CN101218893B - Method for evoking sorghum mature embryo callus and establishing regeneration system - Google Patents
Method for evoking sorghum mature embryo callus and establishing regeneration system Download PDFInfo
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- CN101218893B CN101218893B CN2007100605644A CN200710060564A CN101218893B CN 101218893 B CN101218893 B CN 101218893B CN 2007100605644 A CN2007100605644 A CN 2007100605644A CN 200710060564 A CN200710060564 A CN 200710060564A CN 101218893 B CN101218893 B CN 101218893B
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Abstract
The invention relates to a method for building a callus from mature embryo inducing and regeneration system and the steps are as follows:(1) inducing culture for the callus from mature embryo; husking the seeds and dipping in the alcohol of 75 percent for 0.5 to 1min, in the sodium hypochlorite of 5 to 20 percent to sterilize for 20 to 50min, in the mercuric chloride of 0.1 percent to sterilize for 10 to 20min and then in the aseptic water to wash for 4 to 5 times and being inoculated into an inducing medium after air drying; illuminating for 8 to 12h, and being in dark for 12 to 16h with a temperature of 22 to 30 DEG C; (2) subculturing for the callus; illuminating for 8 to 12h, and being in dark for 12 to 16h with a temperature of 22 to 30 DEG C; (3) differentiation culture; using the massive compact callus obtained through subculture to induce the forming of an embryoid and seedling differentiation in a differentiation medium with a light intensity of 3000 to 4000Lx for each day and illuminating for 10 to16h with a relative humidity of 50 to 70 percent;(4) rooting culture; transplanting the regenerating plants growing to 5 to 8cm and differentiating from the differentiation medium in a rooting medium to carry out rooting; (5) transplanting of the regenerating plant. The method establishes an excellent foundation for utilizing the gene technology to breed.
Description
Technical field
The present invention relates to a kind of method of biotechnology breeding, the method that particularly a kind of evoking sorghum mature embryo callus and regenerating system are set up.
Background technology
The Chinese sorghum tissue is cultivated and regenerated, and its purpose is to allow explant induce callus to high-frequency, sets up somatic cell regenerating system efficiently, for Chinese sorghum genetic transformation and cell engineering research create favorable conditions.Yet Chinese sorghum is that generally acknowledge at present the most difficult set up plant and do not have sex organization or neomorph system and import one of crop of foreign gene.Though the research work of Chinese sorghum heredityization just began before 15 years, but having the cereal crop of important economic worth such as paddy rice, wheat, corn etc. with other compares, set up because high frequency regeneration and efficient transformation system are difficult, therefore its Study on Genetic Transformation job development is slower, and successful examples is also less.Chinese sorghum is compared with other crops, and it is more difficult relatively to obtain regeneration plant by tissue culture.That carry out the Chinese sorghum Study on tissue culture the earliest is (author's names: Strogonov such as nineteen sixty-eight Strogonov B.P, B.P., E.I.Kownzerxo, R.G.Butenko. title of article: Culturing of isolated glasswort, sorghum, sweetclover, and cabbage tissues for comparative study of their salt resistance. periodical title: Soviet Plant Physiol. delivers age and volume phase: 1968.15: the page number: 173～177) utilize Radix Sorghum vulgare Pers and tillering node additional 1mg/L 2 on medium, 4-D, 1mg/L KT, the 5mg/L calcium pantothenate, the 1mg/L ascorbic acid, induce callus, but he does not obtain regeneration plant.After this, the work of Chinese sorghum Study on tissue culture is necessarily paid attention to.The various countries scholar has carried out the work of Chinese sorghum Study on tissue culture in succession, and used explant mainly contains immature embryo, mature embryo, young fringe, stem apex, seed, spire, flower pesticide, seedling etc.At mature embryo tissue culture fermentation, though the relevant report that obtains regeneration plant is also arranged, shoot regeneration frequency is all not high.