CN103120127A - Method for quickly inducing and breeding cotton embryogenic callus - Google Patents
Method for quickly inducing and breeding cotton embryogenic callus Download PDFInfo
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- CN103120127A CN103120127A CN2013100598059A CN201310059805A CN103120127A CN 103120127 A CN103120127 A CN 103120127A CN 2013100598059 A CN2013100598059 A CN 2013100598059A CN 201310059805 A CN201310059805 A CN 201310059805A CN 103120127 A CN103120127 A CN 103120127A
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Abstract
The invention discloses a method for quickly inducing and breeding a cotton embryogenic callus. The method comprises the steps of 1, cutting a hypocotyledonary axis of a cotton aseptic seedling which is cultured to 5-8d into sections with the length of 0.4-0.8cm, then putting the sections into a culture medium A or B to culture for 25-30d at 25-28 DEG C, and thus obtaining the callus; 2, sequentially putting the callus obtained in the culture medium A into culture mediums B and C to be continuously cultured for 25-30d at 25-28 DEG C; and putting the callus obtained in the culture medium B into the culture medium C to be continuously cultured for 25-50d at 25-28 DEG C, and thus obtaining the embryogenic callus. By utilizing the method, the callus and the embryogenic callus of cotton can be quickly obtained; and the embryogenic callus can be quickly produced, and the cotton embryogenic callus can be kept in a good state, so that an excellent and efficient experimental material can be provided for cotton transgenosis.
Description
Technical field
The invention belongs to plant tissue and induce and the culture technique field, be specifically related to a kind of method of rapid induction and breeding cotton embryo callus subculture.
Background technology
Cotton is one of important economic crops, and for a long time, traditional breeding method has been brought into play significant role in New Crop Varieties seed selection work.But conventional breeding is to realize that by the selection to the breeding material phenotypic character selection to the target gene type, people can't directly investigate individual genotype, can only infer genotype from phenotype.And the conventional breeding tool bears the character of much blindness and unpredictability, and along with the development of biotechnology, the problem such as the efficient of conventional breeding is low is also more obvious.
Along with the development of transgenic technology, the new resources of cotton initiative and new quality seed selection also obtain significant achievement, such as: drought resisting, anti-salt, pest-resistant cotton.At present, cotton genetic method commonly used has agrobacterium-mediated transformation, particle bombardment etc.Because other transgenic methods such as particle gun have certain limitation, and can not obtain a large amount of transgene cottons in the short time.And can obtain large batch of transfer-gen plant by agrobacterium-mediated transformation.
Infect embryo callus subculture by Agrobacterium and can obtain fast transfer-gen plant, yet because the explant differentiation capability of cotton is more difficult, be the bottleneck that Agrobacterium is infected mediated method always.In cotton tissue was cultivated, the regenerating system of cotton mainly experiences following process: explant induction became callus, and Calli Differentiation is embryo callus, and embryo callus is divided into somatic embryo, is divided at last cotton seedling.Being the key link of cotton regenerated system from the callus to the embryo callus due to cotton, is also a most difficult step of cotton regenerated system.
Summary of the invention
The object of the invention is to overcome the prior art deficiency, and a kind of method of rapid induction and breeding cotton embryo callus subculture is provided.
For achieving the above object, the present invention adopts following technical scheme:
A kind of method of rapid induction and breeding cotton embryo callus subculture, it comprises the steps:
1) hypocotyl that will cultivate the cotton aseptic seedling of 5-8d cuts into the fragment that length is 0.4-0.8cm, then be placed in culture medium A or medium B and cultivate 25-30d in 25-28 ℃, illumination every day 12-16h, light intensity 1500-1800 Lux obtains callus;
2) callus that obtains in step 1) is placed in successively medium B and C from culture medium A and continues respectively to cultivate 25-30d, illumination every day 12-16h, light intensity 1500-1800 Lux, acquisition embryo callus subculture in 25-28 ℃; The callus that obtains from medium B in step 1) directly is placed in culture medium C continues to cultivate 25-50d, illumination every day 12-16h, light intensity 1500-1800 Lux, acquisition embryo callus subculture in 25-28 ℃.
