CN1209724A - Cytoplasmic male sterile brassica oleracea plants which contain polima CMS cytoplasm and are male sterile at high and low temp. - Google Patents

Abstract

The present invention involves cytoplasmic male sterile Brassica oleracea plants that contain polima CMS cytoplasm. The plants remain sterile at high temperatures. The Brassica oleracea plants of the present invention can be prepared by traditional breeding techniques. Different Brassica types can then be produced using these plants by employing traditional breeding techniques or by protoplast fusion.

Description

Contain polima CMS cytoplasm and male sterile cytoplasmic male sterile brassica plant under high temperature and low temperature

Invention field

The present invention relates to contain the cytoplasmic cytoplasmic male sterile brassica plant of polima CMS, its male sterile under high temperature and low temperature, and show good female educating.

Background of invention and summary of the invention

In the difficult trial of the output that improves plant and food crop, plant breeder's exploitation contains the height that certain gratifying characteristic for example increases, growth rate, the cultivar of higher output or the like usually.One of successful approach is by gratifying characteristic being injected plant, forming good plant lines.Then with good plant lines in conjunction with the F1 hybrid that has gratifying characteristic with formation.Described good hybrid can be with many approach exploitations.

One of general approach that produces good hybrid is by utilizing male sterile plant plant to carry out a kind of plant of required hybridization.The breeder utilizes male sterile line, and the flower by controlling plant carries out crossing pollination and can produce hybrid seed more economically.Can control crossing pollination by the self-pollination that stops female parent.Can remove self-pollination by making plant male sterile.If plant is male sterile, can not produce the pollen that is used to pollinate so.In case present male sterile, just with this plant and genetic donor plant hybridization with desirable characteristics.

Realize that a male sterile approach is by using cytoplasmic male sterile line.Think at present in cytoplasm, to have the hereditary factor of controlling cytoplasmic male sterility (CMS), particularly in the gene of mitochondrial DNA.

Three kinds of the most general in rape kind cytoplasmic male sterile lines are:

(1) the Ogura male sterile cytoplasm of radish sativus;

(2) the polima male sterile cytoplasm of rape; With

(3) the Nap male sterile cytoplasm of rape.

In rape, can hereditary cytoplasmic male sterility by hybridization.Female (egg) parent provide cytoplasm, therefore female with CMS hybridization generation CMS filial generation.But nuclear gene is a heterozygosis.Therefore, " backcrossing " 6-8 generation is necessary, so that produce the breeding of the cell nucleus characteristic CMS strain of isozygotying.As the optional method of another kind, also can produce cytoplasmic male sterile line by the protoplast fusion.In protoplast merged, the protoplast that will have the plant of commercial desirable characteristics combined with the protoplast of CMS strain.Before merging, the nuclear matter of removal or inactivation CMS strain, so it only provides cytoplasm.The cytoplasm hybrid (or cybrid) that obtains has CMS cytoplasm and is male sterile.For example, United States Patent (USP) 5,254,802 disclose and have contained Ogura CMS cytoplasm brassica oleracea plants.These plants can be merged acquisition by protoplast.

Polima CMS cytoplasm has been used to produce for example type rape in winter (Brassica napus) (referring to people such as Barsby, plant science, 53:243-248 (1987)) of CMS strain.But, be the influence that polima cytoplasm is subjected to environmental condition by a significant problem of polima CMS cytoplasmic expression cytoplasmic male sterility.Fan, people such as Z, Can.J.Plant Sci.66:221-227 (1985).More particularly, the cytoplasmic male sterile plants of the known polima of containing CMS becomes and can educate the big Tanaka of high temperature.Id. also referring to Fu, T.D., Encarpia Cruciferea Newsletter 6:6-7 (1981).

The present invention relates to contain the brassica oleracea plants of the cytoplasmic cytoplasmic male sterility of polima CMS, its male sterile under high temperature and low temperature, and show good female educating.Adopt traditional breeding method can prepare brassica oleracea plants of the present invention.By further hybridization or backcross or merge and to develop different Wild cabbage types by protoplast.

In order to obtain brassica oleracea plants of the present invention by traditional breeding technology, make Brassicacampestris cultivar 87110 and wild cabbage cultivar 87101 carry out interspecific cross.Collect the seed that hybridization produces, plantation, and regeneration.The plant that obtains is rape and contains single cover chromosome.By handle this plant with colchicin the chromosome content of described rape is doubled.

By the interspecific cross second time is carried out in rape cultivation strain 87118 and 87101 hybridization of wild cabbage cultivar.Collect the seed that hybridization produces, plantation, and regeneration.The same with the hybridization of front, the plant of acquisition is rape and contains single cover chromosome.Handling this plant with colchicin doubles its chromosome content.

The then rape plant of the generation of interspecific cross for the second time and 87102 hybridization of rape cultivation strain, back one strain contains polima CMS cytoplasm and male sterile.Collect the seed that hybridization produces, plantation, and regeneration.The plant of regeneration is rape and contains polima CMS cytoplasm.

Subsequently plant that obtains and the rape plant that interspecific cross for the first time produces are hybridized.Collect the seed that hybridization produces, plantation, and regeneration.The plant of regeneration is a rape, contains polima CMS cytoplasm and male sterile.

Then with the plant and normal wild cabbage hybridization that obtain.The result of hybridization is that the generation siliqua is collected, and checks its seed.Collect its seed and carry out embryo redemption processing, because the embryo that produces from described interspecific cross was miscarried before maturation usually.But,, can produce the species hybrid plant by using embryo rescue techniques.The F1 plant that obtains contains the polimaCMS cytoplasm that comes from female rape, but examines the combination that the DNA inclusion is rape (N=19) and wild cabbage (N=9).