(author's names: Bhskaran et al. title of article: Control of morphogenesis in sorghum by 2 such as nineteen eighty-three Bhskaran, 4-Dichlorophenoxyacetic acid and cytokinins. periodical title: Plant Physiology, Supplement, deliver age and volume phase: 1983,72: the page number: 142～143) reported first the inducing culture of sorghum mature embryo callus, carried out the research of salt tolerant and anti-aluminium screening and culturing simultaneously, and obtained regeneration plant, but all medium and condition of culture are not done further introduction.(author's names: Tishu Cai such as Tishu Cai in 1987, Barbara Daly, Larry Butler. title of article: Callus induction and plant regeneration from shoot portions of mature embryos of high tannin sorghum. periodical title: Plant Cell.Tissue and Organ Culture, deliver age and volume phase: 1987,9: the page number: 245～252) in " Plant Cell; Tissue and Organ Culture " periodical, reported that the bud-end with sorghum mature embryo is an explant, on the MS medium, induce yellow, granular callus, and on the MS medium, differentiating regeneration plant, regeneration rate also only is 11%～48%.(author's names: Bhat S such as nineteen ninety-five BhatS, Kuruvinashetti M S. title of article: Plant regeneration from tissue cultures of cytoplasmic genetic male-sterility maintainer Lines of sorghum (Sorghum bicolor). periodical title: India Journal of Agricultural Sciences, deliver age and volume phase: 1995,65,20: the page number: 127～129) reported the inducing culture research of Chinese sorghum different explants at periodical " India Journal of Agricultural Sciences ", what wherein mature embryo was cultivated lures the rate of healing also lower, and regeneration is not further introduced.Domestic is that the research report of explant is less relatively with the mature embryo, on domestic several publications sorghum mature embryo is had simple report as explant, but callus of induce rate, differentiation rate are all lower.
Summary of the invention
The technical solution adopted in the present invention is for achieving the above object: the method that a kind of evoking sorghum mature embryo callus and regenerating system are set up is characterized in that implementation step is as follows:
The method that a kind of evoking sorghum mature embryo callus and regenerating system are set up is characterized in that implementation step is as follows:
(1) being carried out to the cooked flake callus induction cultivates
Seed is stripped out from clever shell, select no worm-eaten, seed flawless, complete, big or small uniformity, on superclean bench, use 75% alcohol-pickled 0.5-1min successively, 5-20% clorox sterilization 20-50min, 0.1% mercuric chloride sterilization 10-20min, use aseptic water washing 4-5 time again, blot the moisture of the surface of the seed then with aseptic filter paper, be inoculated in the inducing culture after drying; The callus induction minimal medium is MS, and adding exogenous hormone is 2,4-D and KT; 2, the concentration of 4-D is 1.5-4.0mg/L, and the concentration of KT is 0.0-2.0mg/L, and adds the AgNO of 0-30mg/L in inducing culture
3PH is 5.8-6.0; Inductive condition is: illumination 8-12h, dark 12-16h, temperature 22-30 ℃;
(2) carry out the callus successive transfer culture
Getting the initial faint yellow graininess callus of inducing is seeded in and carries out successive transfer culture in the subculture medium; The successive transfer culture minimal medium is MS, and adding exogenous hormone is 2,4-D, and 2, the concentration of 4-D is 1.0-4.0mg/L, pH is 5.8-6.0; Illumination 8-12h, dark 12-16h, temperature 22-30 ℃;
(3) differentiation culture
Will be through the formation and the seedling differentiation of the bulk that obtains after the successive transfer culture, fine and close callus inducing embryoid body in differential medium; The differential medium minimal medium is MS, and the interpolation exogenous hormone is IAA, 6-BA and KT; The concentration of IAA is 0.5-4.0mg/L, and the concentration of 6-BA is 0.5-2.0mg/L, and the concentration of KT is 0.5-2.0mg/L; Condition of culture is every day light intensity 3000-4000Lx, illumination 10-16h, and relative moisture is 50-70%;
