CN102577980A - Germination and seedling method for somatic embryos of colored cotton - Google Patents
Germination and seedling method for somatic embryos of colored cotton Download PDFInfo
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- CN102577980A CN102577980A CN2012100712933A CN201210071293A CN102577980A CN 102577980 A CN102577980 A CN 102577980A CN 2012100712933 A CN2012100712933 A CN 2012100712933A CN 201210071293 A CN201210071293 A CN 201210071293A CN 102577980 A CN102577980 A CN 102577980A
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Abstract
The invention discloses a germination and seedling method for somatic embryos of colored cotton, which includes the following steps: (1), subculturing an embryo callus obtained from culturing colored cotton hypocotyls for one to two times through an enrichment medium; (2), transplanting the obtained embryo from step (1) to a somatic embryo inducing medium, performing subculture for two to three times, and obtaining a mature cotyledon embryo; (3) transplanting the cotyledon embryo obtained from step (2) into a seedling medium, performing subculture for one time each 20 to 25 days, and obtaining a normal rooting differentiated seedling. According to the germination and seedling method for somatic embryos of colored cotton, the conversion rate from the embryo callus to a somatic embryo reaches 95 %, wherein the differentiated rate of the cotyledon embryo reaches 70%, the differentiated rate of the cotyledon embryo at the developmental stage of the somatic embryo is effectively improved, the problems that the colored cotton has a somatic embryogenesis and is difficult to be seedlings are solved and the technological support for the study and application of the colored cotton transgenic is provided.
Description
Technical field
The present invention relates to the sprouting and the seedling establishment method of plant tissue, be specifically related to the sprouting and the seedling establishment method of color cotton somatic cell embryoid.
Background technology
Development along with gene engineering and Protocols in Molecular Biology; Being the basis with the conventional breeding method, is the GENERALIZATION OF MODERN BREEDING TECHNIQUE system of core technology with the transgenic technology, can the external source desirable genes be imported cotton cells; And make its orientation and stable heredity; Binding molecule marker assisted selection technology can be accelerated the breeding progress, improves efficiency of selection.Cotton tissue is cultivated the development of theory and technology, for the expression and the regulation and control of the mechanism of differentiation of research somatic cell and morphogenesis, cytogene and carry out genetic manipulation and have great importance.Cotton tissue is cultivated has certain degree of difficulty, sprouts long, the characteristics such as the embryo incidence is low, the difficulty of taking root, genotype dependence are strong of cycle that exist through the explant induction somatic embryo.
Mainly comprise in cotton somatic embryos tire differential period: globular embryo; Heart-shape embryo; Four-stages such as torpedo embryo and cotyledonary embryos, wherein globular embryo, heart-shape embryo, torpedo embryo in early stage all can not be sprouted into seedling, have only cotyledonary embryos can sprout into plantlet with the embryo in torpedo later stage.Therefore select suitable culture base and condition of culture, obtaining more cotyledonary embryos and torpedo embryo is the important goal of embryo callus subculture differentiation culture.Research shows: the ripe differentiation of cotton embryo callus rear portion split embryo seedling differentiation, most of embryo callus is converted into the non-embryonic callus tissue, finally loses the generating ability of somatic embryo.At the embryo callus early development, the embryoid differentiation is main with globular embryo mainly, if do not add any hormone; Embryo callus can rest on the globular embryo stage always; Be difficult to seedling differentiation, add cytokinin-like substance not quite, but the sprouting of cotton somatic embryos later stage is had facilitation the influence of cotton embryo callus proliferation function; Therefore to add the exogenous hormone of different components in the inducing culture of cotton somatic embryos, promote the embryo to sprout and differentiation.
Summary of the invention
The object of the invention is exactly to above-mentioned defective of the prior art; Overcome color cotton explant induction somatic embryo sprout exist the cycle long, the embryo incidence is low, the difficulty of taking root, defective that the genotype dependence is strong, and the germination method and the seedling establishment method of color cotton somatic cell embryoid is provided.
To achieve these goals, technical scheme provided by the invention is:
The germination method of color cotton somatic cell embryoid includes following steps:
1) cultivates the embryo callus that obtains with the cotton hypocotyl of coloured silk, through proliferated culture medium successive transfer culture 1-2 time;
2) embryo callus that step 1) is obtained is forwarded to body embryonal induction medium, behind successive transfer culture 2-3 time, obtains ripe cotyledonary embryos.