Then the plant and the wild cabbage that obtain are backcrossed.Produce siliqua again, collect, check its seed.Collect its seed and carry out embryo redemption processing.Then as the hybridization of front, the regeneration embryo.The plant that obtains is the intermediate of chromosome number and contains polima CMS cytoplasm.The nuclear inclusion of this plant is the combination of rape and wild cabbage.

Then the plant and the wild cabbage that obtain are backcrossed.Produce siliqua again, collect, check its seed.With planting seed.The plant that obtains is a wild cabbage, and it is male sterile and contains polima CMS cytoplasm.

Can further hybridize male sterile cabbage plant or backcross to produce different rape types in non-imposed ground.To produce siliqua again, collect, check its seed.With planting seed.The plant that obtains is a wild cabbage, and it is male sterile and has polima CMS cytoplasm.

Can produce dissimilar rapes by the protoplast fusion.The protoplast of protoplast and the male sterile brassica of the nuclear that contains polima CMS cytoplasm and inactivation that will have the rape plant of commercial desirable characteristics merges.After merging, allos cytothesis is CMS rape plant.The plant that obtains is male sterile and has polima cytoplasm.

The CMS rape plant of regeneration contains polima cytoplasm and can be used in other rape type that contains commercial required characteristic and hybridizes.

Accompanying drawing is described

Accompanying drawing 1 is presented in the endonuclease digestive juice of the nuclear DNA that contains polima CMS donor, the DNA of BGL I digestion and the different hybridization mode of probe E5.Be used as the acceptor of CMS proterties with wild cabbage 904005-1 and wild cabbage line of breeding.Swimming lane 1 shows the dna fragmentation (molecular weight standard thing) of phage Hind III digestion.Swimming lane 2 and 3 shows polima CMS wild cabbage 904005- 1DNA.Swimming lane 4 and 5 shows wild cabbage acceptor strain K14.Swimming lane 6 and 7 shows wild cabbage acceptor strain DE70.

Accompanying drawing 2 is presented at the different hybridization mode of DNA with the probe CMS BRAS4 of BGL II digestion in the endonuclease digestive juice of the mitochondrial DNA that contains polima CMS plant and the mitochondrial DNA of acceptor line of breeding plant.Swimming lane 8 and 18 shows the dna fragmentation of phage Hind III digestion.Swimming lane 1-7 shows polima CMS wild cabbage 904005-1 DNA.Swimming lane 9-17 shows wild cabbage acceptor strain K14.Swimming lane 19-24 shows wild cabbage acceptor strain DE70.

Invention is described

The known cytoplasmic cytoplasmic male sterility rapeseed plants of polima that contains is " seepage ". Seepage refers to such fact: plant trends towards losing sterility under certain temperature conditions. For example, the verified cytoplasmic CMS rapeseed plants of the polima partial sterility only when high temperature that contains of research. Referring to Fan, the people such as Z, Can.J.Plant Sci.66:221-227 (1985).

The present invention relates to contain the cytoplasmic CMS brassica oleracea plants of polima. Plant is male sterility and shows good female educating under high temperature and cryogenic conditions. By further hybridization with backcross or can develop different wild cabbage CMS types by protoplast fusion.

The procedure of breeding

Traditional breeding technique is used for developing cabbage plant of the present invention. The inventor has described the procedure of breeding for the preparation of cabbage plant of the present invention. Can realize the described procedure of breeding by making Brassica campestris (=Brassica rapa) entry 87110 (N=10) and wild cabbage entry87101 (N=19) that interspecific hybridization occur. Entry 87110 is from plant research center (CPRO), oily political affairs mailbox 16, and 6700AA, Wageningen, Holland obtains, and is numbered IVT 86007 paar 3MS and is that heredity is upper male sterile. According to the regulation of budapest treaty, be deposited at American type culture collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852 with 87110. 87110 were deposited August 23 nineteen ninety-five, and the ATCC preserving number is 97246. Entry 87101 can be from the Wisconsin, Madison, the department of plant pathology of University of Wisconsin, 1630 Linden Drive, Crucifer Genetics Cooperative (CrGC) obtains, and is numbered CrGC#3-3 (Ccc) and is the wild cabbage of Rapid Circulation. After hybridization, grow acquisition siliqua (seed). Collect siliqua, check its seed and plantation. The plant that obtains is monoploid rape ac (N=19), and making chromosome doubling with colchicine treatment is that amphiploid is counted aacc (2n=38). The plant that obtains is to have aacc genomic " manually " rape. They are labeled as 87116.

With 87,110 second Brassica campestris with identical characteristics, 87118 also hybridize with 87101. 87118 obtain from CPRO, are numbered IVT 86017 paar 3MS. According to the regulation of budapest treaty, be deposited at American type culture collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland, 20852 with 87118. 87118 were deposited August 23 nineteen ninety-five, and the ATCC preserving number is 97247. In hybridization in front, collect seed and plantation. Obtain plant and make chromosome doubling with colchicine treatment. The plant that obtains is to have aacc genomic " manually " rape. They are labeled as 87119.

Then with 87119 plant with contain the cytoplasmic male sterility rape of polima CMS (aacc) (being labeled as 87102) hybridization. The 87102nd, obtain from Crucifer Genetics Cooperative (CrGC), be numbered CrGC#5-4 (AClaacc), it is to have the cytoplasmic Rapid Circulation rape of " polima " CMS. Produce siliqua, collect and check its seed. With the seed plantation, the plant of acquisition is labeled as 88102. These plant are rapes, have the aacc genome, contain polima CMS cytoplasm.

