitude and other ecological extreme ecotypes obtaining after the growth under the unified condition.The data of collecting among the embodiment 3-5 obtain simultaneously with same set of plant.

Female meiosis early stage, dyad, tetrad and degeneration megaspore stage and 1,2,4 and 8 nuclear blastular stages have been carried out cytological analysis, after analyzing each ovule, obtain following data: reduction division or blastular developmental stage, gynoecium length and width, inner integument and external integument length, gonotocont or blastular length and width.According to the method in the following reference: C.F.Crane ﹠amp; J.G.Crman (74 american plants are learned magazine 477-96 (1987)), J.G.Crman etc., the comparative histology of cell wall during two hexaploid Australian Elymus species reduction division and asexual reproduction megaspore are taken place, 31 crops science, 1527-32 (1991) (being incorporated herein by reference); M.D.Peel etc., 37 crops science 724-32 (1997) (being incorporated herein by reference); And M.D.Peel etc., at the unusual A.Love﹠amp of reduction division of wheat and agamete Elumus rectisetus (Nees inLehm.) hybrid; Connor, 37 crops science, 717-23, (1997) (being incorporated herein by reference), the gynoecium that is used for cytological analysis is killed, and is fixing, transparent, observe and measure.The developmental stage data are to (a) gynoecium and integument length and width (initial data) and (b) be expressed as the length of structure and the width (measuring when column cap stretches out) of the percentage of its ripe length and width.By the similitude of the environmental female development time table of variance analysis, and Fig. 3 and 4 and table 2 back produce post analysis figure angle distribution, with the environmental amphiploid (table 1) of cluster the analyses and prediction artificial asexual reproduction sample or the normal development generation of

embodiment

3 and 4.

Table 2 has been represented to disturb the difference microscope to determine gynoecium length and the relation of gynoecium developmental stage of sexual (dliploid) and diploidy sporulation (polyploid) Tripsacum by analyzing transparent ovule with Normurski.

Table 2

The gynoecium number of analyzed gynoecium length (mm) ₁
The gynoecium number of analyzed gynoecium length (mm) ₁			Stage ₂	0.75- 1.00	1.00- 1.25	1.25- 1.50	1.50- 1.75	1.75- 2.00	2.00- 2.25	2.25- 2.50	＞2.50
Sexual 4-nuclear ES 2-nuclear ES	? ????3	????1 ????7	Stage ₂	0.75- 1.00	1.00- 1.25	1.25- 1.50	1.50- 1.75	1.75- 2.00	2.00- 2.25	2.25- 2.50	＞2.50
Sexual 4-nuclear ES 2-nuclear ES	? ????3	????1 ????7	Deqen diad tetrad diad MMC diploidy sporogenesis 8-nuclear ES 4-nuclear big MMC MMC in ES 2-nuclear ES mid-term	? ? ? ????6 ? ? ????17	? ? ????6 ????3 ? ????7 ????29	????3 ????19 ????13 ? ? ????16 ????21	????9 ????17 ????4 ? ????7 ????21 ????5	????18 ????7 ? ????1 ????18 ????11	????10 ????3 ? ????4? ????14 ????3	????6 ????1 ? ????3? ????7	????4 ? ????2 ????5

1 strides species and ploidy level merging gynoecium (seeing Table 1).2 ES: blastular; Degen tetraploid: the tetrad that three megaspores in micropyle district degenerate; Mid-term: the MMC of meiosis metaphase (M.D.Peel etc., 37 crops science 724-32 (1997)).

Embodiment 6

In female development time table, obtain bigger difference

Be to be understood that, (a) to the different photoperiods bloom reaction and (b) the enough difference in the female development time table will can not express all in the existing ecotypes of biological species many, even by having enough hereditary variabilities to set up such difference in their the protogene storehouse of breeding, that is, a certain all in the natural disposition species as a whole.One of feature of the present invention provides the breeding that improves this species diversity and instructs.As D.Wilson, the breeding of form and physiological trait is K.J.Frey (editor), plant breeding II (Iowa State university Press) (1981) (being incorporated herein by reference) pointed out, many forms and physiological trait, comprise that the reaction of blooming to illumination length also is a quantitative inheritance, this means that they are subjected to a plurality of effect genes.Therefore, by to showing the strain cross of some this proterties, and option table reveals the strain system of bigger tendency from its filial generation, is obtaining many progress aspect the duration of day improving, and expects that wherein plant makes a response by blooming.Repeat this process several generations, can expect to obtain bigger progress.By to the strain cross that shows some this proterties and adopt similar repeating to select and breeding system, can obviously reduce in the same way in the duration of day of the plant of making a response by blooming.At present preferably the proterties that can obtain maximum difference by such breeding plan scheme comprises that (a) is to the photoperiodic reaction of blooming of difference, promptly, produce long and the short-day ecotype, (b) with respect to the growth of agametophyte ovule and ovary tissue, accelerate or the archesporium that postpones forms initial, reduction division, blastular growth etc.

