CN103718965A - Method for rapidly and efficiently obtaining regeneration plants by culturing free microspores of brassica vegetables - Google Patents
Method for rapidly and efficiently obtaining regeneration plants by culturing free microspores of brassica vegetables Download PDFInfo
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Abstract
The invention discloses a method for rapidly and efficiently obtaining regeneration plants by culturing the free microspores of brassica vegetables. The method comprises the following steps: transferring microspore embryoids into a shaking table at 2000 lx (Lux), 25 DEG C and 60 r/min (Revolutions Per Minute) for shaking culturing at the time of the generation of the microspore embryoids; after 3 to 7 days, transferring cytoledon-stage embryos turning green into a B5 solid culture medium containing 2.5% of saccharose and 1% of agar powder for continuous culturing so as to obtain regeneration buds; after 15 to 20 days, transferring the regeneration buds into a MS (Murashige Skoog) solid culture medium containing 10% of coconut juice, 3% of saccharose and 0.78% of agar powder; after 20 to 30 days, obtaining results that the majority of plants grow strongly and the rooted seedlings can be domesticated and transplanted, wherein all the culturing conditions are carried out at 90uE/m<2>/s, 12hr/d and 25 DEG C. The average planting percent of the regeneration plants can reach 83.5%. As the method is used, the problem that the microspore embryoids are easy to brown and vitrify is effectively solved; the time of obtaining the DH (Double Haploid) plants by culturing the free microspores is effectively shortened; the DH plants are safer because the addition of any hormones in the DH plants is not required.
Description
Technical field
The present invention relates to a kind of method that Vegetables in Brassica Isolated microspore is cultivated regeneration plant that rapidly and efficiently obtains, belong to field of plant tissue culture technique.
Background technology
Vegetables in Brassica belongs to cross-pollinatd plant, and hybrid vigour is obvious, obtains the time that pure lines need 5～7 years in breeding, and Isolated microspore is cultivated and can be obtained double haploid in 1～2 year, greatly saves purifying time and workload.Because haploid existence in regeneration colony is also conducive to the expression of recessive character, enriched breeding resource simultaneously.In addition, it is unicellular that Isolated microspore belongs to, and being applied to mutation breeding, screening mutant and genetic transformation etc. has its special advantage, and DH colony spontaneous or that artificial doubling obtains carries out especially molecular labeling and draws the ideal material of genome.
Isolated microspore culture technique, it is the unicellular culture technique of being initiated on cabbage type rape by Lichter (1982), the country such as Canadian, German has carried out this research work in succession subsequently, improve the root tips of microspore, in late 1980s, tentatively set up the microspores culture system of cabbage type rape.After this microspore culture belongs in different vegetable and has obtained developing rapidly rape, created from the method for the separated microspore of bud machinery, established and can improve the bud standard of inductivity and heat shock is processed for cultivating, and succeeded on the crops such as leaf mustard, head cabbage, cauliflower, broccoli, Chinese cabbage.China starts late in the research aspect Isolated microspore cultivation, is mainly the experience of foreign, but development rapidly.The nineties in 20th century, Cao Mingqing etc. (1993), Chen Yuping etc. (1998) and Yan Zhun etc. (1999) cultivate and succeed at the Isolated microspore of the crops such as Chinese cabbage, purple tsai-tai and kohlrabi first respectively.2003, Zhu Yunhua etc. (2003) reported first cabbage heart Isolated microspore was cultivated successfully.In addition in Vegetables in Brassica, broccoli, cabbage mustard, cauliflower have also successively obtained sporule regeneration plant.
But forefathers' research mainly focuses on the generation aspect of efficient inducing embryoid body, very few to the research of microspore embryo regeneration frequency, and lack deep research.But for breeder, microspore plant has more practical significance more than microspore embryo.Therefore, how obtaining high-frequency sporule regeneration plant very crucial, is also the key constraints that obtains at present sporule regeneration plant colony (DH).The research discovery of Liu Fan etc. (1997), can microspore embryo develop into plant smoothly, is subject to the restriction of inside and outside two kinds of factors.Microspore embryo exists the phenomenons such as brownization, vitrifying in differentiation, Subculture, directly affects the regeneration rate of microspore plant, so the present invention is intended to set up Chinese cabbage and the broccoli Isolated microspore cultivation regeneration plant system of stablizing high frequency.
