CN102057870B - Method for culturing directly differentiated tissues of cotton true leaf and special culture medium - Google Patents
Method for culturing directly differentiated tissues of cotton true leaf and special culture medium Download PDFInfo
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Abstract
The invention discloses a method for culturing directly differentiated tissues of a cotton true leaf and a special culture medium. The special culture medium provided by the invention is a culture medium which is used when embryogenic callus is directly differentiated from a cotton leaf; and the culture medium is obtained by adding KT, 6-BA, 2,4-D, indole acetic acid (IAA), glucose and gel into marek's disease 1 (MSB1) basic culture solution, wherein the final concentration of the KT, the 6-BA, the 2,4-D, the IAA, the glucose and the Gel rite is 0.005 to 0.15mg/L, 0.005 to 0.15mg/L, 0.0001 to 0.001mg/L, 0.001 to 0.02mg/L, 25 to 35g/L and 1.8 to 2.5g/L respectively. By the method, a tissue culture system is established by taking leaves of field cotton plants as explants; and finally, a stable and high-efficiency tissue culture system of the directly induced embryogenic callus of the leaf is established.
Description
Technical field
The present invention relates to a kind of plant tissue culture field, particularly a kind of land for growing field crops its special culture media of cotton plants true leaf tissue culture method.
Background technology
During cotton tissue was cultivated, somatic embryo took place can be divided into for 4 steps with plant regeneration: the explant dedifferentiation induces callus, callus and is divided into that embryo callus, embryo callus are turned out somatic embryo, somatic embryo is sprouted the acquisition plant regeneration again.Grow the callus ratio from the explant dedifferentiation and be easier to, do not see the report that receives the genotype restriction.Differentiating embryo callus from callus induction, is the bottleneck that cotton tissue is cultivated, and the report that limited by genotype concentrates on the induction period of embryo callus.
The genotype restriction shows that at first the somatic embryo of different cotton seeds in the Gossypium takes place different with the plant regeneration ability.The researchist has only upland cotton, cotton, the Hawaii cotton ability organizer somatic embryo of Lei Mengdeshi through a plurality of cotton seeds are compared discovery.Dong Hezhong and Chen Zhixian have compared the somatic embryo generating ability of 4 culture of cotton kinds, find that upland cotton is prone to the inductor somatic embryo, and Asiatic cotton takes second place, and sea island cotton and african cotton are relatively poor, and (Dong closes loyal 1991; Chen Zhixian, the refined monarch .1987 of Lee).Gossypium has 50 kinds; Comprise 45 wild species; 4 cultivars; Only Ke Laociji is cotton up to now, Dai Weixunshi is cotton, upland cotton, sea island cotton, Asiatic cotton, cotton, cotton 8 kinds of Lei Mengdeshi cotton and Hawaii have obtained somatic embryo, and wherein upland cotton, sea island cotton, Asiatic cotton, cotton and Dai Weixunshi cotton have obtained regeneration plant.
In addition, on somatic embryo generation and plant regeneration ability, also there are differences between each cotton variety.Trolinder studies 28 kinds of upland cotton, and the result has only jade-like stone word 312 and T25 to have higher somatic embryo generating ability, and other kind maybe can not be carried out somatic embryo generation (Trolinder N L, Xhixian C.) than difficulty.Dong Hezhong etc. according to embryo callus become embryo quantity with develop into plant what the upland cotton kind is divided into four types: the first kind is that the somatocyte generating ability is strong, and the kind of Yi Chengmiao is like jade-like stone word 312, jade-like stone word 201 etc.; Second type is to have certain somatic embryo generating ability, becomes seedling quantity medium, through repeatedly obtaining more somatic embryo and partial regeneration plant after the subculture screening, like Mount Tai word 15, Mount Tai word 16 etc.; The 3rd type is that the somatic embryo generating ability is poor, and lopsided embryo is many, after long-time subculture screening, can obtain indivedual plant, like cotton No. 1, No. 7 of Shandong etc.; The 4th type is that somatocyte embryonic pole difficulty maybe can not take place, like that word cotton 215, this word 506 etc.Germplasm resource for cotton is abundant, and only the jade-like stone word is cotton serial at present, and minority kinds such as middle cotton series, the cotton series in Shandong have obtained somatic embryo and regeneration plant.
