CN1552198A - Method for culturing cauliflower regenerative tree by free microspore culture technology - Google Patents
Method for culturing cauliflower regenerative tree by free microspore culture technology Download PDFInfo
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- CN1552198A CN1552198A CNA031299873A CN03129987A CN1552198A CN 1552198 A CN1552198 A CN 1552198A CN A031299873 A CNA031299873 A CN A031299873A CN 03129987 A CN03129987 A CN 03129987A CN 1552198 A CN1552198 A CN 1552198A
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- culturing
- microspore
- cauliflower
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Abstract
A method for culturing the regenerative plant of couliflower by free microspore culturing technique includes such steps as culturing until the microspore is generated, culturing at 25 deg.C and under 4000 LX for illumination (12-16 hr per day) until it becomes green, and culturing on non-hormone solid culture medium MS containing cane sugar (3%) until young seedling is grown up. It features that said solid culture medium contains also three auxins: kinin, 6-benzylpurine and naphthalene acetic acid. Its advantages are broad breeding resources and short breeding time.
Description
Affiliated field:
The invention belongs to a kind of cultural method of vegetable breeding material, particularly a kind of method of utilizing isolated microspore culture technique to cultivate the cauliflower regeneration plant
Background technology:
Isolated microspore culture technique reaches its maturity in the rape genus vegetable breeding at present, adopts Isolated microspore to cultivate as Chinese cabbage, cabbage type rape, stem coconut palm Lay vegetables such as (broccolis) and has all obtained success, and be applied in the breeding work.Cauliflower belongs to Brassicas vegetables Lay, and other brassica vegetable Isolated microspore cultural method roots are difficult succeeds but use.Particularly the plant regeneration of cauliflower microspore using embryo is still unsolved in the world technological difficulties always, the Brassica oleracea var. botrytis L. microspores culture can get the globular stage embryo and heart-shaped phase embryo of some, but the embryo of this moment generally no longer continues arrest of development this period, and brownization is dead gradually even subculture also can not become seedling.
Summary of the invention:
Purpose of the present invention just is to provide a kind of method of utilizing isolated microspore culture technique to obtain the cauliflower regeneration plant, this method has solved the microspore embryo and has stagnated the problem of growing, and makes the microspore embryo that obtains continue the bud into regeneration plant through successive transfer culture.
Technical scheme of the present invention is: a kind of method of utilizing isolated microspore culture technique to cultivate the cauliflower regeneration plant, be when treating that microspore embryo length produces, the illumination condition that forwards 25 ℃ of temperature, illumination 4000LX, photoperiod 12-16hr/d to is cultivated down, transfer to the MS that contains sucrose 3% and do not have and continue on the hormone solid culture medium to cultivate changeing embryo after green, condition of culture is the same, grows up to until seedling; It is characterized in that: do not have at the MS that contains sucrose 3% and add kinetin, 6 benzyl purines and three kinds of growth hormone of methyl in the hormone solid culture medium.
The concentration range of above-mentioned kinetin is that 0.01-3ppm, 6 benzyl purine concentration ranges are that 0.01-3ppm, methyl concentration range are at 0.01-3ppm.
Advantage of the present invention is: 1, can solve the problem of microspore embryonic development stagnation effectively, make the microspore embryo 70 80% energy bud into regeneration plants of acquisition.Because an embryo is being represented a kind of genetic type,, greatly enriched cauliflower breeding resource so the success of the method provides a large amount of new genotype pure lines for the Brassica oleracea var. botrytis L. breeding.2, can accelerate the breeding paces, shorten breeding time greatly, carry out the cauliflower breeding for Applied Biotechnology and established technical foundation.
Embodiment:
A kind of method of utilizing isolated microspore culture technique to cultivate cauliflower, carry out according to the following step: 1. draw materials: get the inflorescence of the long 3-6mm of bud, its pollen development period is monokaryon late period.2. material sterilization:, use aseptic water washing 3 times with the saturated calcium hypochlorite solution of 7% (w/v), surface sterilization 15 minutes.3. collection of microspore and cultivation: aseptic bud is put into mortar, add a small amount of B5 washing medium (prescription is seen attached list), gently press material with mortar, make microspore from flower pesticide, dissociate out, nylon net filter with 45 μ m apertures, collect 1000 rev/mins of filtrates, centrifugal 3 times, each 3 minutes, last centrifugal after, microspore is suspended in the NLN medium (prescription is seen attached list) through the suction filtration sterilization, microspore concentration is adjusted into 1 * 10/ml, adopt the method for liquid culture, put into 2-3ml in each 60 * 15mm culture dish, Parafilm seals; Behind 33 ℃, 24hr high temperature treatment, place 25 ℃ to continue dark the cultivation down, generally through just obtaining the microspore embryo 2-3 week; 4. the acquisition of microspore embryo culture and regeneration plant: when treating that the microspore embryo produces, the illumination condition that forwards 25 ℃ of temperature, illumination 4000LX, photoperiod 12hr/d to is cultivated down, transfer to the MS that contains sucrose 3% and do not have and continue on the hormone solid culture medium to cultivate changeing embryo after green, condition of culture is the same, but 3-4 week just achievement obtain regeneration plant.Adding concentration according to growing state in the MS medium is that the kinetin of 0.01-3ppm, 6 benzyl purines, the concentration that concentration is 0.01-3ppm are the methyl of 0.01-3ppm.5. the domestication of regeneration plant; Open before the transplanting and cultivated bottle cap white silk seedling 3 days, will transplant in the nutritive cube that seedling nutritious soil is housed with the residual medium of root seedling flush away root behind the white silk seedling, transplant early stage and preserve moisture, treat to be colonizated in the greenhouse after the seedling stalwartness with the plastics hut.