Summary of the invention
The object of the invention is the inefficient problem of seedling existing for obtaining isolated microspore regeneration plant in above-mentioned prior art, and a kind of method that Vegetables in Brassica Isolated microspore is cultivated regeneration plant that rapidly and efficiently obtains is provided.
The object of the invention is to realize in the following manner:
Rapidly and efficiently obtain Vegetables in Brassica Isolated microspore and cultivate a method for regeneration plant, the method comprises the steps:
(1) selection of bud, pretreatment and sterilizing: at initial bloom stage and full-bloom stage, choose developmental stage and be the keep to the side bud of phase of monokaryon; By bud moisturizing and be placed in refrigerator and cooled and hide sterilizing after standing 24h;
(2) microspore isolation and purification: in the bud after sterilizing, add B5-13 liquid nutrient medium, adopt extrusion to make microspore free out, then filter, collect filtrate, centrifugal, abandon supernatant, obtain the microspore after purifying; The ratio of described bud and B5-13 liquid nutrient medium is in every 28-32 bud, to add the B5-13 liquid nutrient medium of 1～2ml.
(3) packing of culture fluid, heat-inducible: by the 1/2NLN liquid nutrient medium Eddy diffusion that contains 13% sucrose for the microspore after purifying, then packing, microspore density 1 flower bud/ml, sealing is rear, heat-inducible;
(4) embryoid forms: the microspore suspension after heat shock, in 25 ℃ of standing cultivations of dark, is occurred to macroscopic embryoid after 2～3 weeks;
(5) shaken cultivation: forward above-mentioned embryoid on 2000lx, 25 ℃, 60r/min shaking table shaken cultivation;
(6) regeneration induction plant: transfer to containing sucrose 2.5% turning green cotyledon type embryo after shaken cultivation 3～7d, on the B5 solid culture medium of agar 1%, continue to cultivate and obtain regeneration bud, after 15～20d, proceeded to containing 10% Coconut Juice, 3% sucrose, in the MS solid culture medium of 0.78% agar, cultivate 20～30d.
Above-mentioned monokaryon bud length Chinese cabbage corresponding to phase that keep to the side is 2.0～3.0mm, and broccoli is 3.0mm～4.0mm;
Described B5-13 liquid nutrient medium is microspore extract, prepare by the following method: draw respectively the macroelement 50ml of B5 medium, micro-5ml, molysite 10ml, tetra-kinds of mother liquors of organic element 10ml are mixed in 500ml distilled water, mix, add again 130g sucrose, be stirred to completely and dissolve, be settled to 1000ml, then adjust pH to 6.0～6.2; In 121 ℃, under 1.1Mpa condition, sterilizing is 20 minutes.
Described 1/2NLN liquid nutrient medium prepares by the following method: draw respectively the macroelement 25ml of NLN medium, micro-5ml, molysite 10ml, tetra-kinds of mother liquors of organic element 10ml are mixed in 500ml distilled water, mix, add successively again inositol 0.1g, glutamine 0.8g, serine 0.1g, glutathione 0.03g, sucrose 130g, be stirred to after dissolving completely, be settled to 1000ml, then adjust pH to 6.0～6.2; Adopt the miillpore filter suction filtration sterilizing of 0.22 μ m.
Described B5 solid culture medium prepares by the following method: draw respectively the macroelement 50ml of B5 medium, micro-5ml, molysite 10ml, tetra-kinds of mother liquors of organic element 10ml are mixed in 500ml distilled water, mix, add again 25g sucrose, be stirred to completely and dissolve, be settled to 1000ml, be heated to, after boiling, add 10g agar powder, be stirred to completely and dissolve, adjust pH to 5.8～6.0; At 121 ℃, under 1.1Mpa condition, sterilizing is 20 minutes.
Described MS solid culture medium prepares by the following method: draw respectively the macroelement 50ml of MS medium, micro-5ml, molysite 10ml, tetra-kinds of mother liquors of organic element 10ml, Coconut Juice 100ml is mixed in 500ml distilled water, mix, add again 30g sucrose, be stirred to completely and dissolve, be settled to 1000ml, be heated to after boiling, add 7.8g agar powder, be stirred to completely and dissolve, adjust pH to 5.8～6.0; At 121 ℃, under 1.1Mpa condition, sterilizing is 20 minutes.