Be to be understood that, difference enough in the growth of flower will can not expressed in many non-time existing ecotypes of biological species usually, even by having enough hereditary variabilities to set up such difference in their secondary of breeding and three grades of gene pools, that is, and in same genus, family or section.Can think distant hybridization even can the target species gene be integrated to obtain (a) suitable reaction and (b) in the suitable difference of female development time table of blooming in the future being used for by gene engineering.

Embodiment 7

From the discrepant sexual reproduction strain of flower development system preparation asexual reproduction plant

Technology among the embodiment 1 to 6 is by with instructing to obtain the three or more kind of same species (or the species group that is closely related), obviously be adapted to long illumination (14-20 hour), have the growth with respect to agametophyte ovule and ovary tissue (megarchidium, integument, pericarp etc.), early stage archesporium is grown/early stage reduction division/early stage gametophyte growth.Same technology is by with instructing to obtain the three or more kind of same species (or the species group that is closely related), obviously be adapted to short illumination (10-12 hour), has growth, archesporium reduction division in growth/late period in late period/gametophyte growth in late period with respect to agametophyte ovule and ovary tissue (megarchidium, integument, pericarp etc.).Several strains of each classification (long-day plant and short-day plant etc.) are selected so that their form non-individual body about the derivative illumination length of blooming, that is, have some long, long and very long and have some to lack, lack and very short.These strains are selected, and wherein blastular initial (the degeneration megaspore stage) that form time of (be generally long illumination adapted strain system) in a series of strains system approximately is the time of taking place early stage to mid-term with respect to the female meiosis of agametophyte ovule and ovary tissue development in another serial strain system.

Use above-mentioned standard method (colchicine-induced or B then by repeating _IIIThe generation of hybrid) and other suitable method prepare amphiploid.The cross method of standard is used to prepare the hybrid of Tripsacum species.For Antennaria,, separate female by pollination bag (from the laboratory tissue preparation, as KIMWIPES) is placed on the whole capitulescence.Female inflorescence of acceptance and stamen head are finished pollination by rubbing together in flowering stage.Shou Fen contrast capitulescence is not used to confirm agamic disappearance among each parent.This is even more important for agamic or amphigenetic tetraploid clone occurring.When fruit maturation, in pollination bag, do not need embryo rescue.

At least three in initial 9 possible parental combinations (from each adapt to group) are made into amphiploid: some strain system early and some late strain system, the system of strain early and late strain system, strain system very early and the unusual strain system in evening.According to their asexual reproduction of above-mentioned inspection.If do not express asexual reproduction, then from 9 possibilities, prepare other amphiploid.The genetic background that should be appreciated that strain system origin may influence agamic expression.Therefore, select or prepare other integration the strain system of different genetic backgrounds may be essential sometimes.

Embodiment 8

Preparation asexual reproduction unifacial leaf and dicotyledon

Technology described in embodiment 1-7 can be used for obtaining the asexual reproduction plant from sexual dicotyledon or sexual monocotyledon.

According to the practice of telling about herein, the sexual reproduction plant that can belong to from many angiosperms efficiently obtains the asexual reproduction plant.In the hybrid of artificial unifacial leaf and dicotyledon and amphiploid, can observe asexual reproduction (Linnean once in a while; Asker ﹠amp; Jerling).This occurrent reason be infer and the asynchronous idea of duplicate factor can not be described.And they are not supported by research institute phylogenetic, hereditary, physiological or that grow.The explanation of these researchs is to exist specific asexual reproduction gene still to be suppressed (Asker ﹠amp by dliploid or inappropriate genetic background in the parent; Jerling).This is rational for the embodiment that relates to agamic complex.For example, G.L.Stebbins, the cytology of Antennaria I. normal species, 94 Bot.Gaz.134-51 (1932), the apospory blastular that has write down in sexual Antennaria neglecta and the sexual Antennariaplantaginifolia hybrid forms.Equally, Nordberg, the embryology research of Sanguisorba minor (Rosaceae) complex, 120 BotaniskaNotiser 109-19 (1967) (being incorporated herein by reference).Prepared all hybrid combination between two sexual tetraploids and the sexual octoploid Sanguisorba kind, all tetraploids that obtain and hexaploid F ₁S produces the apospory blastular, and wherein unreduced ovum forms the embryo by parthenogenesis or after fertilization.And, A.Jankun ﹠amp; M.Kovanda, the asexual reproduction of Sorbus eximia (embryology research is carried out in Sorbus 3) dliploid level, 60 Preslia, Praha, 193-213 (1988) (being incorporated herein by reference), write down have fully among dliploid and the tetraploid Sorbus eximia function, high-frequency is aposporous and the diploidy sporulation, Sorbus eximia be geographical go up restriction, from sexual dliploid Sorbus aria and the hybrid species of Sorbus torminalis.These three examples have all comprised the agamic complex that the asexual reproduction gene is not also expressed in sexual dliploid ancestors.On the contrary, the example that comprises the taxon that has nothing to do with agamic complex that also has other.