Then with 87116 and 88102 hybridization, produce siliqua, collect and check its seed. With the seed plantation, the plant of acquisition is rape (aacc), is labeled as 88125. Rape (aacc) is the amphidiploid between wild cabbage N=9 (cc genome) and the Brassica rapa N=10 (aa genome). The 88125th, rape (aacc) contains polima CMS cytoplasm and is male sterility.

With 88125 (aacc) and normal male-fertile broccoli plant, wild cabbage (cc) carries out interspecific hybridization. Those skilled in that art will recognize that any male-fertile cabbage plant can be used in and carry out this hybridization. Grow and obtain siliqua. Then collect siliqua and check its seed. If there is seed, then carries out embryo and save. Because the embryo that usually produces from described interspecific hybridization was miscarried before maturation, therefore carry out embryo and save processing. But, by using embryo rescue techniques, can produce Hybrids Plants of Interspecific.

Embryo is saved to relate to from siliqua and is taken out embryo. Preferably carrying out embryo when embryo is large as far as possible saves. Usually, hybridization was to carry out the good opportunity that embryo is saved in 18-19 days afterwards. But those skilled in that art will recognize that embryo is saved and may occur more early or slower, as long as the seed tunicle does not have hardening.

Save for embryo, at first siliqua was sterilized about 20 minutes in 10% hypochlorite solutions (for example clorox or postassium hypochlorite), in sterilized water, embathe three times with 2.5 and 10 minutes time.

Then, with vertical incision siliqua.Remove young seed, embryo is separated with seed at microscopically.Then embryo is placed proper culture medium to promote growth, for example be described in the BLKC medium among the embodiment 11.But, can use to help any medium of growing.With the embryo at 20 ℃, the fortnight of growing at least under the 4500LUX illumination condition.After root development, as quickly as possible seedling is placed soil.

The F1 plant appointment that obtains is numbered 88132-1 to 88132-84.These plant contain the polima CMS cytoplasm that comes from female rape plant, and still, nuclear DNA inclusion is the combination of rape (N=19) and wild cabbage (N=9).In essence, plant has the genome of mixing.Because their mixed characteristic, plant are that male sterile and part are female educating.Plant is male sterile, comes from female (egg) parent because they contain, rape, 88125 polima CMS cytoplasm.

Utilize and select phenotypic method from 88132-1 to 88132-84, to select to have the almost plant (aacc or cc) of normal DNA inclusion.Utilize and select phenotypic method to make the breeder can select to be similar to most the plant of wild cabbage (cc genome).It is different with the plant with normal DNA inclusion to be observed visually the plant with unusual DNA inclusion.With selected plant and normal siliqua or cauliflower, wild cabbage (cc) backcross (BC1).But those skilled in that art will recognize that and can use any wild cabbage.After hybridization, use embryo rescue techniques again.Be numbered 89015 to 89040 and 89046 to 89056 with so growing the plant appointment that obtains.The chromosome number of these plant mediates the stage, contains polima CMS cytoplasm.Again, nuclear DNA inclusion is the combination of rape and wild cabbage.Plant is male sterile (polima CMS) and part female sterile (chromosome number).

Utilize and select phenotypic method from 89015 to 89040 and 89046 to 89056 to select to have the almost plant of normal DNA inclusion.With selected plant and normal siliqua or cauliflower, wild cabbage (cc) backcross (BC2).But those skilled in that art will recognize that and can use any wild cabbage.Grow siliqua again, but some seeds do not need embryo to save.Collect these seeds and plantation.The plant appointment that obtains from this hybridization is numbered 89070 to 89141.Most these plant have the chromosome set (2N=18) of normal wild cabbage, contain polima cytoplasm and are male sterile.The flower of these plant has reduced flower pesticide and petal.These plant keep sterile under a high temperature low temperature and show normal female educating.

With the plant of these acquisitions and other wild cabbage type siliqua for example, cauliflower, wild cabbage (red, white, savoy cabbage), Sprouts, kohlrabi, kale or the like hybridizes to produce the CMS strain.For example by backcrossing with CMS " polima " plant or can producing different rape types by the protoplast fusion.

For male sterile, the female hybridization subsequently and the system of selection of educating and spending body

Plant that subsequently will from 89015 to 89040 and 89046 to 89056 and 89070 to 89141 is backcrossed with normal siliqua and/or cauliflower and plants in the land for growing field crops.Collect supermature siliqua, check its seed.Collect seed, subsequently sowing.The plant row of polima male sterile line and alternately plantation of siliqua maintenance line plant row.Those skilled in that art understand that maintenance line is meant the strain that is used to hybridize with the generation filial generation, and described filial generation has kept the sterility of male sterile parent material.The maintenance line plant can educate, but is to have identical duplicate with the CMS plant that produces it that grows in the heredity.Usually in the bar that replaces, produce male sterile line seed.After growing period, use natural system of selection (by honeybee and fly fly or the like) to select to have male sterile, good female educating with best floral shape is the plant of good petal size.Selected plant is specified and is numbered CPO-1 ,-2 or the like.These plant are wild cabbages, contain polima CMS cytoplasm, are male sterile and have good female fertility.

After vegetative stage, show male sterile, the plant clone of good female CPO numbering of educating places the cage with typical maintenance line.The preparation of cage comprises a cage a mesh tent (insect screening) being installed or being surrounded plant.This be for stop big Tanaka that cause by insect with crossing pollination other plant.Usually honeycomb or other insect colony are placed in the plant.Collect the siliqua that grows thus, with planting seed.The plant that obtains is to contain the cytoplasmic wild cabbage of polimaCMS, is male sterile, and shows good female educating.

