CN1293800C - Method for breeding somatic embryo of immature soybean cotyledon, preserving succeed generation and revegetating plant - Google Patents
Method for breeding somatic embryo of immature soybean cotyledon, preserving succeed generation and revegetating plant Download PDFInfo
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- CN1293800C CN1293800C CNB021445788A CN02144578A CN1293800C CN 1293800 C CN1293800 C CN 1293800C CN B021445788 A CNB021445788 A CN B021445788A CN 02144578 A CN02144578 A CN 02144578A CN 1293800 C CN1293800 C CN 1293800C
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Abstract
The present invention relates to a method for somatic embryo proliferation, successive transfer preservation and plant regeneration of immaturity cotyledons of soybeans, which belongs to the technical field of a farm crop tissue culture method. The method is performed by the steps of soybean somatic embryogenesis inducement, soybean somatic embryo proliferation and successive transfer preservation, soybean somatic embryo germination and plant regeneration. In the present invention, the somatic embryogenesis is induced by cultivating immaturity cotyledons as explants, and the somatic embryo proliferation is induced on a solid culture medium; meanwhile, the successive transfer preservation is carried out on a solid culture medium, proliferated and preserved somatic embryos can be germinated normally, and plants are further regenerated. The method solves the problem of plant regeneration inducement after proliferation and successive transfer preservation of the soybean somatic embryos for a long term on the solid culture medium.
Description
Technical field:
The present invention relates to crops method for tissue culture technical field, is that a kind of soybean unmature subleaf somatic embryo propagation, subculture are preserved and plant regeneration method.
Background technology:
Soybean is important economic crops, is the main source of human edible oil and vegetable protein.The yield and quality that has a strong impact on soybean that in soybean produces, usually does harm to because of sick grass-and-insect painting.Breeding method with routine improves soybean varieties, because of the deficient general improved effect in anti-source not good.
Along with the development of molecular biology and molecular genetics, utilizing transgenic technology to change in the plant foreign gene over to improvement its some bad proterties has become one of important method of modern molecular breeding.Tissue culture technique is the basis of transgenosis work, takes place and plant regeneration succeed (Christianon, 1983 through tissue culture inductor cell stage although be explant with the immature embryo of the cultivated soybean and unmature subleaf; Lazzeri, 1985; Barwale, 1986), but the tissue culture of soybean can not resemble other crops, behind formation embryo callus subcultures such as wheat, paddy rice, corn, repeatedly shoot proliferation and preservation, and induce differentiation and regeneration plant, and be considered to the crop that is difficult to cultivate always, limited that soyabean tissue cultivates and the application of transgenic technology.
Finer and Nagasawa (1988) have reported the system that breed with suspension culture the living back of somatic embryos of soybean fetal hair, the surface of spherical blast constantly produces the spherical blast of time one-level in the liquid medium within, forms the cells,primordial group suspension culture of being made up of globular embryo.Go in the new liquid nutrient medium after cells,primordial group suitably cut apart, can continue propagation, form new cells,primordial group.Somatic embryo change over to again on the solid MS medium can further grow, ripe, sprout, form whole plant.This system has improved the tissue culture efficient of soybean greatly, but liquid suspension is cultivated equipment such as needing the constant temperature shaking table, operating technology is numerous and diverse, be difficult to grasp, only in indivedual the cultivated soybean kinds such as Fayette, obtain cells,primordial group, and only have the Individual testwas chamber to use this system (Bailey, 1993), be not used widely as yet.
Summary of the invention:
The technical problem to be solved in the present invention is to disclose a kind of unmature subleaf somatic embryo propagation, subculture simple to operate, that be suitable for producing main most of soybean varieties of planting to preserve and plant regeneration method.
The scheme of technical solution problem of the present invention is finished by following steps:
What one, the somatic embryos of soybean fetal hair was given birth to induces:
With the additional 10-40mg/L 2 of MS medium, 4-D (pH=5.8-6.2) inducing soybean unmature subleaf somatic embryo under 25 ± 1 ℃ of dark conditions takes place, and cultivates 30-60 days visible volume cell stages and takes place.