Concrete, described culture medium A is by carbon source, MSB5 medium, IAA(indole-3-acetic acid), the KT(6-furfuryladenine), 2,4-D(2,4-dichlorphenoxyacetic acid) and coagulating agent composition; IAA concentration 0.05-0.15mg/L in culture medium A, KT concentration 0.05-0.15 mg/L, 2,4-D concentration 0.05-0.15 mg/L.
Described medium B is by carbon source, MSB5 medium, IAA, KT, 2, and 4-D and coagulating agent form; IAA concentration in medium B is 0.05-0.15mg/L, and KT concentration is 0.01-0.1 mg/L, and 2,4-D concentration is 0.01-0.1mg/L.
Described culture medium C is by carbon source, MSB5 medium, IAA, KT, 2, and 4-D and coagulating agent form; IAA concentration in culture medium C is 0.05-0.15mg/L, and KT concentration is 0.01-0.1 mg/L, and 2,4-D concentration is 0.01-0.1mg/L.
The method of described rapid induction and breeding cotton embryo callus subculture, preferably, can be with step 2) embryo callus subculture that obtains first is placed in medium D and is incubated at 25-28 ℃ and cultivates 15-25d and obtain lurid embryo callus subculture, illumination every day 12-16h, light intensity 1500-1800 Lux; If differentiating phenomenon do not occur, continue to be placed in medium D and cultivate, every 15-20d is a subculture cycle; If differentiating phenomenon occurs, be placed in medium E and continue to cultivate 15-25d, illumination every day 12-16h, light intensity 1500-1800 Lux in 25-28 ℃.
Described medium D is by carbon source, MSB5 medium, IAA, 6-BA(6-benzyl aminoadenine) and the Phytagel(plant gel, available from Solarbio company) form; IAA concentration in medium D is 0.001-0.01 mg/L, and 6-BA concentration is 0.001-0.01 mg/L, and Phytagel concentration is 2.0-3.0g/L.
Described medium E is comprised of carbon source, MSB5 medium, IAA, KT and Phytagel; IAA concentration in medium E is 0.001-0.01mg/L, and KT concentration is 0.001-0.01 mg/L, and Phytagel concentration is 2.0-3.0g/L.
Cotton used is selected from common upland cotton coker413, coker201 or coker310.
In the present invention, described carbon source is glucose, and working concentration is 20-30g/L.MSB5 medium composition used sees Table 1.
Culture medium A in the present invention, B and C all adjust the pH value to 6.0-6.5 with KOH, and in 121 ℃ of autoclaving 15min; Coagulating agent described in culture medium A, B and C is selected the agar powder (Agar Powder) of Solarbio company, and working concentration is 6.5-7.5g/L.Medium D and E all adjust the pH value to 6.4-6.8 with KOH; And in 121 ℃ of autoclaving 15min.
Cotton Hypocotyl described in the present invention preferably obtains as follows: at first the cotton seeds of lint is peelled off shell, use 0.1% HgCl
2Then sterilization 5min uses aseptic water washing 2-3 time, drains away the water.Seed is placed in a seedling medium sprouts, Cotton Hypocotyl is cut into the fragment of length 0.4-0.8cm under aseptic condition after growth 5-8d.Described seedling medium consists of: sucrose 12.5g/L, agar powder (Agar Powder) 6.0g/L and water; And will send out seedling and cultivate based on 121 ℃ of autoclaving 15min.
Compared to the prior art, the advantage of the inventive method is: in conjunction with medium of the present invention and method, can obtain fast callus and the embryo callus of cotton; Can the Fast-propagation embryo callus subculture, and can keep the kilter of cotton embryo callus subculture, can be cotton transgenic good, efficient experiment material is provided.
Description of drawings
Fig. 1 is obtained hypocotyl segment by cotton seeds after sprouting 7d on medium;
Fig. 2 is that hypocotyl is induced and cultivates the callus that tangent plane produces after 7d;
Fig. 3 is that hypocotyl is induced and cultivates after 25d formed callus group;
Fig. 4 mainly is formed at the junction of callus and medium for by inducing the embryo callus subculture that obtains;
The Cotton Embryogenic Callus of Fig. 5 for obtaining by Fast-propagation.