Protoplast merges

Also can prepare different CMS rape types by the protoplast fusion.From containing CMSpolima cytoplasm and being that male sterile wild cabbage can obtain protoplast.For example, can use the cabbage plant that is obtained by the previously described procedure of breeding, for example 89070 plant are to the plant of 89111 plant and CPO series.Protoplast merges can be by being that use causes the polyethylene glycol (PEG) of aggegation so that the film fusion is finished in the presence of the high pH solution merging in the presence of the buffer solution.Under by people such as Sundberg (plant science 43 (1986) 155) (being incorporated herein by reference) disclosed condition, realize described somatic hybridization, to produce species hybrid or its modified forms.But those skilled in that art will recognize that the mode that can use except that polyethylene glycol (PEG) finishes protoplast and merge.For example, protoplast can be merged by the integration technology of using electric field to induce, " fusion that electric field is induced and rebuild the sub-protoplast of cell and higher plant with the single protoplast of pre-selected " described as people such as Koop, the electroporation and the electricity that are published in cell biology merge, people such as Neuman, editors, 355-365 (1989) is incorporated herein by reference.In addition, can finish protoplast with glucan and polyvinyl alcohol and merge, as people such as Hauptmann, " replenish selection carrot * tobacco burdo ", the 6th international protoplast annual meeting, Basel by the amino acid analogue resistance, August nineteen eighty-three, 12-16 was incorporated herein by reference.

Merge if finish protoplast, can use the program that describes below with polyethylene glycol.

In the wash solution of Miao Shuing (W5 '), be convenient to realize the fusion of protoplast hereinafter, described wash solution contains for example carbohydrate mannitol for example of bleeding agent, sorbierite, glucose or sucrose and potassium and calcium salt.PH can be in the scope of 5.2-10, preferably about 5.7.The protoplast of separate sources is mixed, and being concentrated into final densities easily is 10 ⁵With 10 ⁸Protoplast/milliliter.

The protoplast mixture should be placed static at least 10 minutes so that make protoplast be deposited in the bottom of culture dish then.Use polyethylene glycol then, preferably have the polyethylene glycol that molecular weight is 1500-6000 (PEG) and handle this mixture.Usually, can obtain good result when use contains the aqueous solution (PFS) of the PEG of 18.8% weight, described W5 ' is 10: 1 to 1: 1 with the volume ratio of PFS.PFS generally includes bleeding agent and calcium salt.According to the fragility of cell, the time that protoplast is present among the PFS is 15-20 minute.

By with containing bleeding agent (for example glucose) and potassium, the wash solution of sodium and calcium salt (W5 ') washing protoplast is for example finished fusion three times, and the concentration of described bleeding agent is for obtaining the osmotic pressure lower than PFS.In 16-20 ℃ temperature range, preferably 18 ℃ are carried out the fusion program.The concentration of PEG reduces with each continuous washing step (referring to embodiment 6) in the integrative mixture.Each washing step should carry out 15 minutes at least so that make the osmotic pressure of the reduction of the slow acclimatizing culture medium of protoplast, to avoid the explosion of cell.After washing step was finished, the protoplast of fusion should be 10 ⁵-10 ⁶In the scope of protoplast/milliliter.In the presence of nonfused parent's protoplast or after from culture, selecting, make the product regeneration of the fusion of acquisition non-imposedly.Microoperation by cell is for example according to by people such as Patnaik, and Plantscience Letter 24 (1982) 105, are incorporated herein by reference, and carry out described non-imposed selection, so that manual the separation and the heterocaryon of differential plant.

When using selection scheme, parent's protoplast is for example with for example fluorescein diacetin dyeing of fluorescence.When the fluorescein diacetin was used with the hypocotyl source point, it showed yellow under UV light.The protoplast of leaf contains the chloroplast that becomes red AF under UV light.

The fusion product that obtains is cultivated in proper culture medium, and described medium contains the good nutrient solution of balance with the growth of supply protoplast.Medium contains trace and macroelement, vitamin, and amino acid and carbohydrate in a small amount, for example various sugar are glucose for example.Glucose has the effect as carbon source and bleeding agent.Medium also comprises plant hormone (auxin and cell factor), and this medium can be regulated cell division and young shoot regeneration.The example of suitable auxin comprises methyl (NAA), and the 2,4 dichloro benzene ethoxyacetic acid (2,4-D) and heteroauxin (IAA).The example of the suitable basic element of cell division comprises benzyl aminopyrine (BAP), zeatin (Zea) and gibberellic acid (GA3).Common NAA and 2,4-D uses to start cell division with BAP.The ratio of the auxin and the basic element of cell division must be high, for example is higher than 1.Fusion treatment 2-3 days afterwards, described medium are almost completely cultivated medium (BP) and are replaced, and described cultivation medium contains agarose, its embedding fusion product and parent's protoplast.