In step (6), the condition of culture of regeneration plant is all 90uE/m
2/ s, 12hr/d, 25 ℃.
The NaClO solution disinfection of the alcohol and 7% (v/v) that described sterilization steps is is 75% by volumetric concentration by pretreated bud successively, aseptic water washing.
Described heat-inducible is that microspore suspension is placed in to dark standing 24h under 33 ℃ of high temperature.
The selection of described bud be by aceto-camine pressed disc method microscopy determine microspore developmental stage and with the corresponding relation of bud length, accurately finding developmental stage is the keep to the side bud of phase of monokaryon.
Macroelement in medium can be NH
4nO
3, KNO
3, MgSO
47H
2o, (NH
4)
2sO
4, NaH
2pO
4h
2o, Ca (NO
3)
24H
2o, KH
2pO
4, CaCl
2in one or more.Trace element can be MnSO
4h
20, H
3bO
3, ZnSO
47H
2o, KI, Na
2moO
42H
2o, CuSO
45H
2o, CoCl
26H
2one or more in O.Fe salt can be: Na
2-EDTA, FeSO
47H
2one or more in O.Organic element can be inositol, nicotinic acid, VB
6, VB
1, one or more in glycine, glutamine, serine, glutathione, folic acid, vitamin h.
The above-mentioned method that rapidly and efficiently obtains Vegetables in Brassica Isolated microspore cultivation regeneration plant, specifically comprises the steps:
(1) selection of bud: at initial bloom stage and full-bloom stage, by aceto-camine pressed disc method microscopy determine microspore developmental stage and with the corresponding relation of bud length, accurately finding developmental stage is the keep to the side bud of phase of monokaryon;
(2) bud pretreatment: by the suitable bud moisturizing of choosing and be placed in 4 ℃ of refrigerators and process 24h;
(3) sterilizing: 75% alcohol disinfecting 30s for the bud of choosing, then use the NaClO solution disinfection 15min of 7% (v/v), finally use aseptic water washing 3 times, each 3～5min;
(4) microspore isolation and purification: the bud after sterilization is placed in to mortar, adds 1～2ml B
5-13 liquid nutrient mediums, adopt extrusion to make microspore free out, then with 300 mesh filter screens, filter, and filtrate is collected in to 10ml centrifuge tube, and the centrifugal 3min of 1000rpm, abandons supernatant, repeats 3 times;
(5) packing of culture fluid: by the 1/2NLN liquid nutrient medium Eddy diffusion that contains 13% sucrose for the microspore after purifying, be then sub-packed in 60 * 15mm culture dish, every ware 3ml, microspore density is 1 flower bud/ml approximately, with the sealing of Parafilm film;
(6) heat-inducible: above-mentioned microspore suspension is placed in to dark processing 24h under 33 ℃ of high temperature;
(7) embryoid forms: the microspore suspension after heat shock, in 25 ℃ of standing cultivations of dark, can be occurred to macroscopic embryoid after 2～3 weeks;
(8) shaken cultivation: forward above-mentioned embryoid on 2000lx, 25 ℃, 60r/min shaking table shaken cultivation;
(9) regeneration induction plant: transfer to containing sucrose 2.5% turning green cotyledon type embryo after shaken cultivation 3～7d, on the B5 solid culture medium of agar 1%, continue to cultivate and obtain regeneration bud, after 15～20d, proceeded to containing 10% Coconut Juice, 3% sucrose, in the MS solid culture medium of 0.78% agar, the seedling that 20～30d can grow up to stalwartness and take root, can rooting culture;
The various mother liquor formulas that Isolated microspore is cultivated are as follows:
Beneficial effect of the present invention compared with the prior art:
The method that the present invention rapidly and efficiently obtains Vegetables in Brassica Isolated microspore cultivation regeneration plant has obtained sporule regeneration plant colony (DH).
