CN103053424A - Method for culturing black mustard microspore to obtain regenerated plantlet - Google Patents
Method for culturing black mustard microspore to obtain regenerated plantlet Download PDFInfo
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- CN103053424A CN103053424A CN2013100139236A CN201310013923A CN103053424A CN 103053424 A CN103053424 A CN 103053424A CN 2013100139236 A CN2013100139236 A CN 2013100139236A CN 201310013923 A CN201310013923 A CN 201310013923A CN 103053424 A CN103053424 A CN 103053424A
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Abstract
The invention discloses a method for culturing a black mustard microspore to obtain a regenerated plantlet, and belongs to the technical field of plant tissue culture. The method comprises the following steps of firstly, preparing a culture medium; secondly, selecting and carrying out pretreatment on plantlets and flower buds; thirdly, carrying out induction culture on a microspore embryoid; fourthly, separating and carrying out germination culture on regenerated plantlets; fifthly, carrying out rooting culture and transplanting the regenerated plantlets; and sixthly, detecting the ploidy of the regenerated plantlets. According to the method, by culturing the black mustard microspore, the germ extraction rate can be 15 embryos per bud at most, the emergence rate is 50% on average, and double-ploid regenerated plantlet materials which are pure in heredity and accordant in phenotype can be obtained in batch. The method can be popularized and applied in research fields such as black mustard breeding, genetic maps, resistance gene positioning, clone, transgenosis and the like.
Description
Technical field
The present invention relates to field of plant tissue culture technique, be specifically related to a kind of black mustard microspore tissue and cultivate the cultural method that obtains regeneration plant.
Background technology
It is individual that monoploid refers to only carry the gametic chromosome number purpose, all significant in crop breeding practice and genetic breeding theoretical research.Isolated microspore is cultivated as the haploid effective way of artificial induction, has practicality.In the practices of breeding, it is double haploid colony that monoploid is carried out just obtaining pure lines in chromosome doubling present age, than the traditional selfing purification process of employing about 3-5 that saves time, greatly improves breeding efficiency, has shortened breeding cycle.In the genetic breeding theoretical research, double haploid has absolute homozygosity in heredity, being the good material of carrying out AFLP, RFLP and SSR equimolecular mark, genetic map drafting and important resistant gene Position Research, also is the ideal material that carries out spore research, genetic analysis and Cloning Plant Genes screening.
Black mustard (
Brassica nigra, be that the Cruciferae rape belongs to one of 3 elementary species BB), mostly be wild distribution.There are some researches show, exist in the BB genome at black mustard place mainly the cause a disease resistance of biological strain 1 and 4 of black rot, to the high resistance of club root, and to the resistance in the time of infertility of balck shank.And to Vegetables in Brassica, particularly be subjected in the comparatively serious wild cabbage of these three kinds of harm influences and the Chinese cabbage to shift these good resistant genes, will to cultivate many anti-, the quality vegetables new varieties are significant.But the disease-resistant research of black mustard is confined to the material Disease Resistance Identification of various breeding times at present, and further molecular biology research relatively lags behind, and makes slow progress.Particularly few to the Position Research of the drafting of correlated inheritance collection of illustrative plates and important resistant gene, one of reason be lack in the heredity isozygoty, Research Group that phenotype is consistent, and microspore culture provides quick, an effective approach for obtaining this colony.
Black mustard is to carry out least easily one of material that Isolated microspore cultivates in the crop in cruciferae, has realized in a lot of crops successfully obtaining monoploid by microspores culture although belong to rape.Relevant black mustard Isolated microspore culture studies, the present domestic relevant report that yet there are no, external also rarely seen one piece of early stage report, and its germ extraction rate is low, and regeneration strain obtains quantity very limited (Lichter 1989).This research is by cultivating the black mustard Isolated microspore, and successfully obtain the double haploid regeneration plant of some, for carrying out the genetic research of black mustard germ plasm resource, the breeding method of setting up high-efficient simple provides important theory and technological guidance, provides good research material for the correlated inheritance collection of illustrative plates is drawn, important resistant gene is located and spore research etc. simultaneously.