After 14 days, do not contain or be substantially devoid of the concentration that the culture fluid of auxin dilutes auxin by adding other.Usually grow star-like small callus after week at 3-4.Then small callus is transferred to regeneration culture medium so that start the formation of young shoot, after preferably in the intermediate regeneration medium, adapting to, so that adapt to the different of the composition of cultivating medium and regeneration culture medium and physical characteristic.Form for young shoot, the ratio of auxin/basic element of cell division should be low in the regeneration culture medium, for example is lower than 1: 10.Usually preferably with basic element of cell division Zea and BAP and auxin NAA in conjunction with being used for young shoot regeneration.Regeneration culture medium, it is relative poor medium that BR compares with the cultivation medium with K3.They contain less vitamin, and the content of carbon source reduces, and they only comprise that sucrose and wood sugar as carbon source, do not contain amino acid.Regeneration culture medium also contains and is higher than the viscosity of cultivating medium.Regeneration culture medium Br is solid culture medium and contains growth regulator 2,4-D, and NAA and BAP, the ratio of auxin/basic element of cell division is lower than 1.Medium K3 contains Zea and GA3, also contains silver nitrate and grows to promote young shoot.

After regeneration fortnight, the callus that diameter is about 3 millimeters is transferred to the K3 regeneration culture medium that contains low concentration sucrose on the Br medium.In this stage, young shoot is grown in week at 2-3.The young shoot that is obtained is for example taken root on the B5 at the minimal medium that does not add other hormone then.Differentiate nuclear DNA and the mitochondrial DNA of the plantling that is obtained then in the known mode of those skilled in that art, for example use suitable restriction endonuclease and the DNA digestion method and the parental cell line of the fusion product that obtains compared.

Obtain to be used as in this article the protoplast of the inactivation of the wild cabbage protoplast of starting material and polima CMS cabbage plant from corresponding plant cell in the known mode of those skilled in that art.According to by Glimelius, the method that Physiologia Plantarum 61 (1984) 38 (being incorporated herein by reference) describes, from green plant material leaf material and the cell that obtains not have cell wall from for example weak seedling of white plant material protoplast for example for example, the hypocotyl protoplast is used to regenerate.When the non-imposed selection of fusion product is carried out in plan, be convenient to select starting material from green plant or from white plant material, preferably their dyeing is selected so that help.

In the known mode of those skilled in that art, obtain protoplast corresponding to the inactivation of the polima CMS cabbage plant of polimaCMS cabbage plant cell or protoplast by radiation for example.

By means of γ, UV or X-ray can be realized causing examining inactivation by radiation.When carrying out radiation with X-ray source, examining inactivation usually can be by using for example 10krad. minute dosage radiation acquisition in 3-20 minute.For example make the basic horizontal of the x-ray radiation that protoplast group 100% causes death set up suitable x-ray dose: the percentage of the number assessment dead cell by calculating the bacterium colony that forms in the culture after 10-20 days by mensuration.

As mentioned above, after merging, allos cytothesis forms and contains the cytoplasmic cabbage plant of CMS.Subsequently can be with these plant and the hybridization of other cabbage plant.

Will recognize that cabbage plant of the present invention can be used as starting material, be used to prepare other head cabbage varieties by external and/or hybridization technique with polima CMS.Described external and hybridization technique is that the breeder is known in this area.

Employed solution comprises:

A. enzyme solutions (1L)
	90g mannitol 0.0272g KH 2PO 40.1g?KNO 30.246g?MgSO 4·7H 2O 0.0008g?KI 0.00025g?CuSO 4·5H 2O 1.4g?CaCl 2·2H 2The O 1.1g MES 6g cellulase R10 1g huge plain enzyme of pH5.8 (Macarozyme)

B. washing lotion (W5) (1L)
	18.4g?CaCl 2·2H 2O 4.91g NaCl 0.372g KCl 0.901g glucose pH=5.8

C. washing lotion (W5 ') (1L)
	18.4g?CaCl 2·2H 2O 4.91g NaCl 0.372g KCl 0.901g glucose 9.76g MES pH=5.8

D．CPW?16S(1L)
	160g sucrose 0.0272g KH 2PO 40.1g?KNO 31.45g?CaCl 2·2H 2O 0.246g?MgSO 4·2H 2O 0.0008g?KI 0.00025g?CuSO 4·5H 2O pH=5.5-5.8

E. merge liquid (PFS)
	18.8％PEG(MW?4000) 0.06M?CaCl 2·2H 2O 0.1M mannitol 0.025M glucin 10% (v/v) DMSO

F.SPA solution (1L)
	20g SeaPlaque agarose 100g sucrose

The composition of table 1:MS medium (MG/L)

	????8P	????MAC	????Br	????K3	????B5	????MS
							KNO 2	????956	????956	????1556	????1556	????3000	????1900
NH4NO 3	????600	????600	????250	????250	????--	????1650
							MgSO 4·2H 2O	????300	????300	????250	????250	????250	????370
KH 2PO 4	????164	????164	????--	????--	????--	????170
							CaCl 2·2H 2O	????600	????600	????300	????300	????150	????440
KCl	????300	????300	????--	????--	?????--	?????--
							(NH 4) 2SO 4	????--	?????--	????134	????134	????134	?????--
K	????0.75	????0.75	????0.75	????0.75	????0.75	????0.83
							MnSO 4·4H 2O	????10	????10	????10	????10	????10	????22.3
H 2BO 3	????3	????3	????3	????3	????3	????6.2
							ZnSO 4·2H 2O	????2	????2	????2	????2	????2	????8.6