Acquisition Isolated microspore after improveing by the present invention is cultivated (IMC) regeneration plant method, the Vegetables in Brassica that make easy brownization of conventional method, vitrifying, is difficult for taking root, be difficult to obtain regeneration plant has obtained high-frequency regeneration plant, the highest plumule inductivity reaches more than 90%, and the average planting percent of regeneration plant reaches 83.5%; In addition, great majority are only used the MS medium regeneration plant that adds Coconut Juice to take root, and average rooting rate reaches 75% left and right, need not add any hormone and use root media, has saved the time and safer.
Accompanying drawing explanation
Fig. 1 is the upgrowth situation of cotyledon type embryo in containing the B5 solid culture medium of variable concentrations agar powder in the present invention: in figure, A, B represent respectively the upgrowth situation of cotyledon type embryo in the B5 solid culture medium of 0.8% and 1% concentration agar powder.In easy brownization of cotyledon type embryo containing in the B5 solid culture medium of 0.8% agar, plumule incidence is extremely low; Can induce regeneration bud the cotyledon type embryo containing in 1% B5 solid culture medium is most of, plumule incidence improves.
Fig. 2 is that in the present invention, Vegetables in Brassica Isolated microspore is cultivated growth and the condition of rooting of regeneration plant in different MS solid culture mediums: A representative plant strain growth situation in not containing the MS solid culture medium of Coconut Juice in figure; B representative plant growth condition in containing the MS solid culture medium of Coconut Juice 10% in figure; In figure, C represents plant strain growth and condition of rooting contrast in two kinds of different MS solid culture mediums.Better containing the regeneration plant growing way on the MS solid culture medium of 10% Coconut Juice, and the root of inducing is thick and close.
Embodiment
The preparation of embodiment 1 medium
1, the preparation of mother liquor
Before experiment, first macroelement, trace element, organic element and the molysite of the required various medium of using in the inventive method being mixed with to mother liquor is stored in 4 ℃ of refrigerators, compound concentration is respectively: 20 times (with 20X, representing below), 200 times (with 200X, representing below), 100 times (with 100X, representing below) and 100 times (with 100X, representing below), concrete composition is in Table 1.
The various mother liquor formulas that table 1 Isolated microspore is cultivated
2, the preparation of medium and sterilizing
1. microspores culture base (1/2NLN-13): 1/2NLN+13% sucrose, pH is 6.0～6.2;
The miillpore filter suction filtration sterilizing of 0.22 μ m for medium;
2. microspore extract (B
5-13): B
5minimal medium+13% sucrose, pH is 5.8;
3. B5 solid culture medium:
A, B
5minimal medium+2.5% sucrose+0.8% agar (ck), pH is 5.8;
B, B
5minimal medium+2.5% sucrose+1% agar, pH is 5.8;
4. MS solid culture medium:
A, MS minimal medium+3% sucrose+0.78% agar (ck), pH is 5.8;
B, MS minimal medium+3% sucrose+0.78% agar+10% Coconut Juice, pH is 5.8;
More than cultivate based on 121 ℃, under 1.1Mpa condition, sterilizing is 20 minutes.
Embryo is cultivated and induced to embodiment 2 Vegetables in Brassica Isolated microspores
1, genotype: Chinese cabbage NHCC-549; Broccoli A36-5, A47-4, A48-3.
The crossbreed of Chinese cabbage NHCC-549---" Ma Ertou " and " little Song dish " (be maternal by " Ma Ertou ", " little Song dish " gets for male parent adopts conventional method hybridization); Broccoli A36-5---commodity are called " outstanding No. 1 "; Broccoli A47-4---commodity are called " green for a long time "; Broccoli A48-3---commodity are called " rich island ".All purchase the sensible seed business department in academy of agricultural sciences, Jiangsu Province.