Medium is formed (mh/l)
Chemical name B
5NLN MS
KNO
3 300.0 125.0 1900.0
MgSO
4·7H
2O 500.0 125.0 370.0
Ca(NO
3)
2·4H
2O / 500.0 /
CaCl
2·2H
2O 750.0 / 440.0
KH
2PO
4 / 125.0 170.0
NaH
2PO
4·H
2O 150.0 / /
(NH
4)
2·SO
4 134.0 / /
NH
4NO
3 / / 1650.0
Fe-EDTA 40.0 40.0 40.0
MnSO
4·4H
2O 10.0 22.3 22.3
H
3BO
3 3.0 6.2 6.2
ZnSO
4·7H
2O 2.0 8.6 8.6
Na
2MoO
4·2H
2O 0.25 0.25 0.25
CuSO
4·5H
2O 0.025 0.025 0.025
CoCl·6H
2O 0.025 0.025 0.025
KI 0.75 0.83 0.83
Glycine/2.0 2.0
Inositol 100.0 100.0 100.0
Nicotinic acid 1.0 0.5 0.5
B
6 1.0 0.5 0.5
B
1 1.0 0.5 0.4
Vitamin h/0.05/
Folic acid/0.5/
Glutamine/800.0/
Serine/100.0/
Glutathione/30.0/
Sucrose 130000.0 130000.0 30000.0
Agar // 10000.0
Claims (2)
1, a kind of method of utilizing isolated microspore culture technique to cultivate the cauliflower regeneration plant, be when treating that microspore embryo length produces, the illumination condition that forwards 25 ℃ of temperature, illumination 4000LX, photoperiod 12-16hr/d to is cultivated down, transfer to the MS that contains sucrose 3% and do not have and continue on the hormone solid culture medium to cultivate changeing embryo after green, condition of culture is the same, grows up to until seedling; It is characterized in that: do not have at the MS that contains sucrose 3% and add kinetin, 6 benzyl purines and three kinds of growth hormone of methyl in the hormone solid culture medium.
2, the method for utilizing isolated microspore culture technique to cultivate the cauliflower regeneration plant according to claim 1, it is characterized in that: the concentration range of above-mentioned kinetin is that 0.01-3ppm, 6 benzyl purine concentration ranges are that 0.01-3ppm, methyl concentration range are at 0.01-3ppm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031299873A CN1552198A (en) | 2003-06-05 | 2003-06-05 | Method for culturing cauliflower regenerative tree by free microspore culture technology |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA031299873A CN1552198A (en) | 2003-06-05 | 2003-06-05 | Method for culturing cauliflower regenerative tree by free microspore culture technology |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1552198A true CN1552198A (en) | 2004-12-08 |
Family
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|---|---|---|---|
| CNA031299873A Pending CN1552198A (en) | 2003-06-05 | 2003-06-05 | Method for culturing cauliflower regenerative tree by free microspore culture technology |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101617631B (en) * | 2009-08-13 | 2011-09-07 | 浙江省农业科学院 | Culture method of high diplont rate sporule regeneration plant of broccoli |
| CN102726300A (en) * | 2012-07-06 | 2012-10-17 | 福建农林大学 | Anther culture method of cauliflower |
| CN103421842A (en) * | 2013-09-05 | 2013-12-04 | 南开大学 | Method and application for obtaining cauliflower materials with different phenotypes |
| CN103416309A (en) * | 2013-08-20 | 2013-12-04 | 甘肃农业大学 | Quick multiplication method of broccoli isolated cells |
-
2003
- 2003-06-05 CN CNA031299873A patent/CN1552198A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101617631B (en) * | 2009-08-13 | 2011-09-07 | 浙江省农业科学院 | Culture method of high diplont rate sporule regeneration plant of broccoli |
| CN102726300A (en) * | 2012-07-06 | 2012-10-17 | 福建农林大学 | Anther culture method of cauliflower |
| CN103416309A (en) * | 2013-08-20 | 2013-12-04 | 甘肃农业大学 | Quick multiplication method of broccoli isolated cells |
| CN103416309B (en) * | 2013-08-20 | 2016-03-30 | 甘肃农业大学 | A kind of broccoli cells in-vitro quick proliferation method |
| CN103421842A (en) * | 2013-09-05 | 2013-12-04 | 南开大学 | Method and application for obtaining cauliflower materials with different phenotypes |
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