NaMoO 4·2H 2O	??0.25	?0.25	?0.25	?0.25	?0.25	?0.25
							C 4SO 4·4H 2O	??0.025	?0.025	?0.025	?0.025	?0.025	?0.025
CoCl 2·6H 2O	??0.025	?0.025	?0.025	?0.025	?0.025	?0.025
							Fe-EDTA	????40	????40	????40	????40	????40	????43
Thiamine HCl	????10	????10	????10	????10	????10	????0.1
							Pyridoxine HCl	????1	????1	????1	????1	????1	????0.5
Nicotinic acid	????1	????1	????1	????1	????1	????0.5
							Sodium Pyruvate	????20	????20	???--	????--	???--	????--
Citric acid	????40	????40	???--	????--	???--	????--
							Maleic acid	????40	????40	???--	????--	???--	????--
Fumaric acid	????40	????40	???--	????--	???--	?????--
							Glycine	????--	????--	???--	????--	???--	????2
Wood sugar	????--	????--	??250	????--	???--	????--
							Inositol	????100	????--	??--	????--	???--	????100
Sucrose	???50,000	?80,000	?40,000	?10,000	?20,000	????**
							Glucose	???40,000	????--	??--	????--	???--	?????--
Casein hydrolysate	????250	???250	??--	????--	???--	?????--
							Agarose	????--	????--	16,000	????--	???--	?????--
Agar	????--	????--	?--	???8,000	?8,000	?8,000
							NAA	????0.1	????0.1	?0.1	????--	???--	?????--
2,4-D	????1	????0.2	?0.1	????--	???--	?????--
							Zeatin	????--	????--	??--	??0.26	???--	?????--
BAP	????0.5	????0.5	?0.5	????--	???--	?????--
							GA 3	????--	????--	???--	?0.006	???--	?????--
AgNO 3	????--	????--	???--	????5	???--	?????--

* is referring to embodiment

For example, rather than, provide embodiments of the invention now for any restriction.Embodiment 1: seed sterilization and breeding

The wild cabbage siliqua seed (this paper called after wild cabbage 904005-1) that will have a polima CMS was dipped in 70% ethanol about 10 seconds, and at 20 ℃, sterilized twice in 1.5% liquor natrii hypochloritis, each 10 minutes.Need subsequently fully to embathe with sterile distilled water.Seed placed have 3% sucrose, do not have the MS medium (referring to table 1) of hormone.In order to obtain green aseptic plant, under 20 ℃ the temperature seed to be grown in the vial of illumination (8000lux) at 25 ℃ and night by day, illumination period is 16 hours.Under identical condition, in plastic containers, go down to posterity and cultivate aseptic young shoot.Organize for example hypocotyl in order to obtain to be used for the white that protoplast separates, under 25 ℃ of dark conditions with seed growth in culture dish.Embodiment 1a: the hybridization that causes producing 904005-1

904005-1 is the seed of No. 1 plant collection from entry numbering 904005.The 904005th, the backcrossing of the CMS polima 892731-2 and the siliqua that can educate normally.892731-2 selects 2 seeds of collecting from the plant of entry892731.This is backcrossing of the CMS polima89094-2 and the siliqua that can educate normally.

89094-2 selects 2 seeds of collecting from the plant of entry89094.This is backcrossing of the CMSpolima 89022 and the siliqua that can educate normally.

89022-4 is the seed of collecting from the plant 4 of entry 89022.Embodiment 2

Be similar to the program of embodiment 1, with wild cabbage, sterilization of the seed of cultivar DE70 and breeding.Cultivar DE70 is the inbred line cauliflower strain that can educate fully, and it is used as parental line.But can use typical cabbage plant to be used for protoplast merges.Embodiment 3: the separation of protoplast

The blade of the young shoot that four week of the vegetable material of embodiment 1 is big is cut into pieces, at 25 ℃, on the gyratory shaking table, cultivates in enzyme solutions 16 hours with 40rpm.With nylon wire (40 microns) with suspension filtered, with the CPW 16S solution of 2/3 volume, by embathing secondary in centrifugal 5 minutes with 817rpm.This can floatingly select complete protoplast.Collect protoplast, use W5 solution, by embathing in centrifugal 5 minutes with 708rpm.Before being used for test for fusion protoplast being diluted to density is 1 * 10 ⁵Protoplast/milliliter W5 solution.Embodiment 4

According to the method for embodiment 3, separate the hypocotyl of 6-8 days vegetable material, different is during enzyme is handled, and adds the fluorescein diacetin of 3 mcg/ml.Acquisition is used for the protoplast that the dyeing of fusion frequency mensuration was selected and was used in picking.Embodiment 5: the radiation of protoplast

The protoplast of the fresh separated that embodiment 3 is obtained is incubated in 6 centimetres the culture dish that contains W5 solution (2-3 milliliter).Utilize X-ray (Baltograph CE100), with the dosage radiation protoplast of 3500Gy 20 minutes.After radiation, use W5 solution, by washing protoplast in centrifugal 5 minutes with 817rpm.Before being used for test for fusion, protoplast is diluted to 1 * 10 ⁵The density of protoplast/milliliter W5 solution.Embodiment 6: the fusion program

Being mixed to total concentration according to the protoplast of embodiment 4 and 5 with 1: 3 ratio is 1 * 10 ⁵Protoplast/milliliter W5 solution.The mixture of three droplets, 100 microlitres is placed 6 centimetres of culture dishes that do not wrap quilt, make protoplast precipitation 5-10 minute.Central authorities at three droplets add the PFS solution of 300 microlitres to induce aggegation 15 minutes.Afterwards, the W5 solution that adds 300 microlitres adds after 10 minutes once more to this mixture, adds once more after 5 minutes.The 8P solution that concentrates with twice substitutes the W5 medium, and at 25 ℃, the cultivation protoplast is 1-3 days in the dark.Embodiment 7