2, the selection of bud: at initial bloom stage and full-bloom stage, by aceto-camine pressed disc method microscopy determine microspore developmental stage and with the corresponding relation of bud length, accurately finding developmental stage is the keep to the side bud of phase of monokaryon, wherein the suitable bud length Chinese cabbage that carries out microspores culture is 2.0～3.0mm, and broccoli is 3.0mm～4.0mm;
3, bud pretreatment: by the suitable bud moisturizing of choosing and be placed in 4 ℃ of refrigerators and process 24h;
4, sterilizing: 75% alcohol disinfecting 30s for the bud of choosing, then use the NaClO solution disinfection 15min of 7% (v/v), finally use aseptic water washing 3 times, each 3～5min;
5, microspore isolation and purification: the bud after sterilization is placed in to mortar, adds 1～2ml B5-13 medium, adopt extrusion to make microspore free out, then with 300 mesh filter screens, filter, filtrate is collected in to 10ml centrifuge tube, the centrifugal 3min of 1000rpm, abandon supernatant, repeat 3 times; The ratio of described bud and B5-13 liquid nutrient medium is in every 28-32 bud, to add the B5-13 liquid nutrient medium of 1～2ml.
6, the packing of culture fluid: by the 1/2NLN medium Eddy diffusion that contains 13% sucrose for the microspore after purifying, be then sub-packed in 60 * 15mm culture dish, every ware 3ml, microspore density is 1 flower bud/ml approximately, with the sealing of Parafilm film;
7, high temperature induction: above-mentioned microspore suspension is placed in to dark processing 24h under 33 ℃ of high temperature;
8,25 ℃ of standing cultivations of dark;
9, embryoid forms: dark processing can occur macroscopic embryoid after 2～3 weeks.
Result of implementation is as follows:
The Vegetables in Brassica Isolated microspore of different genotype is cultivated goes out embryo situation: darkroom is cultivated after 2 weeks Isolated microspore culture materials is added up, and sees if there is graininess embryoid and occurs.Result shows as table 2, and in four kinds of genotypic Vegetables in Brassicas, broccoli A47-4 germ extraction rate is the highest, up to 1.49.
The Vegetables in Brassica Isolated microspore of table 2 different genotype is cultivated goes out embryo situation
Embodiment 3 inducing embryoid bodies obtain regeneration bud
1, shaken cultivation: forward the embryoid obtaining in embodiment 2 on 2000lx, 25 ℃, 60r/min shaking table shaken cultivation; 2, inducing embryoid body obtains regeneration bud: equal number of days has been turned to green cotyledon type embryo and transferred to respectively containing sucrose 2.5%, agar powder 1% with containing continuing to cultivate 15～20d on two kinds of B5 solid culture mediums of sucrose 2.5%, agar powder 0.8% (ck), condition of culture is 90uE/m2/s, 12hr/d, the culturing room of 25 ℃.Observe its upgrowth situation and carry out statistic record.
Result of implementation is as follows:
Table 3 result shows: 1. NHCC-549 and A48-3 are containing almost whole brownization on the B5 solid culture medium of 0.8% agar, plumule incidence is extremely low, but containing on the B5 solid culture medium of 1% agar, all induce regeneration bud, plumule incidence is respectively 0.50 and 0.33; 2. A36-5 and A47-4, having brought up to 0.92 by 0.75 respectively containing the plumule incidence on the B5 solid culture medium of 1% agar, have brought up to 0.91 by 0.71.
Four kinds of genotype Vegetables in Brassicas of table 3 are in the contrast situation of the plumule incidence of the B5 solid culture medium containing variable concentrations agar
Embodiment 4 obtains Vegetables in Brassica Isolated microspore and cultivates regeneration plant
1, genotype: broccoli A36-5, A47-4;
2, obtain regeneration plant: the good somatic embryogenesis bud of upgrowth situation in two kinds of genotype that obtain in embodiment 3 is proceeded to respectively containing in 10% Coconut Juice, 3% sucrose, 0.78% agar and two kinds of MS solid culture mediums without Coconut Juice, 3% sucrose, 0.78% agar (ck), and condition of culture is all 90uE/m2/s, 12hr/d, the culturing room of 25 ℃.After 20～30d, observe its upgrowth situation and carry out statistic record.The seedling that plant to be planted grows up to 4～6 true leaves, stalwartnesses and takes root, can rooting culture.
Result of implementation is as follows:
Table 4 result shows: two kinds of A36-5 and A47-4 than all high without the planting percent on the MS solid culture medium of Coconut Juice and rooting rate in routine, have improved respectively 1.46 times on the MS solid culture medium that adds 10% Coconut Juice, 2.15 times and 1.87 times, and 3.36 times.And by accompanying drawing explanation 2, can be found out better containing the regeneration plant growing way on the MS solid culture medium of 10% Coconut Juice, and the root of inducing is thick and close.