(4) culture of rootage
The regeneration plant length that will differentiate from differential medium moves into root media during to 5-8cm and takes root; The root media minimal medium is MS, and the interpolation exogenous hormone is IAA, 6-BA; The concentration of IAA is 1.0-4.0mg/L, and the concentration of 6-BA is 0.5-2.0mg/L; Condition of culture is every day light intensity 3000-4000Lx, illumination 10-16h, and relative moisture is 50-70%;
(5) transplanting of regeneration plant
When treating that 2-3 week root is long slightly, long close, removed the decap hardening 5-7 days, take out then, clean the residual medium of root, be transplanted to and carry out the transition cultivation in the sterile soil, and increase intensity of illumination gradually; After the transplanting preceding 3 days are watered once permeablely every day, and later soil conservation certain humidity prevents that seedling from rotting root; Temperature keeps 22-28 ℃ to be suitable for the cauline leaf growth daytime, maintains 16-21 ℃ night, helps the growth of root system; 1-2 is after week, and plant grows young leaves, means transplant survival; Seedling is transplanted in the greenhouse after growing two young leaves again.
The invention has the beneficial effects as follows: utilize this method that the inbred line kind of seed selection is experimentized, obtained 58 Chinese sorghum tissue cultivating seedling.The highest callus induction rate of example 1 mature embryo wherein of the present invention is 48.3%, and the highest seedling differentiation rate is 80.0%; The highest callus induction rate of example 2 mature embryos of the present invention is 45.8%, and the highest seedling differentiation rate is 38.6%; The highest callus induction rate of example 3 mature embryos of the present invention is 32.5%, and the highest seedling differentiation rate is 66.7%.
The present invention cultivates the Chinese sorghum tissue and regenerates, make explant can induce to high-frequency callus, set up somatic cell regenerating system efficiently,, have laid a good foundation for utilizing the transgenic technology breeding for Chinese sorghum genetic transformation and cell engineering research create favorable conditions.This method implementation step is simple, and implementation condition is loose, the effect highly significant.
Description of drawings
Fig. 1 is the callus figure that example 1 of the present invention forms in callus inducing medium; Formed faint yellow, the block callus of inducing culture of example 1 mature embryo process 25-30d on callus inducing medium.
Fig. 2 is the callus figure that example 2 of the present invention forms in callus inducing medium; Formed faint yellow, the block callus of inducing culture of example 2 mature embryos process 25-30d on callus inducing medium.
Fig. 3 is the II type embryo callus figure that the callus of example 1 of the present invention forms in subculture medium; Example 1 faint yellow, granular II type callus occurs through the callus surface of successive transfer culture, occurs green bud point sometimes.
Fig. 4 is the II type embryo callus figure that example 3 callus of the present invention form in subculture medium; Example 3 faint yellow, granular II type callus occurs through the callus surface of successive transfer culture.
Fig. 5 is the example 1 of the present invention figure of taking root in root media; Example 1 differentiation seedling is taken root easily, and the growing way of differentiation seedling is better.
Fig. 6 is the differentiation seedling figure that example 1 of the present invention forms in differential medium; Differentiation seedling after example 1 is taken root goes to nutrition soil and cultivates, and grows new blade.
Fig. 7 is the tissue cultivating seedling of the example 3 of the present invention figure of growing in nutrition soil; Differentiation seedling after example 3 is taken root goes to nutrition soil and cultivates, and grows new blade.
Fig. 8 be the tissue cultivating seedling of example 1 of the present invention forward in the greenhouse cultivate, the pollination figure of normally blooming.