The germination method of the cotton somatic cell embryoid of above-mentioned coloured silk, said step 2) in, the consisting of of body embryonal induction medium: remove NH
4NO
3The MSB medium in add the N6-isopentennyladenine and the indolebutyric acid of 0.3-0.5mg/L of 0.1-0.3mg/L and the KNO of interpolation 1.9-3.8g/L
3, the plant gel of 1.5-2.0g/L, the glutamine of 0.1-0.5g/L and the asparagine of 0.1-0.5g/L.
Second purpose of the present invention provided: the seedling establishment method of color cotton somatic cell embryoid includes following steps:
1) cultivate with the cotton hypocotyl of coloured silk and obtain ripe cotyledonary embryos and be forwarded on the differential medium, behind successive transfer culture 2-3 time, seedling differentiation;
2) regrowth that step 1) is obtained is forwarded in the subculture medium, and subculture was 1 time in 20-25 days, until taking root;
The seedling establishment method of the cotton somatic cell embryoid of above-mentioned coloured silk in the said step 3), becomes consisting of of seedling medium: the MSB medium of 1/4-1/2, plant gel, the asparagine of 0.1-0.5g/L and the glutamine of 0.1-0.5g/L of interpolation 2.0-2.5g/L.
Somatic embryo is the committed step that cotton tissue is cultivated; If the successive transfer culture that on the medium that does not add hormone, continues; Be that the embryo that can realize cell is taken place theoretically, but the quantity of somatic embryo seldom, and most embryos can continue to rest on the stage of globular embryo.In the sprouting and process of growth of the somatic embryo of maturation, high salinity is ripe, the maximum obstacle of sprouting and take root of embryoid in the medium, and low salt is the essential condition that cotton somatic embryos shape body is effectively sprouted.Therefore, in the cotton process of somatic culture, according to the body embryo culture developmental stage suitably reduce in the medium salt in the macroelement concentration, could obtain good result.This method has also confirmed this point, removes NH
4NO
3Help the generation of somatic embryo, effective embryo's of later stage generation and cotyledonary embryos are sprouted into seedling then need reduce the mineral salt composition among the MS.Simultaneously according to the research of Zhang Baohong etc., the material material that life has a facilitation to the cotton somatic embryos fetal hair has IBA, ABA, IAA, BA, KT, ZT and ZiP, and its effect increases progressively according to above-mentioned order.This research has confirmed the IBA of high concentration through IBA concentration is provided with gradient, and the combination of the KT of low concentration helps the embryo callus subculture differentiation most.
Beneficial effect of the present invention is:
The cotton somatic cell embryoid of coloured silk provided by the present invention is effectively sprouted and seedling establishment method, is to sprout into the optimum condition that seedling works out to color cotton strain embryo callus subculture to the differentiation and the later stage of body embryo.The method makes embryo callus reach 95% to the conversion ratio of body embryo through regulating exogenous hormone, and wherein the cotyledonary embryos differentiation rate has effectively improved the differentiation rate of cotyledonary embryos in the body embryonic development stage up to 70%.Becoming the seedling medium then is through reducing inorganic salt concentration, change the plant gel usage amount, make cotyledonary embryos normally sprout into seedling, solved the difficult problem of taking root, effectively having shortened the incubation time of regenerated transgenic seedling.This method is mainly colored cotton somatic cell embryoid sprouting and later stage Cheng Miao provides a practicable cultural method, lays the foundation for carrying out colored cotton transgenic breeding work.
Embodiment
Mentioned MSB medium in the technical scheme material of the present invention is a kind of conventional medium, and its main component and consumption are as shown in table 1:
Table 1
Wherein, consisting of of 1/2-1/4MS germination medium: 1/2-1/4MS (macroelement)+15g glucose+4.5g agar powder, distilled water constant volume.
KT is the N6-isopentennyladenine; IBA is an indolebutyric acid, is to analyze pure level reagent.
Gelrite is a plant gel, and the source is the import packing of Sigma company; Asparagine and glutamine are and analyze pure level reagent.
The blake bottle number of globular embryo inductivity=induce globular embryo/total inoculation bottle number * 100%.