Summary of the invention
The objective of the invention is, to be difficult for carrying out Isolated microspore in the crop in cruciferae to cultivate for black mustard, the defective of the double haploid regeneration plant material that thereby also being difficult to obtain isozygotys in the heredity, phenotype is consistent, provide a kind of and enable to obtain in batches to isozygoty in the heredity, the black mustard microspores culture of the consistent double haploid regeneration plant of phenotype material obtains the method for regeneration plant, to set up black mustard microspores culture regenerating system, accelerate breeding process and the efficient of black mustard; The researchs such as accurate location, clone and transgenosis that can be simultaneously the drafting of black mustard genetic map and resistant gene provide material.
The object of the invention is achieved through the following technical solutions.
A kind of black mustard microspores culture obtains the method for regeneration plant, and the method is carried out as follows:
(1) preparation of medium: comprise the medium in each stage of microspores culture, their component and the weight of each component in every liter of medium are:
1) embryoid induction medium: NLN-13 liquid nutrient medium+sucrose 130 g/L, pH 5.6 ~ 6.0, filtration sterilization;
2) embryoid differential medium: B
5Medium+ZT 0.5 ~ 2.0 mg/L+NAA 0.01 ~ 0.1 mg/L+BAP 0.5 ~ 2.0 mg/L+sucrose 20 ~ 30 g/L+agar 7 ~ 9 g/L, pH 5.6 ~ 6.0, high-temperature sterilization;
3) sprouting, root media: MS medium+NAA 0.05 ~ 0.1 mg/L+sucrose 30 g/L+agar 8 g/L, pH 5.8, high-temperature sterilization;
(2) selection of donor plant, bud and preliminary treatment:
1) selection of donor plant, bud: select the black mustard of healthy growth as donor plant; And therefrom select petal and anther length than between 0.7 ~ 1.0, the bud that is in the monokaryon middle and advanced stage is the donor bud;
2) bud sterilization: with behind the pure water cleaning bud 3 times, put it into by 0.1% HgCl first
2In the sterilized solution that+0.1% polysorbas20 is made into, after shaking table carried out surface sterilization in 12 minutes by 50-80 rpm slight wobble, in super-clean bench sterile water wash 4 times, for subsequent use again;
3) separation of bud microspore, be mixed and packing: bud places aseptic beaker after will sterilizing on super-clean bench, add behind the 1 mL embryoid induction medium with the tack glass rod crush bud, add 9 mL same medium and stir into suspension again; Filter in centrifuge tube with the aseptic nylon leaching net of 40 μ m, centrifugal 4 minutes of 1000rpm abandons supernatant; Add after the embryoid induction medium contains the contained microspore amount of 1.0 buds to average every 4mL medium, again with the medium in this liquid with by NLN-13 liquid nutrient medium+1g/L active carbon, through the active carbon mixed liquor of high-temperature sterilization by volume 0.05 mL ∕ 4mL ratio be mixed into microspore suspension; And be sub-packed in the sterile petri dish that diameter is 6 cm, add a cover rear with the sealing of parafilm film, for subsequent use;
(3) the microspore induction of embryoid is cultivated: above-mentioned culture dish is placed 31-33 ℃ of incubator, secretly cultivated 24-48 hour; Be placed in 25 ℃ of constant incubators, secretly cultivate and occurred to embryoid in 15-20 days; 50rpm shaken cultivation 7-10 days under 25 ℃ of dark again forms to cotyledonary embryos shape body;
(4) differentiation of regeneration plant with sprout to cultivate: the cotyledon embryoid is lain against on the embryoid differential medium, and 25 ℃ of illumination every day was cultivated 15 ~ 20 days in 16 hours to the embryoid plant that makes new advances of expanding and regenerate;
(5) culture of rootage of regeneration plant and transplanting: cutting to sprout has the regeneration plant of normal growth point to insert in the root media, and 25 ℃ of illumination every day were cultured in 16 hours takes root; The regeneration plant of will taking root moves in the matrix by peat and by volume 2 ︰, 1 preparation of perlite, water permeable, behind the plastic covering film 25 ℃ of 1 weeks of lower cultivation;
(6) ploidy of regeneration plant detects: get the tender leaf of transplant survival regeneration plant, detect the dna ploidy of this regeneration plant with the partec PA the ploidy analyser of German Partec company production.