Planting percent and the rooting rate situation of two kinds of genotype of table 4 isolated microspore regeneration plant on different MS solid culture medium
Claims (10)
1. rapidly and efficiently obtain Vegetables in Brassica Isolated microspore and cultivate a method for regeneration plant, it is characterized in that: comprise the steps:
(1) selection of bud, pretreatment and sterilizing: at initial bloom stage and full-bloom stage, choose developmental stage and be the keep to the side bud of phase of monokaryon; By bud moisturizing and be placed in refrigerator and cooled and hide sterilizing after standing 24h;
(2) microspore isolation and purification: in the bud after sterilizing, add B5-13 liquid nutrient medium, adopt extrusion to make microspore free out, then filter, collect filtrate, centrifugal, abandon supernatant, obtain the microspore after purifying; The ratio of described bud and B5-13 liquid nutrient medium is in every 28-32 bud, to add the B5-13 liquid nutrient medium of 1～2ml.
(3) packing of culture fluid, heat-inducible: by the 1/2NLN liquid nutrient medium Eddy diffusion that contains 13% sucrose for the microspore after purifying, then packing, microspore density 1 flower bud/ml, sealing is rear, heat-inducible;
(4) embryoid forms: the microspore suspension after heat shock, in 25 ℃ of standing cultivations of dark, is occurred to macroscopic embryoid after 2～3 weeks;
(5) shaken cultivation: forward above-mentioned embryoid on 2000lx, 25 ℃, 60r/min shaking table shaken cultivation;
(6) regeneration induction plant: transfer to containing sucrose 2.5% turning green cotyledon type embryo after shaken cultivation 3～7d, on the B5 solid culture medium of agar 1%, continue to cultivate and obtain regeneration bud, after 15～20d, proceeded to containing 10% Coconut Juice, 3% sucrose, in the MS solid culture medium of 0.78% agar, cultivate 20～30d.
2. according to claim 1ly rapidly and efficiently obtain the method that Vegetables in Brassica Isolated microspore is cultivated regeneration plant, it is characterized in that: monokaryon bud length Chinese cabbage corresponding to phase that keep to the side is 2.0～3.0mm, and broccoli is 3.0mm～4.0mm.
3. according to claim 1ly rapidly and efficiently obtain the method that Vegetables in Brassica Isolated microspore is cultivated regeneration plant, it is characterized in that: described B5-13 liquid nutrient medium is microspore extract, prepare by the following method: draw respectively the macroelement 50ml of B5 medium, micro-5ml, molysite 10ml, tetra-kinds of mother liquors of organic element 10ml are mixed in 500ml distilled water, mix, add again 130g sucrose, be stirred to completely and dissolve, be settled to 1000ml, then adjust pH to 6.0～6.2; In 121 ℃, under 1.1Mpa condition, sterilizing is 20 minutes.
4. according to claim 1ly rapidly and efficiently obtain the method that Vegetables in Brassica Isolated microspore is cultivated regeneration plant, it is characterized in that: described 1/2NLN liquid nutrient medium prepares by the following method: the macroelement 25ml that draws respectively NLN medium, trace element 5ml, molysite 10ml, tetra-kinds of mother liquors of organic element 10ml are mixed in 500ml distilled water, mix, add successively again inositol 0.1g, glutamine 0.8g, serine 0.1g, glutathione 0.03g, sucrose 130g, be stirred to after dissolving completely, be settled to 1000ml, then adjust pH to 6.0～6.2, adopt the miillpore filter suction filtration sterilizing of 0.22 μ m.
5. according to claim 1ly rapidly and efficiently obtain the method that Vegetables in Brassica Isolated microspore is cultivated regeneration plant, it is characterized in that: described B5 solid culture medium prepares by the following method: draw respectively the macroelement 50ml of B5 medium, micro-5ml, molysite 10ml, tetra-kinds of mother liquors of organic element 10ml are mixed in 500ml distilled water, mix, add again 25g sucrose, be stirred to completely and dissolve, be settled to 1000ml, be heated to after boiling, add 10g agar powder, be stirred to completely and dissolve, adjust pH to 5.8～6.0; At 121 ℃, under 1.1Mpa condition, sterilizing is 20 minutes.