Embodiment
Below in conjunction with accompanying drawing and preferred embodiment, to details are as follows according to concrete implementation step provided by the invention:
Referring to Fig. 1-Fig. 8, the method that a kind of evoking sorghum mature embryo callus and regenerating system are set up is characterized in that implementation step is as follows:
(1) being carried out to the cooked flake callus induction cultivates
Seed is stripped out from clever shell, select no worm-eaten, seed flawless, complete, big or small uniformity, on superclean bench, use 75% alcohol-pickled 0.5-1min successively, 5-20% clorox sterilization 20-50min, 0.1% mercuric chloride sterilization 10-20min, use aseptic water washing 4-5 time again, blot the moisture of the surface of the seed then with aseptic filter paper, be inoculated in the inducing culture after drying; The callus induction minimal medium is MS, and adding exogenous hormone is 2,4-D and KT; 2, the concentration of 4-D is 1.5-4.0mg/L, and the concentration of KT is 0.0-2.0mg/L, and adds the AgNO of 0-30mg/L in inducing culture
3PH is 5.8-6.0; Inductive condition is: illumination 8-12h, dark 12-16h, temperature 22-30 ℃;
(2) carry out the callus successive transfer culture
Getting the initial faint yellow graininess callus of inducing is seeded in and carries out successive transfer culture in the subculture medium; The successive transfer culture minimal medium is MS, and adding exogenous hormone is 2,4-D, and 2, the concentration of 4-D is 1.0-4.0mg/L, pH is 5.8-6.0; Illumination 8-12h, dark 12-16h, temperature 22-30 ℃;
(3) differentiation culture
Will be through the formation and the seedling differentiation of the bulk that obtains after the successive transfer culture, fine and close callus inducing embryoid body in differential medium; The differential medium minimal medium is MS, and the interpolation exogenous hormone is IAA, 6-BA and KT; The concentration of IAA is 0.5-4.0mg/L, and the concentration of 6-BA is 0.5-2.0mg/L, and the concentration of KT is 0.5-2.0mg/L; Condition of culture is every day light intensity 3000-4000Lx, illumination 10-16h, and relative moisture is 50-70%;
(4) culture of rootage
The regeneration plant length that will differentiate from differential medium moves into root media during to 5-8cm and takes root; The root media minimal medium is MS, and the interpolation exogenous hormone is IAA, 6-BA; The concentration of IAA is 1.0-4.0mg/L, and the concentration of 6-BA is 0.5-2.0mg/L; Condition of culture is every day light intensity 3000-4000Lx, illumination 10-16h, and relative moisture is 50-70%;
(5) transplanting of regeneration plant
The seedling root cultivated of approximately taking root in 2-3 week just can remove decap hardening 5-7d when long thick, long close, takes out then, and the careful residual medium of root of cleaning moves into and carries out the transition cultivation in the nutritive cube that vermiculite is housed, and increases intensity of illumination gradually.Preceding 3d after the transplanting waters once permeablely every day, and later soil conservation certain humidity prevents that seedling from rotting root.Temperature keeps 22-28 ℃ to be suitable for the cauline leaf growth daytime, maintains 16-21 ℃ night, helps the growth of root system.1-2 is after week, and plant grows young leaves, means transplant survival.Seedling is transplanted in the greenhouse after growing two young leaves again.
Above-mentioned detailed description of the method for evoking sorghum mature embryo callus and regenerating system foundation being carried out with reference to embodiment; be illustrative rather than determinate; therefore in the variation and the modification that do not break away under the general plotting of the present invention, should belong within protection scope of the present invention.