The invention has the beneficial effects as follows:
1, the present invention cultivates the microspore of black mustard, and the double haploid regeneration plant material of can obtain in batches to isozygoty in the heredity, phenotype is consistent to set up black mustard microspores culture regenerating system, is accelerated breeding process and the efficient of black mustard; The researchs such as accurate location, clone and transgenosis that can be simultaneously the drafting of black mustard genetic map and resistant gene provide material.
2, the germ extraction rate of black mustard microspores culture of the present invention reaches as high as 15 embryo/flower buds;
3, the emergence rate of embryoid of the present invention, average out to 50%.
Embodiment
The present invention is described in further detail by following examples, but content of the present invention is not limited to this.
The method 1 of embodiment 1:(black mustard microspores culture regeneration plant)
(1) preparation of medium: comprise the medium in each stage of microspores culture, their component and the weight of each component in every liter of medium are:
1) embryoid induction medium: NLN-13 liquid nutrient medium+sucrose 130 g/L, pH 6.0, filtration sterilization (wherein the NLN-13 culture medium prescription sees Table 1); 2) embryoid differential medium: B
5Medium+ZT 1.5 mg/L+NAA 0.05 mg/L+BAP 0.8 mg/L+sucrose 20 g/L+agar 7 g/L, pH 6.0, high-temperature sterilization, (B wherein
5Culture medium prescription sees Table 1);
3) sprouting, root media: MS medium+NAA 0.08 mg/L+sucrose 30 g/L+agar 7g/L, pH 5.8, high-temperature sterilization (wherein the MS culture medium prescription sees Table 1);
(2) selection of donor plant, bud and preliminary treatment:
1) selection of donor plant, bud: it is healthy with flower bud growth to be grown in 5 ~ 25 ℃, day illumination about 14 hours, NPK abundance balanced in nutrition, plant when selecting bolting to bloom, and makes donor without the black mustard of the obvious state of an illness; And therefrom to select petal and anther length ratio be 0.9, is in 10 in the bud of monokaryon middle and advanced stage;
2) bud sterilization: with behind the pure water cleaning bud 3 times, put it into by 0.1% HgCl first
2In the sterilized solution that+0.1% polysorbas20 is made into, after shaking table carried out surface sterilization in 12 minutes by 50-80 rpm slight wobble, in super-clean bench sterile water wash 4 times, for subsequent use again;
3) separation of bud microspore, be mixed and packing: bud places aseptic beaker after will sterilizing on super-clean bench, add behind the 1 mL embryoid induction medium with the tack glass rod crush bud, add 9 mL same medium and stir into suspension again; Filter in centrifuge tube with the aseptic nylon leaching net of 40 μ m, centrifugal 4 minutes of 1000rpm abandons supernatant; It is centrifugal and abandon supernatant by same method to add 10 mL embryoid induction medium again; Add embryoid induction medium 40 mL, make contain the contained microspore amount of 1.0 buds in average every 4mL medium after, with the medium in this liquid with by NLN-13 liquid nutrient medium+1g/L active carbon, through the active carbon mixed liquor of high-temperature sterilization 4mL:0.05 mL ratio by volume, namely add 0.5 mL active carbon mixed liquor and be mixed into microspore suspension again; And be sub-packed in the sterile petri dish that diameter is 6 cm, add a cover rear with the sealing of parafilm film, for subsequent use;
(3) the microspore induction of embryoid is cultivated: will divide the culture dish that installs to place 33 ℃ of constant incubators, secretly cultivate 24 hours; Be placed on 25 ℃ of constant incubators, secretly cultivate and occurred to embryoid in 15-20 days; 50 rpm shaken cultivation 7-10 days under 25 ℃ of dark again form to cotyledonary embryos shape body;
(4) differentiation of regeneration plant with sprout to cultivate: on super-clean bench, the cotyledon embryoid is lain against on the embryoid differential medium, cultivated 15 days in 16 hours to the embryoid plant that makes new advances of expanding and regenerate in 25 ℃ of illumination every day;
(5) culture of rootage of regeneration plant and transplanting: cutting to sprout has the regeneration plant of normal growth point to insert in the root media, is cultured in 16 hours in 25 ℃ of illumination every day and takes root; The regeneration plant of will taking root moves in the matrix by peat and by volume 2 ︰, 1 preparation of perlite, water permeable, behind the plastic covering film 25 ℃ of 1 weeks of lower cultivation;
(6) ploidy of regeneration plant detects: get the tender leaf of transplant survival regeneration plant, detect the dna ploidy of this regeneration plant with the partec PA the ploidy analyser of German Partec company production.