At 25 ℃, cultivation is whole integrative mixture 1-3 days of embodiment 6 in the dark.By with 548 centrifugal 5 minutes collecting cells, being diluted to concentration is that the 8P medium that every milliliter of twice concentrates is 1 * 10 ⁵Protoplast.Add isopyknic warm SPA medium of hand of using, cell is placed 5 droplets, 100 microlitres of the culture dish of bag quilt.Add 5 100 microlitres with feeder cell.After 2 weeks, remove the droplet with feeder cell, each culture dish adds 2 milliliters of MAC medium.After 2 weeks, droplet is scattered in the solid Br medium.At 2-3 after week, with single colony lift in the culture dish that contains solid K 3 medium.At 25 ℃, according to intensity (2500lux), small callus is cultivated in illumination 18 hours in low light.Embodiment 18: the selection of fusion product and growth

Adopt micromanipulator to provoke in the presence of double fluorescein the cell of the fusion that can naked eyes recognizes.Hybrid cell is incubated at the agarose droplet (1%SeaPlaque) of 100 microlitres with the density of 2000-5000 protoplast/microlitre.Place the liquid that contains feeder cell to cultivate culture systems (Costar-Transwell col) droplet, at 25 ℃, dark culturing.After 2 weeks, droplet is scattered in the solid Br medium.Small callus is transferred to solid K 3 medium.At 25 ℃, to cultivate according to intensity (2500lux) in low light, light application time is 16 hours.Embodiment 9: plant regeneration

With diameter is the embodiment 7 of 2-5 millimeter size and the medium that 8 callus is transferred to fresh K3, and condition is normal light intensity (8000lux), and 25 ℃, light application time is 16 hours.Little young shoot transferred to have 1% sucrose, do not have the B5 medium of hormone, on identical medium, take root.Embodiment 10: the analysis of molecules of fusion product

A) nuclear DNA forms

Use specific dna probe to carry out qualitative to the nuclear DNA composition of fusion product.To contain the clone of 820 kilobase of cauliflower nuclear DNA to the Bgl1 fragment, probe ES and polimaCMS donor, each bands of a spectrum of the endonuclease digestion method of the nuclear DNA of wild cabbage line of breeding are hybridized, and described wild cabbage line of breeding is used as the acceptor (referring to accompanying drawing 1) of CMS proterties.

B) composition of mitochondrial DNA

With the DNA of CMS Bras4, contain the clone of 1950 base-pair Bgl II fragments of cauliflower mitochondrial DNA, carry out qualitative.This clone produces the right bands of a spectrum of 4 kilobase with CMS polima mitochondrial DNA and comes from for example right bands of a spectrum (referring to accompanying drawing 2) of 6 kilobase of the mitochondrial DNA of DE70 of acceptor line of breeding with having.By utilizing primer 94RSO1 and 94RSO2, the dna fragmentation of 500 base-pairs is carried out pcr amplification also can realize qualitative.The sequence of primer 94RSO1 is that the sequence of 5 '-GAA CCA ACT GCT TTC ACA CCG-3 ' and reverse primer 94RSO2 is 5 '-CTT GGC TCT CTG CGA ATG TC-3 ' forward.The fragment of this 500 base-pair is from CMS polima mitochondrial DNA amplification, rather than from for example mitochondrial DNA amplification of DE70 of acceptor strain.Embodiment 11: embryo is saved

Usually carrying out embryo after hybridizing in 18-19 days saves.In order to realize the embryo redemption, need siliqua.Needed siliqua is those siliquas with embryo of the big as far as possible seed tunicle that has as yet not hardening.

Before taking out embryo, at first siliqua must be not pollute.For example siliqua was carried out disinfection in 20 minutes in sodium chloride or the potassium chloride by being placed on 10% chloride solution.With 2,5 and 10 minutes siliqua is embathed in sterile water three times.

By siliqua is vertically cut, take out the seed of growing, break away from the embryo of seed and save embryo in the microscopically preparation then.

After from seed, taking out embryo, be placed in the suitable growth medium.Can use the medium of any promotion embryonic development.For example can use and have 3﹠amp; The MS medium of the IDA of sucrose and 0.4 mg/litre thiamine and 0.2 mg/litre.Embodiment 12: keep the sterile of CMS cabbage plant when high temperature and low temperature

To contain the cytoplasmic cytoplasmic male sterility plant of polima CMS and whether keep sterile when high temperature and the low temperature in order to measure, with siliqua cultivar 915095,915107,915100 and 915144 and red cabbage hybridization check whether it obtains 100% hybridization.Cultivar 915095-915144 and red cabbage are hybridized.When the seed of these cultivars and normal siliqua maintenance line pollination, and when it sow, the hypocotyl color of seedling and the color of leaf normally were greens.If the hybridization of these cultivars and red cabbage, hybrid seed is an intermediate so, and seedling shows that strong authocyan (purple) expresses, and inbred line keeps green.With 915095,915107,915100 and 915144 planting seed, the cage that places plastics to cover with the red cabbage plant 20 plant of each cultivar.In summer, the temperature of cage changed between 15-40 ℃ in one day.The temperature of cage writes down once weekly.Will be from 915095,915107, the sample sowing of 915100 and 915144 seeds of collecting, the plant of all acquisitions is hybrids, shows not have selfing and do not have polima CMS cytoplasm seepage.Whenever any plant can not produce part male sterile.

Causing producing 915095,915107,915100 and 915144 hybridization shows below.

(promptly 88132 * bc) when producing 2 or 3 arrows, and each arrow representative is once hybridized with different siliquas is genotypic so when a hybridization.Embodiment 13: female educating

What list below is the data of the seed production of acquisition after CMS " polima " type and siliqua are with hand hybridization.The seed production of data show CMS " polima " is higher than the siliqua inbred line.This is to have influenced fertility because repeat selfing.Hybridization between siliqua F1 ' s and CMSOgura can better be compared.The result shows that the seed production of CMS polima is in the scope that is same as other hybridization of two types.