Claims (1)
1. the method set up of evoking sorghum mature embryo callus and regenerating system is characterized in that implementation step is as follows:
(1) being carried out to the cooked flake callus induction cultivates
Seed is stripped out from clever shell, select no worm-eaten, seed flawless, complete, big or small uniformity, on superclean bench, use 75% alcohol-pickled 0.5-1min successively, 5-20% clorox sterilization 20-50min, 0.1% mercuric chloride sterilization 10-20min, use aseptic water washing 4-5 time again, blot the moisture of the surface of the seed then with aseptic filter paper, be inoculated in the inducing culture after drying; The callus induction minimal medium is MS, and adding exogenous hormone is 2,4-D and KT; 2, the concentration of 4-D is 1.5-4.0mg/L, and the concentration of KT is 0.0-2.0mg/L, and adds the AgNO of 0-30mg/L in inducing culture
3PH is 5.8-6.0; Inductive condition is: illumination 8-12h, dark 12-16h, temperature 22-30 ℃;
(2) carry out the callus successive transfer culture
Getting the initial faint yellow graininess callus of inducing is seeded in and carries out successive transfer culture in the subculture medium; The successive transfer culture minimal medium is MS, and adding exogenous hormone is 2,4-D, and 2, the concentration of 4-D is 1.0-4.0mg/L, pH is 5.8-6.0; Illumination 8-12h, dark 12-16h, temperature 22-30 ℃;
(3) differentiation culture
Will be through the formation and the seedling differentiation of the bulk that obtains after the successive transfer culture, fine and close callus inducing embryoid body in differential medium; The differential medium minimal medium is MS, and the interpolation exogenous hormone is IAA, 6-BA and KT; The concentration of IAA is 0.5-4.0mg/L, and the concentration of 6-BA is 0.5-2.0mg/L, and the concentration of KT is 0.5-2.0mg/L; Condition of culture is every day light intensity 3000-4000Lx, illumination 10-16h, and relative moisture is 50-70%;
(4) culture of rootage
The regeneration plant length that will differentiate from differential medium moves into root media during to 5-8cm and takes root; The root media minimal medium is MS, and the interpolation exogenous hormone is IAA, 6-BA; The concentration of IAA is 1.0-4.0mg/L, and the concentration of 6-BA is 0.5-2.0mg/L; Condition of culture is every day light intensity 3000-4000Lx, illumination 10-16h, and relative moisture is 50-70%;
(5) transplanting of regeneration plant
When treating that 2-3 week root is long slightly, long close, removed the decap hardening 5-7 days, take out then, clean the residual medium of root, be transplanted to and carry out the transition cultivation in the sterile soil, and increase intensity of illumination gradually; After the transplanting preceding 3 days are watered once permeablely every day, and later soil conservation certain humidity prevents that seedling from rotting root; Temperature keeps 22-28 ℃ to be suitable for the cauline leaf growth daytime, maintains 16-21 ℃ night, helps the growth of root system; 1-2 is after week, and plant grows young leaves, means transplant survival; Seedling is transplanted in the greenhouse after growing two young leaves again.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2010011175A1 (en) * | 2008-07-23 | 2010-01-28 | Temasek Life Sciences Laboratory Limited | Method of high frequency regeneration of sorghum |
| CN109609545A (en) * | 2019-01-24 | 2019-04-12 | 安徽科技学院 | Sorghum mature seed transgenic method |
| CN110699379B (en) * | 2019-12-02 | 2023-09-29 | 山西省农业科学院生物技术研究中心 | Agrobacterium-mediated genetic transformation method for inducing callus by taking sorghum mature embryo as explant |
| CN114467760B (en) * | 2022-04-15 | 2022-07-26 | 三亚南京农业大学研究院 | Water lily mature embryo callus induction and proliferation method |
| CN116584391A (en) * | 2023-06-09 | 2023-08-15 | 辽宁省农业科学院 | Induction method of mature sorghum seed somatic embryo |
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Non-Patent Citations (3)
| Title |
|---|
| 刘明等.诱导突变高粱愈伤组织初探.《生物技术通报》.2004,(第4期),47-52. * |
| 张明洲等.抗生素对高粱愈伤组织诱导和生长的影响.《浙江大学学报(农业与生命科学版)》.2004,第30卷(第21期),221-225. * |
| 郭建华.高粱幼胚小盾片愈伤组织的诱导及其再生植株性状的变异分析.《辽宁农业科学》.1989,(第3期),7-13. * |
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