Cultivate black mustard sporule regeneration plant totally 192 strains that obtain by the example method, detect through ploidy, wherein diplontic regeneration plant has 131 strains, and the dliploid rate accounts for 68.2% of total strain number.
The method 2 of embodiment 2:(black mustard microspores culture regeneration plant)
In the present embodiment, the preparation of step (1) medium: 1) PH in the embryoid induction medium is 5.6; 2) ZT in the embryoid differential medium is 0.5 mg/L, and NAA is 0.01 mg/L, and BAP is 2.0 mg/L, and sucrose is 30 g/L, and agar is 8 g/L, and pH is 5.8; 3) NAA in sprouting, the root media is 0.05mg/L; Selection and the preliminary treatment of step (2) donor plant, bud: wherein 1) select petal and anther length than being 10 in 0.9 bud; Step (3) microspore induction of embryoid is cultivated: will divide the culture dish that installs to place 32 ℃ of constant incubators, secretly cultivate 36 hours; The differentiation of step (4) regeneration plant with sprout to cultivate: on super-clean bench, the cotyledon embryoid is lain against on the embryoid differential medium, cultivated 18 days in 16 hours to the embryoid plant that makes new advances of expanding and regenerate in 25 ℃ of illumination every day; All the other process all are same as embodiment 1.
The method 3 of embodiment 3:(black mustard microspores culture regeneration plant)
In the present embodiment, the preparation of step (1) medium: 1) PH in the embryoid induction medium is 5.8; 2) ZT in the embryoid differential medium is 2.0 mg/L, and NAA is 0.1 mg/L, and BAP is 0.5 mg/L, and sucrose is 25 g/L, and agar is 9 g/L, and pH is 5.6; 3) NAA in sprouting, the root media is 0.1mg/L; Selection and the preliminary treatment of step (2) donor plant, bud: wherein 1) select petal and anther length than being 10 in 1.0 bud; Step (3) microspore induction of embryoid is cultivated: will divide the culture dish that installs to place 31 ℃ of constant incubators, secretly cultivate 48 hours; The differentiation of step (4) regeneration plant with sprout to cultivate: on super-clean bench, the cotyledon embryoid is lain against on the embryoid differential medium, cultivated 20 days in 16 hours to the embryoid plant that makes new advances of expanding and regenerate in 25 ℃ of illumination every day; All the other process all are same as embodiment 1.
The application of embodiment 4:(dliploid material in the theoretical research of black mustard genetic breeding)
The dliploid regeneration plant colony that microspores culture is obtained carries out AFLP, SRAP, SSR equimolecular labeled analysis, makes up the dense genetic map spectrum.And its self progeny is deceived the inoculated identification of the disease resistances such as rotten, club root and balck shank, wherein M1108 and M1139 be to the high sense of black rot, and hybridize offspring F with high resistance individual plant M1103
2Colony's black rot anti-the sense plant substantially present the ratio of 3:1, thereby show that tentatively the black rot resistance is by Dominant gene in the high resistance black mustard, the work well afoots such as the screening of resistant gene, Cloning and sequencing.