6. according to claim 1ly rapidly and efficiently obtain the method that Vegetables in Brassica Isolated microspore is cultivated regeneration plant, it is characterized in that: described MS solid culture medium prepares by the following method: draw respectively the macroelement 50ml of MS medium, micro-5ml, molysite 10ml, tetra-kinds of mother liquors of organic element 10ml, Coconut Juice 100ml is mixed in 500ml distilled water, mix, add again 30g sucrose, be stirred to completely and dissolve, be settled to 1000ml, be heated to after boiling, add 7.8g agar powder, be stirred to completely and dissolve, adjust pH to 5.8～6.0; At 121 ℃, under 1.1Mpa condition, sterilizing is 20 minutes.
7. the method that rapidly and efficiently obtains Vegetables in Brassica Isolated microspore cultivation regeneration plant according to claim 1, is characterized in that: in step (6), the condition of culture of regeneration plant is all 90uE/m
2/ s, 12hr/d, 25 ℃.
8. according to claim 1ly rapidly and efficiently obtain the method that Vegetables in Brassica Isolated microspore is cultivated regeneration plant, the NaClO solution disinfection of the alcohol and 7% (v/v) that the sterilization steps described in it is characterized in that is is 75% by volumetric concentration by pretreated bud successively, aseptic water washing.
9. the method that rapidly and efficiently obtains Vegetables in Brassica Isolated microspore cultivation regeneration plant according to claim 1, is characterized in that described heat-inducible is that microspore suspension is placed in to dark standing 24h under 33 ℃ of high temperature.
10. according to claim 1ly rapidly and efficiently obtain the method that Vegetables in Brassica Isolated microspore is cultivated regeneration plant, the selection that it is characterized in that described bud be by aceto-camine pressed disc method microscopy determine microspore developmental stage and with the corresponding relation of bud length, accurately finding developmental stage is the keep to the side bud of phase of monokaryon.
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| CN105154383A (en) * | 2015-09-07 | 2015-12-16 | 镇江瑞繁农艺有限公司 | Separation method of microspores developed and enlarged by broccoli |
| CN108207628A (en) * | 2018-01-08 | 2018-06-29 | 江苏省农业科学院 | A kind of method of broccoli Plantlet Regeneration by Isolated Microspore Culture |
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| CN110731271A (en) * | 2019-12-02 | 2020-01-31 | 海南省农业科学院蔬菜研究所 | method for increasing embryoid output number of flowering cabbage isolated microspore culture embryoid |
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| CN104871958A (en) * | 2015-06-09 | 2015-09-02 | 西南大学 | Method for breeding cabbage type yellow-seeded rape recessive genic male sterility temporary maintainer line |
| CN105154383A (en) * | 2015-09-07 | 2015-12-16 | 镇江瑞繁农艺有限公司 | Separation method of microspores developed and enlarged by broccoli |
| CN108207628A (en) * | 2018-01-08 | 2018-06-29 | 江苏省农业科学院 | A kind of method of broccoli Plantlet Regeneration by Isolated Microspore Culture |
| CN110495393A (en) * | 2019-08-12 | 2019-11-26 | 浙江省农业科学院 | A method of obtaining purple cauliflower microspore DH regeneration plant |
| CN110731271A (en) * | 2019-12-02 | 2020-01-31 | 海南省农业科学院蔬菜研究所 | method for increasing embryoid output number of flowering cabbage isolated microspore culture embryoid |
| CN111869566A (en) * | 2020-08-01 | 2020-11-03 | 梁江 | Ginseng free microspore induction culture method |
| CN115777528A (en) * | 2023-02-09 | 2023-03-14 | 广州市农业科学研究院 | Culture method for improving microspore embryo emergence rate of hybrid seeds of flowering cabbage and brassica oleracea |
| CN115777528B (en) * | 2023-02-09 | 2023-04-21 | 广州市农业科学研究院 | Culture method for improving embryo emergence rate of microspores of hybrid seeds of flowering cabbage and laver |
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