Two, the propagation of somatic embryos of soybean and subculture are preserved:
In the somatic embryo that produces, do not select and eaten by the Annexation of callus institute on every side, diameter is at the yellow green of 0.9-1.1 millimeter, globular embryo fine granularity, cluster, switching is at the additional 10-40mg/L 2 of MS medium, 4-D (pH=5.8-6.2) is inductor blast propagation under 16 hours conditions of 25 ± 1 ℃ of low light levels, changed once fresh medium every 12-16 days, continue to select globular embryo yellow green, fine granularity, cluster and cultivate, remove callus and large granular spherical embryo.Can directly sprout after the somatic embryo propagation, also can preserve by subculture, when the needs subculture is preserved, the globular embryo of cluster is connected to the additional 10-40mg/L2 of MS medium, 4-D (pH=5.8-6.2) cultivated under the condition at 20 ± 1 ℃ of low light levels in 16 hours, 15-30 days subcultures once, subculture repeatedly induces globular embryo to sprout when needs make its regeneration plant.
Three, the sprouting of somatic embryos of soybean and regeneration plant:
Somatic embryo is transferred in the MS medium (active carbon, 5-10% sucrose, the pH=5.8-6.2 that add 0.5-1%) 25 ± 1 ℃ of illumination to be cultivated under the condition in 16 hours, 15-20 days subcultures once, somatic embryo was sprouted in 30-60 days, the somatic embryo of sprouting changed in 1/2MS or MS (2 times molysite, the pH=7.0) medium again and cultivated under the condition in 23 hours 25 ± 1 ℃ of illumination, through 25-35 days regeneration plants.
The present invention is to be that the explant induction somatic embryo takes place with the cultivated soybean unmature subleaf, inductor blast propagation on solid culture medium, simultaneously, subculture is preserved on solid culture medium, propagation can normally be sprouted with the somatic embryo of preserving, further regeneration plant, solved that somatic embryos of soybean breed on solid culture medium and the long-term subculture preservation after induce the plant regeneration problem.Induced 16 kinds in 30 soybean varieties of utilization this method establishing in large scale on producing and produced the spherical blast that to breed with the long-term subculture preservation, accounted for and induced 53.33% of kind.Method of operating is simple and easy to grasp, and has saved equipment such as constant temperature shaking table, and production cost is low.
Embodiment:
Embodiment 1:
Close rich 25 soybean varieties somatic embryos generation, globular embryo propagation, subculture preservation and plant regeneration
It is rich 25 that field planting closes, and wins the 16-20 days soybean children pods of blooming through 75% ethanol disinfection 2 minutes, 0.1%HgCl
2Sterilized 15 minutes, sterile water is towards Xian 3 times, strip off kind skin, choose the 3-5mm cotyledon and remove plumular axis, with the cotyledon adaxial and its surface upwards be inoculated in the MS medium (additional 20mg/L 2,4-D, pH=6.0) in, cultivate under 25 ℃ of dark conditions, incidence rate of somatic embryo in the time of 42 days (somatic embryo generation cotyledon number/inoculation cotyledon number * 100%) is 41.36%.
In somatic embryo, do not select and eaten by the Annexation of callus institute on every side, diameter is at the yellow green of 0.9-1.1 millimeter, globular embryo fine granularity, cluster, switching is at MS medium (additional 20mg/L 2,4-D) under 16 hours conditions of 25 ℃ of low light levels (200Lux) inductor blast propagation, changed a subculture in 15 days, induced the globular embryo of propagation through 60 days, inducing percentage (cotyledon number/somatic embryo generation cotyledon number * 100% that fertile globular embryo takes place) is 18.40%.
Fertile globular embryo the MS medium (additional 20mg/L 2,4-D, pH=6.0) under 16 hours conditions of 20 ℃ of low light levels (200Lux) subculture preserve, 20 days subcultures once, subculture was preserved 19 months.