In Triticeae, reported low-frequency asexual reproduction (from the not parthenogenesis of subtrahend the ovum) (A.Mujeeb-Kazi in three the three species hybrids, from the asexual reproduction filial generation that Hordem-Triticum is bigenered, 72 genetics magazine 284-85 (1981) (being incorporated herein by reference); A.Mujeeb-Kazi, the asexual reproduction in Triticum aestivum/Leymus racemosus//Thinopyrum elongatum three intergeneric cross, 61 Cytologia 15-18 (1996) (being incorporated herein by reference); R.Von Bothmer etc., aggregate species intermolecular hybrid of barley (Hordeum vulgare L.) and agamic may the generation, 76 theoretical and applied genetics 681-90 (1988) (being incorporated herein by reference), wherein neither one relates to Elymusrectisetus, unique agamete among the Triticeae, even other Elymus sp.

In sexual amphiploid Raphanus sativs and the hybrid between the Brassica oleraceae, observed the high many asexual reproduction of frequency.From 10 hybrids that produce, comprise 1.6-2.9 apospory blastular (S.Ellerstrom ﹠amp in the ovule of 6 36-70%; L.Zagorcheva, sterile and asexual reproduction blastular among the Raphanobrassica forms, 87Hereditas 107 (1977) (being incorporated herein by reference), an and maternal filial generation (S.Ellerstrom who has write down apospory Raphanobrassica, the asexual reproduction filial generation of Raphanobrassica, 99 Hereditas 315 (1983) (being incorporated herein by reference).Other in parent's species, genus, section or whole Brassicales (Linnean) is local expresses in apospory, and this illustrates that forcefully apospory is not to grow the asexual reproduction gene that suppresses to accumulate just gradually and show in this case.At this effect, S.Ellerstrom ﹠amp; L.Zagorcheva, 87 Hereditas 107 (1977) tell about:

" we think, it seems that more Impartial Findings are Raphanobrassica

In the formation of apospory blastular cause by physiologic derangement, in the hybrid two

The damaged result of cooperation between the individual parental gene group, rather than there is control in supposition

The special genes that this blastular forms.”

Sven Ellerstrom is just dead soon after carrying out this research, does not carry out follow-up research.S.Ellerstrom ﹠amp; L.Zagorcheva, 87 Hereditas, 107 (1977) (being incorporated herein by reference) and S.Ellerstrom, the asexual reproduction filial generation of Raphanobrassica, the essence or the reason of physiologic derangement between the aposporous genus of the responsible Raphanobrassica beyond hybridize in the source far away that 99 Hereditas 315 (1983) (being incorporated herein by reference) do not think deeply Ernst to be provided.They do not determine how such disorder explains that evidence and some agamete of quite simple Mendellian heredity in many agametes may be derived from autopolyploid yet.These defectives are bred in the research that unusual kind is and genome is formed angiosperm (Linnean) and are resolved, and wherein the asynchronous expression of duplicate factor obtains identifying.This idea explained basically all main inconsistencies in the asexual reproduction document and by countless system take place, genetic, genomic and physiological research institute supports (Linnean; M.D.Peel etc., 37 crops science 724-32 (1997); M.D.Peel etc., crops science 37,717-23 (1997)).

Be to be understood that the present invention can embody with other specific forms not breaking away under its spirit or the situation of its essential characteristic, its basis is to have found that asexual reproduction is that asynchronous expression by duplicate factor in the female sexual development path causes.Above-mentioned step and material all should be counted as indicative rather than restrictive, and scope of the present invention is illustrated by additional claim rather than by the description of front.The institute of the meaning and scope changes and is also contained in its scope in the suitable thing of claim.

Claims

1. one kind obtains the method for asexual reproduction plant from the sexual reproduction plant, may further comprise the steps:

(a) obtain the described strain of at least two covers system from plant species or akin plant species colony, these strains systems are by being distinguished various photoperiodic reaction female time started developmental stage and the duration relevant with the ovary tissue development with them and agametophyte ovule of blooming; And

(b) make described strain cross and select the hybrid strain system of those genetic stockss that comprise each described strain system so that it is endowed nonsynchronous flower development and the asexual reproduction hybrid strain that causes thus is.

2. method according to claim 1, the response difference of wherein blooming are present in short-day plant, long-day plant, dual day long plant, day intermediate plant or the osculant photoperiod plant.