Claims (1)
1. a black mustard microspores culture obtains the method for regeneration plant, it is characterized in that carrying out as follows:
(1) preparation of medium: comprise the medium in each stage of microspores culture, their component and the weight of each component in every liter of medium are:
1) embryoid induction medium: NLN-13 liquid nutrient medium+sucrose 130 g/L, pH 5.6 ~ 6.0, filtration sterilization;
2) embryoid differential medium: B
5Medium+ZT 0.5 ~ 2.0 mg/L+NAA 0.01 ~ 0.1 mg/L+BAP 0.5 ~ 2.0 mg/L+sucrose 20 ~ 30 g/L+agar 7 ~ 9 g/L, pH 5.6 ~ 6.0, high-temperature sterilization;
3) sprouting, root media: MS medium+NAA 0.05 ~ 0.1 mg/L+sucrose 30 g/L+agar 8 g/L, pH 5.8, high-temperature sterilization;
(2) selection of donor plant, bud and preliminary treatment:
1) selection of donor plant, bud: select the black mustard of healthy growth as donor plant; And therefrom select petal and anther length than between 0.7 ~ 1.0, the bud that is in the monokaryon middle and advanced stage is the donor bud;
2) bud sterilization: with behind the pure water cleaning bud 3 times, put it into by 0.1% HgCl first
2In the sterilized solution that+0.1% polysorbas20 is made into, after shaking table carried out surface sterilization in 12 minutes by 50-80 rpm slight wobble, in super-clean bench sterile water wash 4 times, for subsequent use again;
3) separation of bud microspore, be mixed and packing: bud places aseptic beaker after will sterilizing on super-clean bench, add behind the 1 mL embryoid induction medium with the tack glass rod crush bud, add 9 mL same medium and stir into suspension again; Filter in centrifuge tube with the aseptic nylon leaching net of 40 μ m, centrifugal 4 minutes of 1000rpm abandons supernatant; Add after the embryoid induction medium contains the contained microspore amount of 1.0 buds to average every 4mL medium, again with the medium in this liquid with by NLN-13 liquid nutrient medium+1g/L active carbon, through the active carbon mixed liquor of high-temperature sterilization by volume 0.05 mL ∕ 4mL ratio be mixed into microspore suspension; And be sub-packed in the sterile petri dish that diameter is 6 cm, add a cover rear with the sealing of parafilm film, for subsequent use;
(3) the microspore induction of embryoid is cultivated: above-mentioned culture dish is placed 31-33 ℃ of incubator, secretly cultivated 24-48 hour; Be placed in 25 ℃ of constant incubators, secretly cultivate and occurred to embryoid in 15-20 days; 50rpm shaken cultivation 7-10 days under 25 ℃ of dark again forms to cotyledonary embryos shape body;
(4) differentiation of regeneration plant with sprout to cultivate: the cotyledon embryoid is lain against on the embryoid differential medium, and 25 ℃ of illumination every day was cultivated 15 ~ 20 days in 16 hours to the embryoid plant that makes new advances of expanding and regenerate;
(5) culture of rootage of regeneration plant and transplanting: cutting to sprout has the regeneration plant of normal growth point to insert in the root media, and 25 ℃ of illumination every day were cultured in 16 hours takes root; The regeneration plant of will taking root moves in the matrix by peat and by volume 2:1 preparation of perlite, water permeable, behind the plastic covering film 25 ℃ of 1 weeks of lower cultivation;
(6) ploidy of regeneration plant detects: get the tender leaf of transplant survival regeneration plant, detect the dna ploidy of this regeneration plant with the partec PA the ploidy analyser of German Partec company production.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101617630A (en) * | 2009-08-13 | 2010-01-06 | 浙江省农业科学院 | Culture method of high diplont rate sporule regeneration plant of broccoli |
| CN101617631A (en) * | 2009-08-13 | 2010-01-06 | 浙江省农业科学院 | Culture method of high diplont rate sporule regeneration plant of broccoli |
| CN102362579A (en) * | 2011-11-10 | 2012-02-29 | 浙江大学 | Method for cultivating artificial hybridization hexaploid rape microspore regeneration plants |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101617630A (en) * | 2009-08-13 | 2010-01-06 | 浙江省农业科学院 | Culture method of high diplont rate sporule regeneration plant of broccoli |
| CN101617631A (en) * | 2009-08-13 | 2010-01-06 | 浙江省农业科学院 | Culture method of high diplont rate sporule regeneration plant of broccoli |
| CN101617630B (en) * | 2009-08-13 | 2011-08-10 | 浙江省农业科学院 | Culture method of high diplont rate sporule regeneration plant of broccoli |
| CN102362579A (en) * | 2011-11-10 | 2012-02-29 | 浙江大学 | Method for cultivating artificial hybridization hexaploid rape microspore regeneration plants |
Non-Patent Citations (3)
| Title |
|---|
| E.HETZ AND O.SCHIEDER: "Direct embryogenesis and plant regeneration through microspore culture of Brassica nigra", 《CRUCIFERAE NEWSLETT》 * |
| E.MARGALE AND A.M.CHEVTE: "Factors affecting embry production from microspore culture of Brassica nigra(Koch)", 《CRUCIFERAE NEWSLETT》 * |
| S.GOVIL等: "Plant Regeneration from in vitro Cultured Anthers of Black Mustard（Brassica nigra Koch）", 《PLANT BREEDING》 * |
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