The blake bottle quantity of cotyledonary embryos inductivity=induce cotyledonary embryos/total inoculation bottle number * 100%.
The blake bottle number that induces globular embryo is meant that globular embryo accounts for 1/2 of total inoculation callus amount in the middle of one bottle; The blake bottle number that induces cotyledonary embryos is meant that cotyledonary embryos accounts for 1/10 of total inoculation callus amount in the middle of one bottle.
Embodiment 1:
(1) supplies the examination material: the embryo callus of green cotton No. 3 of Gansu Province, green cotton G3-1 and brown cotton BC05.
(2) with the embryo callus of three kinds of materials
Be positioned over MSB and (remove NH
4NO
3), add 0.1mg/L KT and 0.3mg/LIBA, and add 1.9g/L KNO
3, 1.5g/L plant gel gelrite, on the differential medium of 0.1g/L glutamine and 0.1g/L asparagine, subculture 2 times.Experimental result shows, green cotton No. 3 embryo callus with green cotton G3-1 in Gansu Province have 100% and 97% to be divided into globular embryo respectively, and the quantity of differentiation that growth is divided into cotyledonary embryos accounts for 73% and 69% of total inoculation embryo callus bottle number respectively.The globular embryo differentiation rate of the cotton BC05 of palm fibre is relatively low, reaches 90%, and the cotyledonary embryos differentiation rate accounts for 65%.
(3) three kinds of materials are sprouted the cotyledonary embryos that on above-mentioned medium and pick out and be positioned over the 1/4MSB medium, and add 2.0g/L plant gel gelrite, 0.1g/L asparagine and 0.1g/L glutamine.Through behind 2 subcultures, the cotyledonary embryos of three kinds of materials all can be sprouted into seedling, and takes root gradually.
Embodiment 2:
(1) supplies the examination material: the embryo callus of green cotton No. 3 of Gansu Province, green cotton G3-1 and brown cotton BC05.
(2) embryo callus with three kinds of materials is positioned over MSB (removal NH
4NO
3), add 0.3mg/L KT and 0.5mg/LIBA, and add 3.8g/L KNO
3, 2.0g/L plant gel gelrite, on the differential medium of 0.5g/L glutamine and 0.5g/L asparagine, subculture 2 times.Experimental result shows, green cotton No. 3 embryo callus with green cotton G3-1 in Gansu Province have 99% and 98% to be divided into globular embryo respectively, and the quantity of differentiation that growth is divided into cotyledonary embryos accounts for 72.5% and 70% of total inoculation embryo callus bottle number respectively.The globular embryo differentiation rate of the cotton BC05 of palm fibre is relatively low, reaches 93%, and the cotyledonary embryos differentiation rate accounts for 56%.
(3) three kinds of materials are sprouted the cotyledonary embryos that on above-mentioned medium and pick out and be positioned over the 1/2MSB medium, and add 2.5g/L plant gel gelrite, 0.5g/L asparagine and 0.5g/L glutamine.Through behind 1 subculture, the cotyledonary embryos of three kinds of materials all can be sprouted into seedling, and takes root gradually.
Embodiment 3:
(1) supplies the examination material: the embryo callus of green cotton No. 3 of Gansu Province, green cotton G3-1 and brown cotton BC05.
(2) embryo callus with three kinds of materials is positioned over MSB (removal NH
4NO
3), add 0.2mg/L KT and 0.4mg/LIBA, and add 2.9g/L KNO
3, 1.7g/L plant gel gelrite, on the differential medium of 0.3g/L glutamine and 0.3g/L asparagine, subculture 2 times.Experimental result shows, green cotton No. 3 embryo callus with green cotton G3-1 in Gansu Province have 99% and 96% to be divided into globular embryo respectively, and the quantity of differentiation that growth is divided into cotyledonary embryos accounts for 74% and 68% of total inoculation embryo callus bottle number respectively.The globular embryo differentiation rate of the cotton BC05 of palm fibre is relatively low, reaches 88%, and the cotyledonary embryos differentiation rate accounts for 55.5%.