Embodiment
The present invention is further illustrated by the following examples, but protection scope of the present invention is not limited to this.
The method of embodiment 1. rapid induction coker413 cotton embryo callus subcultures
(1) aseptic hypocotylar acquisition
1) lint of cotton seeds: get 100 coker413 cotton seeds and carry out quick lint with 98% concentrated sulfuric acid, obtain clean lint seed;
2) hypocotylar acquisition: the coker413 cotton seeds of lint is peelled off the cotton shell and used 0.1%(w/v) HgCl
2Then sterilization 5min uses the sterile water wash seed 3 times, drains away the water.Cotton seeds after sterilization is placed in sends out seedling medium (consisting of: sucrose 12.5g/L, agar powder 6.0g/L and water) cultivation 7d, Cotton Hypocotyl is cut into the fragment (see figure 1) of length 0.5cm left and right under gnotobasis.
(2) acquisition of embryo callus subculture
1) acquisition of callus: the hypocotyl fragment of coker413 cotton aseptic seedling is inoculated in culture medium A in 26 ℃ cultivates (placing approximately 8-9 root hypocotyl fragment in each blake bottle), illumination every day 14h, light intensity 1600Lux cultivates 7d; The callus (see figure 2) can occur at hypocotylar tangent plane, continue to cultivate, 25d obtains lurid callus and rolls into a ball (see figure 3).
2) acquisition of embryo callus subculture: (color is light yellow with the callus group of above-mentioned acquisition, gloss is better, and is softer) be placed in successively medium B and C and continue respectively to cultivate 30 days in 26 ℃, illumination every day 14h, light intensity 1600Lux obtains the embryo callus subculture (see figure 4).
Culture medium A in embodiment 1 is by glucose, MSB5 medium, IAA, KT, 2, and 4-D and agar powder form; Concentration of glucose 25g/L in culture medium A, IAA concentration 0.1mg/L, KT concentration 0.1 mg/L, 2,4-D concentration is 0.1 mg/L, agar powder concentration 7.0g/L.Medium B is by glucose, MSB5 medium, IAA, KT, 2, and 4-D and agar powder form; Concentration of glucose 25g/L in medium B, IAA concentration 0.1mg/L, KT concentration 0.09mg/L, 2,4-D concentration 0.09mg/L, agar powder concentration 7.0g/L.Culture medium C is by glucose, MSB5 medium, IAA, KT, 2, and 4-D and agar powder form; Concentration of glucose 25g/L in culture medium C, IAA concentration 0.1mg/L, KT concentration 0.08 mg/L, 2,4-D concentration 0.08mg/L, agar powder concentration 7.0g/L.
The method of embodiment 2. rapid induction coker201 cotton embryo callus subcultures
(1) aseptic hypocotylar acquisition
1) lint of cotton seeds: get 100 coker201 cotton seeds and carry out quick lint with 98% concentrated sulfuric acid, obtain clean lint seed;
2) hypocotylar acquisition: the coker201 cotton seeds of lint is peelled off the cotton shell and used 0.1%(w/v) HgCl
2Then sterilization 5min uses the sterile water wash seed 3 times, drains away the water.Cotton seeds after sterilization is placed in sends out seedling medium (consisting of: sucrose 12.5g/L, agar powder 6.0g/L and water) cultivation 6d, Cotton Hypocotyl is cut into the fragment of length 0.5cm left and right under gnotobasis.
(2) acquisition of embryo callus subculture
1) acquisition of callus: the hypocotyl fragment of coker201 cotton aseptic seedling is inoculated in medium B in 26 ℃ cultivates (placing approximately 8-9 root hypocotyl fragment in each blake bottle), illumination every day is 14h, and light intensity 1600Lux cultivates 25d.
2) acquisition of embryo callus subculture: the callus (color is light yellow, and gloss is better, and is softer) of above-mentioned acquisition is placed in culture medium C in 26 ℃ of continuation cultivations 40 days, illumination every day 14h, light intensity 1600Lux obtains embryo callus subculture.The embryo callus subculture of this embodiment is induced and is better than above-described embodiment 1.