3. method according to claim 1, the response difference of blooming wherein be present in short-day plant, long-day plant, dual day long plant, day intermediate plant, osculant photoperiod plant and at least one member of the colony that forms of day-neutral plant in.

4. method according to claim 1 wherein obtains by plant breeding various photoperiodic differences of blooming reaction.

5. method according to claim 1, wherein the difference of the time started of female developmental stage and duration is present in archesporium formation, megaspore generation, megagametophyte generation or the early embryonic development.

6. method according to claim 1, wherein the difference of the time started of female developmental stage and duration is present among at least one member who is selected from archesporium formation, megaspore generation, megagametophyte generation and early embryonic development.

7. method according to claim 1 wherein obtains by plant breeding in the time started of female developmental stage and the difference of duration.

8. method according to claim 1, agametophyte ovule wherein and ovary tissue comprise at least one member of the colony that is made up of megarchidium, integument, pericarp, hypanthium and gynoecium wall.

9. method according to claim 1, wherein genetic stocks comprises the genome that can express the described strain of every suit system of the asynchronism of measuring by asexual reproduction of giving suitable degree.

10. method according to claim 1, wherein genetic stocks comprises the chromosome that can express the described strain of every suit system of the asynchronism of measuring by asexual reproduction of giving suitable degree.

11. method according to claim 1, wherein genetic stocks comprises the gene that can express the described strain of every suit system of the asynchronism of measuring by asexual reproduction of giving suitable degree.

12. method according to claim 1, it is unusual that the performance of wherein said selected hybrid strain system is selected from the breeding of apospory, diploidy sporulation and polyembriony.

13. method according to claim 1, wherein said selected hybrid strain system is the polyploid in the heredity.

14. method according to claim 1, wherein said selected hybrid strain system is the triploid in the heredity.

15. method according to claim 1, wherein said selected hybrid strain system is the aneuploid in the heredity.

16. method according to claim 1, wherein said selected hybrid strain system is the dliploid in the heredity.

17. the method from sexual reproduction plant acquisition asexual reproduction plant comprises step:

(a) differences to the various photoperiodic reactions of blooming of natural generation in plant identification species or the akin plant species colony;

(b) obtain two covers described by them to the strain system in various photoperiodic the bloom plant species distinguished of reaction or the akin plant species colonies;

(c) identify in described strain system or between female developmental stage time started relevant with the ovary tissue development and the difference of duration with the agametophyte ovule;

(d) obtain the described strain of at least two covers system of described species or akin species colony, they are by being distinguished with female developmental stage time started and the duration relevant with the ovary tissue development with the agametophyte ovule the various photoperiodic reactions of blooming; And

(e) the preparation hybrid strain system that comprises the genetic stocks of each described strain system makes it be endowed nonsynchronous flower development and the agamic hybrid strain that causes thus is.

18. a method that obtains apospory, diploidy sporulation or polyembryony plant from sexual reproduction monocotyledon or dicotyledon may further comprise the steps:

(b) obtaining two described strains of the various photoperiodic reactions of blooming being distinguished among plant species or the akin plant species group by them of cover is;

(c) identify in described strain system or between relevant with the growth of agametophyte ovule that is selected from megarchidium, integument, pericarp, hypanthium and gynoecium wall and ovary tissue archesporium formation, megaspore generation, megagametophyte generation and female time started developmental stage of early embryonic development and the difference aspect the duration of being selected from;

(d) obtain the described strain of at least two covers system of described species or species colony, the differentiation of these strains systems owing to they:

(i) to the various photoperiodic reactions of blooming, difference occur in be selected from short-day plant, long-day plant, dual day long plant, day intermediate plant, osculant photoperiod plant and a member of day-neutral plant in or cross among the above member, and

(ii) relevant with the growth of agametophyte ovule that is selected from megarchidium, integument, pericarp, hypanthium and gynoecium wall and ovary tissue be selected from that archesporium forms, megaspore is taken place, megagametophyte takes place and female time started developmental stage and the duration of early embryonic development, difference occurs in the member of so female developmental stage or crosses over an above member;

(e) the preparation polyploid, triploid, dliploid or the aneuploid strain system that comprise genome, chromosome or the gene of each described strain system makes and expresses asexual reproduction.

19. prepare the method for asexual reproduction plant from two or more sexual reproduction plants identical or akin species, may further comprise the steps:

(a) obtain two sexual reproduction strain systems that the female reproduction phenotype is different, at the asynchronism that between two strain systems, has female development time table under the similar environmental condition;

(b) if described strain system also is not a polyploid, be that chromosome doubling prepares amphiploid by the different sexual reproduction strain of female development time table; And

(c) make these two sexual reproduction strain crosses to induce asexual reproduction.

20. method according to claim 19, step wherein (c) are in step (b) before.