Siliqua seed production 1991-1994 (using hand-polliante)

The seed number of the each hybridization of year corssing form

1991 (CMS polima * siliqua) F1 114 (n=4)

(siliqua inbred line * siliqua) F1 64 (n=23)

(siliqua F1 * siliqua) F1 116 (n=45)

1992 (CMS polima * siliqua) F1 146 (n=66)

(siliqua inbred line * siliqua) F1 100 (n=76)

(siliqua F1 * siliqua) F1 135 (n=28)

(CMS Oguar * siliqua) F1 140 (n=32)

1993 (CMS polima * siliqua) F1 79 (n=41)

(siliqua inbred line * siliqua) F1 53 (n=70)

(siliqua F1 * siliqua) F1 80 (n=35)

(CMS Oguar * siliqua) F1 144 (n=5)

1994 (CMS polima * siliqua) F1 93 (n=9)

(siliqua inbred line * siliqua) F1 47 (n=50)

(siliqua F1 * siliqua) F1 84 (n=35)

(CMS Oguar * siliqua) F1 74 (n=9) weighed average

(CMS polima * siliqua) F1 115 (n=120)

(siliqua inbred line * siliqua) F1 60 (n=227)

(siliqua F1 * siliqua) F1 103 (n=143)

(CMS Oguar * siliqua) F1 (127) (n=40)

Though described the present invention with embodiment preferred, will be appreciated that it is not modified and can depart from the scope of the present invention in conjunction with specific.Following what is claimed is in order to cover common all variations of observing its principle, the application or the variation of invention, and be included in big Tanaka and carry out the situation that deviates from disclosed content that relevant known or usual practice of the present invention produces, or to the conspicuous situation of those skilled in that art.

Claims (9)

1. be used to produce the method for cytoplasmic male sterile brassica plant, described plant is sterile under high temperature and low temperature, and contains polima CMS cytoplasm, and this method comprises the following steps:

A) seed of described hybridization generation is hybridized and collected to Brassica campestris cultivar 87110 and wild cabbage cultivar 87101;

B) described seed is regenerated as the rape plant, it contains single cover chromosome;

C) make the chromosome doubling of step b) plant, so that the rapes that contain two cover chromosomes to be provided;

D) Brassica campestris cultivar 87118 and wild cabbage cultivar 87101 are hybridized and collect the described seed of making deposits yields;

E) described seed is regenerated as the rape plant, it contains single cover chromosome;

F) make the chromosome doubling of step e) plant, so that the rapes that contain two cover chromosomes to be provided;

G) step f) plant and rape cultivation strain 87102 are hybridized and collect the seed of described hybridization generation, described rape is male sterile, and contains polima CMS cytoplasm;

H) be the rape plant with described plant regeneration, it contains polima CMS cytoplasm;

I) making step c) plant and step h) plant hybridizes and collects the seed that described hybridization produces;

J) be the rape plant with described plant regeneration, it is male sterile, and contains polimaCMS cytoplasm;

K) with step j) plant and wild cabbage cultivar hybridize and collect the seed that described hybridization produces;

1) take out embryo from seed, make described embryo be regenerated as plant, this plant is male sterile, and contains polima CMS cytoplasm;

M) plant and the wild cabbage cultivar of step 1) are backcrossed, collect the seed of backcrossing and producing from described;

N) take out embryo from seed, make described embryo be regenerated as the F1 plant, this plant is male sterile, and contains polima CMS cytoplasm;

P) with step n) plant and wild cabbage cultivar backcross, collect the seed of backcrossing and producing from described; And

Q) make described seed be regenerated as cabbage plant, this plant is male sterile, and contains polima CMS cytoplasm.

2. method according to claim 1 further comprises: with step q) plant and wild cabbage hybridization; Collect the seed that described hybridization produces; And making described seed be regenerated as cabbage plant, this plant is male sterile, and contains polima CMS cytoplasm.

3. method according to claim 1, wherein plant is selected from following group: cauliflower, Sprouts, wild cabbage, kale, kohlrabi and siliqua.

4. be used to produce the method for cytoplasmic male sterile brassica plant, described plant is sterile under high temperature and low temperature, and contains polima CMS cytoplasm, and this method comprises the following steps:

A) protoplast of the male sterile brassica that the described method of claim 1 is prepared merges, and described wild cabbage contains polima CMS cytoplasm, keeps sterile under high temperature and low temperature; With

B) with the allos cytothesis that obtains for containing the cytoplasmic CMS cabbage plant of polima.

5. method according to claim 4, wherein wild cabbage is selected from following group: cauliflower, Sprouts, wild cabbage, kale, kohlrabi and siliqua.

6. method according to claim 4 further comprises: the step of the CMS wild cabbage that will contain the cytoplasmic regeneration of polima and the cabbage plant hybridization that contains commercial required characteristic.

7. the cytoplasmic male sterile brassica plant that is obtained by the method for claim 1, it keeps sterile and contains polima CMS cytoplasm under high temperature and low temperature.

8. the cytoplasmic male sterile brassica plant that is obtained by the method for claim 4, it keeps sterile and contains polima CMS cytoplasm under high temperature and low temperature.

9. according to claim 7 or 8 described plant, wherein plant is selected from following group: cauliflower, Sprouts, wild cabbage, kale, kohlrabi and siliqua.

Images