Medium B in embodiment 2 is by glucose, MSB5 medium, IAA, KT, 2, and 4-D and agar powder form; Concentration of glucose 25g/L in medium B, IAA concentration 0.1mg/L, KT concentration 0.09mg/L, 2,4-D concentration 0.09mg/L, agar powder concentration 7.0g/L.Concentration of glucose 25g/L in culture medium C, IAA concentration 0.1mg/L, KT concentration 0.08 mg/L, 2,4-D concentration 0.08mg/L, agar powder concentration 7.0g/L.
Interpretation: find cotton embryo callus subculture preparation method of the present invention, obtain embryo callus subculture short required experimental period, the shortest need 50 days, compare with existing cotton tissue culture method, the present invention has shortened to a great extent and has induced the embryo callus subculture required time, can be that the transgenosis Quick is for a large amount of donor materials.
Below in conjunction with embodiment, the method for Fast-propagation embryo callus subculture is further detailed and confirms.
Embodiment 3: the method for Fast-propagation coker413 cotton embryo callus subculture
The embryo callus subculture that embodiment 1 is obtained is placed in medium D in 26 ℃ of continuation cultivation 25d, illumination every day 14h, light intensity 1600Lux, can obtain a large amount of embryo callus (color is light yellow, and gloss is bright-coloured, graininess), a small amount of differentiating phenomenon appears simultaneously, so be placed in medium E and continue to cultivate 20d, illumination every day 14h, light intensity 1600Lux in 26 ℃.
Medium D in embodiment 3 is comprised of glucose, MSB5 medium, IAA, 6-BA and Phytagel; Concentration of glucose 27g/L in medium D, IAA concentration 0.005 mg/L, 6-BA concentration 0.003 mg/L, Phytagel concentration 2.0g/L.Medium E is comprised of glucose, MSB5 medium, IAA, KT and Phytagel; Concentration of glucose 27g/L in medium E, IAA concentration is 0.005 mg/L, KT concentration 0.003mg/L, Phytagel concentration 2.0g/L.
Embodiment 4: the method for Fast-propagation coker201 cotton embryo callus subculture
The embryo callus subculture that embodiment 2 is obtained is placed in medium D in 26 ℃ of continuation cultivation 15d, illumination every day 14h, and light intensity 1600Lux, (color is light yellow, and gloss is bright-coloured, graininess can to obtain a large amount of embryo callus; See Fig. 5); Produce without differentiating phenomenon this moment, cultivates therefore continue to be placed in medium D, and every 15-20d is a subculture cycle.
Medium D in embodiment 4 is comprised of glucose, MSB5 medium, IAA, 6-BA and Phytagel; Concentration of glucose 23g/L in medium D, IAA concentration 0.005mg/L, 6-BA concentration 0.003mg/L, Phytagel concentration 2.0g/L.
Result: by the cultivation of medium D and E, embryo callus subculture can keep good embryo callus subculture state, and can obtain more embryo callus subculture by Fast-propagation.By to the keeping of embryo callus subculture state, can utilize these embryo callus subcultures directly to carry out the genetic transformation that Agrobacterium infects to realize cotton, shortened to a great extent the time that transgene cotton obtains.
Claims (8)
1. the method for a rapid induction and breeding cotton embryo callus subculture, is characterized in that, comprises the steps:
1) hypocotyl that will cultivate the cotton aseptic seedling of 5-8d cuts into the fragment that length is 0.4-0.8cm, then be placed in culture medium A or medium B and cultivate 25-30d in 25-28 ℃, illumination every day 12-16h, light intensity 1500-1800 Lux obtains callus;
2) callus that obtains in step 1) is placed in successively medium B and C from culture medium A and continues respectively to cultivate 25-30d, illumination every day 12-16h, light intensity 1500-1800 Lux, acquisition embryo callus subculture in 25-28 ℃; The callus that obtains from medium B in step 1) directly is placed in culture medium C continues to cultivate 25-50d, illumination every day 12-16h, light intensity 1500-1800 Lux, acquisition embryo callus subculture in 25-28 ℃.
2. the method for rapid induction cotton embryo callus subculture as claimed in claim 1, is characterized in that, described culture medium A is by carbon source, MSB5 medium, IAA, KT, 2, and 4-D and coagulating agent form; IAA concentration 0.05-0.15mg/L in culture medium A, KT concentration 0.05-0.15 mg/L, 2,4-D concentration 0.05-0.15 mg/L.