(3) three kinds of materials are sprouted the cotyledonary embryos that on above-mentioned medium and pick out and be positioned over the 1/2MSB medium, and add 2.2g/L plant gel gelrite, 0.3g/L asparagine and 0.3g/L glutamine.Through behind 2 subcultures, the cotyledonary embryos of three kinds of materials all can be sprouted into seedling, and takes root gradually.
What should explain at last is: the above is merely the preferred embodiments of the present invention; Be not limited to the present invention; Although the present invention has been carried out detailed explanation with reference to previous embodiment; For a person skilled in the art, it still can be made amendment to the technical scheme that aforementioned each embodiment put down in writing, and perhaps part technical characterictic wherein is optimized.All within spirit of the present invention and principle, any modification of being done, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (4)
1. the germination method of color cotton somatic cell embryoid is characterized in that, includes following steps:
1) cultivates the embryo callus that obtains with the cotton hypocotyl of coloured silk, through proliferated culture medium successive transfer culture 1-2 time;
2) embryo callus that step 1) is obtained is forwarded to body embryonal induction medium, behind successive transfer culture 2-3 time, obtains ripe cotyledonary embryos.
2. the germination method of the cotton somatic cell embryoid of coloured silk according to claim 1 is characterized in that: said step 2), and the consisting of of body embryonal induction medium: remove NH
4NO
3The MSB medium in add the N6-isopentennyladenine and the indolebutyric acid of 0.3-0.5mg/L of 0.1-0.3mg/L and the KNO of interpolation 1.9-3.8g/L
3, the plant gel of 1.5-2.0g/L, the glutamine of 0.1-0.5g/L and the asparagine of 0.1-0.5g/L.
3. the seedling establishment method of color cotton somatic cell embryoid is characterized in that, includes following steps:
1) cultivates the embryo callus that obtains with the cotton hypocotyl of coloured silk, through proliferated culture medium successive transfer culture 1-2 time;
2) embryo callus that step 1) is obtained is forwarded to body embryonal induction medium, behind successive transfer culture 2-3 time, obtains ripe cotyledonary embryos;
3) with step 2) cotyledonary embryos that obtains is forwarded to into the seedling medium, and subculture was 1 time in 20-25 days, until seedling differentiation.
4. the seedling establishment method of the cotton somatic cell embryoid of coloured silk according to claim 3; It is characterized in that: in the said step 3); Become consisting of of seedling medium: the MSB medium of 1/4-1/2, plant gel, the asparagine of 0.1-0.5g/L and the glutamine of 0.1-0.5g/L of interpolation 2.0-2.5g/L.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1034298A (en) * | 1987-11-18 | 1989-08-02 | 泛多金公司 | Regeneration of cotton and conversion |
| US20030143744A1 (en) * | 2000-03-15 | 2003-07-31 | Dunwell James Martin | Method for the production of cotton somatic embryos |
| WO2005063002A1 (en) * | 2003-12-31 | 2005-07-14 | Council Of Scientific And Industrial Research | A tissue culture process for producing cotton plants |
| CN101707963A (en) * | 2009-12-30 | 2010-05-19 | 河南省农业科学院 | Cotton breeding method |
-
2012
- 2012-03-16 CN CN2012100712933A patent/CN102577980A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1034298A (en) * | 1987-11-18 | 1989-08-02 | 泛多金公司 | Regeneration of cotton and conversion |
| US20030143744A1 (en) * | 2000-03-15 | 2003-07-31 | Dunwell James Martin | Method for the production of cotton somatic embryos |
| WO2005063002A1 (en) * | 2003-12-31 | 2005-07-14 | Council Of Scientific And Industrial Research | A tissue culture process for producing cotton plants |
| CN101707963A (en) * | 2009-12-30 | 2010-05-19 | 河南省农业科学院 | Cotton breeding method |
Non-Patent Citations (3)
| Title |
|---|
| E.FIROOZABADY,ET AL.: "PLANT REGENERATION VIA SOMATIC EMBRYOGENESIS IN MANY CULTIVARS OF COTTON(GOSSYPIUM HIRSUTUM L.)", 《IN VITRO CELL DEVELOPMENT BIOLOGY》 * |
| 刘方等: "棉花组织培养高效植株再生体系的建立", 《棉花学报》 * |
| 孟庆玉等: "新疆彩色棉体细胞胚状体的发生及植株再生", 《中国棉花》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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: 20120718 |