During inducing soybean somatic embryo regeneration plant, somatic embryo is transferred in the germination medium of active carbon (10% sucrose, pH=6.0) of MS minimal medium additional 0.5% 25 ℃ of illumination (3200Lux) cultivated under the condition in 16 hours, changed once fresh medium in per 18 days, somatic cell was sprouted in 45 days, and germination rate (sprout embryo number/quilt and induce embryo number * 100% of sprouting) is 30%.Change over to after somatic embryo is sprouted in MS (2 times molysite, the pH=7.0) medium and cultivated under the condition in 23 hours in 25 ℃ of illumination (3200Lux), changed once fresh medium in per 15 days, 30 days plant regenerations, regeneration rate (regeneration plant number/induced embryo number * 100% of sprouting) is 60%.
Embodiment 2:
The agricultural 40 soybean varieties somatic embryos in east take place, globular embryo is bred, subculture is preserved and plant regeneration
Field planting east farming 40 is won the 16-20 days soybean children pods of blooming through 75% ethanol disinfection 2 minutes, 0.1%HgCl
2Sterilized 12 minutes, sterile water is towards Xian 3 times, strip off kind skin, choose the 3-5mm cotyledon and remove plumular axis, with the cotyledon adaxial and its surface upwards be inoculated in the MS medium (additional 40mg/L 2,4-D, pH=6.0) in, cultivate under 25 ℃ of dark conditions, incidence rate of somatic embryo in the time of 50 days (somatic embryo generation cotyledon number/inoculation cotyledon number * 100%) is 40.34%.
In somatic embryo, do not select and eaten by the Annexation of callus institute on every side, diameter is at the yellow green of 0.9-1.1 millimeter, globular embryo fine granularity, cluster, switching is at MS medium (additional 20mg/L 2,4-D) under 16 hours conditions of 25 ℃ of low light levels (200Lux) inductor blast propagation, changed a subculture in 15 days, induced the globular embryo of propagation through 50 days, inducing percentage (cotyledon number/somatic embryo generation cotyledon number * 100% that fertile globular embryo takes place) is 16.50%.
Fertile globular embryo the MS medium (additional 20mg/L 2,4-D, pH=6.0) under 16 hours conditions of 20 ℃ of low light levels (200Lux) subculture preserve, 20 days subcultures once, subculture was preserved 13 months.
During inducing soybean somatic embryo regeneration plant, somatic embryo is transferred in the germination medium of active carbon (10% sucrose, pH=6.0) of MS minimal medium additional 0.5% 25 ℃ of illumination (3200Lux) cultivated under the condition in 16 hours, changed once fresh medium in per 15 days, somatic cell was sprouted in 45 days, and germination rate (sprout embryo number/quilt and induce embryo number * 100% of sprouting) is 20%.Change over to after somatic embryo is sprouted in MS (2 times molysite, the pH=7.0) medium and cultivated under the condition in 23 hours in 25 ℃ of illumination (3200Lux), changed once fresh medium in per 15 days, 30 days plant regenerations, regeneration rate (regeneration plant number/induced embryo number * 100% of sprouting) is 45%.