3. the method for rapid induction and breeding cotton embryo callus subculture as claimed in claim 1, is characterized in that, described medium B is by carbon source, MSB5 medium, IAA, KT, 2, and 4-D and coagulating agent form; IAA concentration 0.05-0.15mg/L in medium B, KT concentration 0.01-0.10 mg/L, 2,4-D concentration 0.01-0.10 mg/L.
4. the method for rapid induction and breeding cotton embryo callus subculture as claimed in claim 1, is characterized in that, described culture medium C is by carbon source, MSB5 medium, IAA, KT, 2, and 4-D and coagulating agent form; IAA concentration 0.05-0.15mg/L in culture medium C, KT concentration 0.01-0.10 mg/L, 2,4-D concentration 0.01-0.10 mg/L.
5. the method for rapid induction and breeding cotton embryo callus subculture as claimed in claim 1, it is characterized in that, with step 2) embryo callus subculture that obtains first is placed in medium D and is incubated at 25-28 ℃ and cultivates 15-25d, illumination every day 12-16h, light intensity 1500-1800 Lux; If differentiating phenomenon do not occur, continue to be placed in medium D and cultivate, every 15-20d is a subculture cycle; If differentiating phenomenon occurs, be placed in medium E and continue to cultivate 15-25d, illumination every day 12-16h, light intensity 1500-1800 Lux in 25-28 ℃.
6. the method for rapid induction and breeding cotton embryo callus subculture as claimed in claim 5, is characterized in that, described medium D is comprised of carbon source, MSB5 medium, IAA, 6-BA and Phytagel; IAA concentration 0.001-0.01 mg/L in medium D, 6-BA concentration 0.001-0.01 mg/L, Phytagel concentration 2.0-3.0g/L.
7. the method for rapid induction and breeding cotton embryo callus subculture as claimed in claim 5, is characterized in that, described medium E is comprised of carbon source, MSB5 medium, IAA, KT and Phytagel; IAA concentration 0.001-0.01 mg/L in medium E, KT concentration 0.001-0.01 mg/L, Phytagel concentration 2.0-3.0g/L.
8. as described in as arbitrary in claim 1 to 7, the method for rapid induction and breeding cotton embryo callus subculture, is characterized in that, cotton used is selected from common upland cotton coker413, coker201 or coker310.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104004783A (en) * | 2014-05-04 | 2014-08-27 | 河北农业大学 | Method for improving cotton transgenosis efficiency |
| CN106258953A (en) * | 2016-07-18 | 2017-01-04 | 运城学院 | A kind of processing method of inducing cotton wound healing fast-growth |
| CN106305415A (en) * | 2016-07-18 | 2017-01-11 | 山西省农业科学院棉花研究所 | Treatment method for quick induced proliferation of cotton calli |
| CN108271692A (en) * | 2018-02-11 | 2018-07-13 | 中国农业科学院棉花研究所 | Cotton explant is directly divided into the method and culture medium of embryo callus |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104004783A (en) * | 2014-05-04 | 2014-08-27 | 河北农业大学 | Method for improving cotton transgenosis efficiency |
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| CN106258953A (en) * | 2016-07-18 | 2017-01-04 | 运城学院 | A kind of processing method of inducing cotton wound healing fast-growth |
| CN106305415A (en) * | 2016-07-18 | 2017-01-11 | 山西省农业科学院棉花研究所 | Treatment method for quick induced proliferation of cotton calli |
| CN106305415B (en) * | 2016-07-18 | 2019-09-03 | 山西省农业科学院棉花研究所 | A kind of processing method of cotton callus rapid induction proliferation |
| CN108271692A (en) * | 2018-02-11 | 2018-07-13 | 中国农业科学院棉花研究所 | Cotton explant is directly divided into the method and culture medium of embryo callus |
| CN108271692B (en) * | 2018-02-11 | 2021-06-01 | 中国农业科学院棉花研究所 | Method for directly differentiating cotton explant into embryogenic callus and culture medium |
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Application publication date: 20130529 |