Table 1 part soybean varieties incidence rate of somatic embryo with can breed the globular embryo inductivity
| Sequence number | Soybean varieties | Incidence rate of somatic embryo (%) | Can breed globular embryo inductivity (%) |
| 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 | Black farming 35 black farmings 40 are closed rich 25 and are closed rich 39 east farmings agricultural 40567 Jilin 26 Jilin, 35 Jilin 36 Jilin, the 38 lucky farmings 9 in 40 eastern agricultural L13 east farming 163 east farmings 434 east farmings 1168 east farmings, 32310 east and open and educate 10 and open and educate 11 iron rich 29 interior beans No. 4 | 25.51 57.14 41.36 55.87 40.34 42.71 0.29 3.15 77.62 32.47 0.00 2.50 9.43 16.54 58.70 23.27 35.47 2.36 18.00 32.72 | 10.2 15.0 18.4 15.6 16.5 22.1 0.0 0.0 18.3 6.5 0.0 0.0 0.0 16.8 0.0 0.0 0.0 5.2 0.0 0.0 |
Claims (2)
1, a kind of soybean unmature subleaf somatic embryo propagation, subculture are preserved and plant regeneration method, it is characterized in that: may further comprise the steps:
What (1), the somatic embryos of soybean fetal hair was given birth to induces:
With the additional 10-40mg/L 2 of MS medium, 4-D, its pH=5.8-6.2, inducing soybean unmature subleaf somatic embryo takes place under 25 ± 1 ℃ of dark conditions, cultivates 30-60 days visible volume cell stages and takes place;
(2), the propagation of somatic embryos of soybean and subculture are preserved:
In the somatic embryo that produces, do not select and eaten by the Annexation of callus institute on every side, diameter is at the yellow green of 0.9-1.1 millimeter, globular embryo fine granularity, cluster, switching is at the additional 10-40mg/L 2 of MS medium, 4-D, its pH=5.8-6.2, inductor blast propagation was changed once fresh medium every 12-16 days under 16 hours conditions of 25 ± 1 ℃ of low light levels, continue to select globular embryo yellow green, fine granularity, cluster and cultivate, remove callus and large granular spherical embryo;
(3), the sprouting of somatic embryos of soybean and regeneration plant:
Somatic embryo is transferred in the MS medium, and active carbon, the 5-10% sucrose of adding 0.5-1%, pH=5.8-6.2,25 ± 1 ℃ of illumination were cultivated under the condition in 16 hours, 15-20 days subcultures once, 30-60 days somatic embryos are sprouted, and the somatic embryo of sprouting changed in the MS medium of 2 times of molysite, pH=7.0 again and cultivated under the condition in 23 hours 25 ± 1 ℃ of illumination, through 25-35 days regeneration plants.
2, soybean unmature subleaf somatic embryo propagation according to claim 1, subculture are preserved and plant regeneration method, it is characterized in that: when subculture is preserved, the globular embryo of cluster is connected to the additional 10-40mg/L 2 of MS medium, 4-D, its pH=5.8-6.2 cultivated under the condition at 20 ± 1 ℃ of low light levels in 16 hours, and 15-30 days subcultures once, repeatedly subculture induces globular embryo to sprout when needs make its regeneration plant.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB021445788A CN1293800C (en) | 2002-11-05 | 2002-11-05 | Method for breeding somatic embryo of immature soybean cotyledon, preserving succeed generation and revegetating plant |
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| CNB021445788A CN1293800C (en) | 2002-11-05 | 2002-11-05 | Method for breeding somatic embryo of immature soybean cotyledon, preserving succeed generation and revegetating plant |
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| CN1415191A CN1415191A (en) | 2003-05-07 |
| CN1293800C true CN1293800C (en) | 2007-01-10 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100559933C (en) * | 2003-09-30 | 2009-11-18 | 中国农业大学 | A kind of method that obtains a large amount of Festuca Arundinacea regeneration plants by tissue culture |
| CN103168684B (en) * | 2012-12-18 | 2014-07-09 | 新疆农业大学 | Tissue culture intermediate propagation method of chickpeas |
| CN107182786A (en) * | 2017-06-09 | 2017-09-22 | 吉林省农业科学院 | The cultural method with succeeding preservation is continued to multiply for somatic embryos of soybean shape body |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US584877A (en) * | 1897-06-22 | Mawuke spreadee | ||
| CN87106206A (en) * | 1986-08-04 | 1988-07-27 | 卢布瑞遗传公司 | The sex change of Glycine species, the method that the body embryo is taken place and whole plant is regenerated |
| US5569834A (en) * | 1988-07-22 | 1996-10-29 | Monsanto Company | Method for soybean transformation and regeneration |
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2002
- 2002-11-05 CN CNB021445788A patent/CN1293800C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US584877A (en) * | 1897-06-22 | Mawuke spreadee | ||
| CN87106206A (en) * | 1986-08-04 | 1988-07-27 | 卢布瑞遗传公司 | The sex change of Glycine species, the method that the body embryo is taken place and whole plant is regenerated |
| US5569834A (en) * | 1988-07-22 | 1996-10-29 | Monsanto Company | Method for soybean transformation and regeneration |
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