Research shows between the upland cotton different explants type and has very big difference aspect callus induction and the differentiation.Therefore, the improvement of cotton tissue culture system and the screening of explant always are one of research emphasis of tissue culture worker.The cotton hypocotyl makes explant superior (Troloinder etc. 1988) than cotyledon or leaf.It near the cotyledonary node part (Finer1984, Zhang Xianlong etc. 1990) of the easiest formation callus of hypocotyl.Finer etc. (1984) find to make explant and compare with aseptic seedling with stem, leaf, the petiole of ripe cotton plant, are difficult to evoked callus.Tan Xiao even waits (1988) to study potted plant plan like cotton leaf, petiole and the stem reaction in isolated culture, and discovery employing stem inductor cell stage the most easily takes place and plant regeneration.The callus that the ovule of not pollinating produces is not as pollination ovule (Song Ping, 1987).The placement direction of explant is very important, when utilizing hypocotyl to make explant, epidermis is contacted with substratum, if otch is contacted with substratum, can produce poky red callus (Price etc.).Hypocotyl is the easiest, and mesocotyl and epicotyl take second place, and cotyledon is relatively poor, and blade and stem section are the poorest, and the Recent study hair root can be regenerated, but complexity lacks comparison (Jiao Gaili etc., 2002); Zhang Hai etc. (2002) have reported cotton cotyledon isolated culture and plant regeneration, but regeneration frequency is not high.
The conclusion that different investigators draw has certain difference, possibly exist bigger difference relevant to the inducing culture system with different genotype materials.And generally speaking, (Chi Jina waits 2004) takes place than the easy inducing cotton somatic embryo of mature tissue in tender tissue.Zhang Chaojun etc. have set up cotton land for growing field crops petiole tissue culturing system, and the high differentiation rate material (patent No. ZL200610089439.1) that utilized the seed selection of petiole tissue culturing system.Utilizing ripe plant tissue in land for growing field crops or organ to set up tissue culturing system as explant, is one of focus of cotton tissue culture studies.
It is a lot of to be applied to cotton healing tissue's inductive minimum medium at present, like MS, and LS, White, BT, BS, Hitsch, CM-1, SM, KM, P etc., but use more with MS and LS.The tissue culture of cotton different explants is used substratum, mainly is based on the MS substratum and improves.
Summary of the invention
The object of the present invention is to provide the substratum of the direct induced embryonic callus of a kind of cotton leaf.
The substratum of the direct induced embryonic callus of cotton leaf provided by the invention is in the MSB1 basic culture solution, to add the substratum that following material obtains: KT, 6-BA, 2,4-D, IAA, carbon source and gelifying agent;
The final concentration of KT is that 0.005-0.15mg/L, 6-BA final concentration are 0.005-0.15mg/L, 2 in the said substratum, and the final concentration of 4-D is that the final concentration of 0.0001-0.001mg/L, IAA is 0.001-0.02mg/L.
Above-mentioned MSB1 basic culture solution is that the organic composition of inorganic salt that an ammonium nitrate reduces by half, saltpetre increases half the MS substratum and B5 medium is formed, and its solvent is a water, and solute is seen table 1.
The solute of table 1.MSB1 basic culture solution
Above-mentioned gelifying agent can be tissue culture such as agar powder, carrageenin or Gel rite solidifying agent commonly used, preferably the Gel rite of U.S. Sigma company production (sigma company produces, and article No. is G1910).Can certainly be agar powder, consumption be 6.5-7.0 gram/L, also can be tissue culture gelifying agents commonly used such as carrageenin, and consumption is the basis with the substratum hardness that 2.2g/L Gel rite can reach, and suitably adjusts consumption.
Above-mentioned carbon source can be glucose or sucrose, and the embryonic callus induction stage is used glucose, and the embryoid sprouting stage is used sucrose.
Further; KT is that 0.005-0.15mg/L, 6-BA are 0.005-0.15mg/L, 2 in the substratum of the direct induced embryonic callus of above-mentioned cotton leaf; 4-D is 0.0001-0.001mg/L, and IAA is 0.001-0.02mg/L, and glucose is that 25-35g/L and Gel rite are 1.8-2.5g/L.
Optimally, KT is that 0.05mg/L, 6-BA are 0.1mg/L, 2 in the substratum of the direct induced embryonic callus of cotton leaf, and 4-D is 0.005mg/L, and IAA is 0.005mg/L, and glucose is that 25g/L and Gel rite are 2.2g/L.
Another object of the present invention is to provide a kind of method of producing cotton regenerated seedling.
The method of the cotton regenerated seedling of production provided by the invention may further comprise the steps:
1) places the substratum of the above-mentioned direct induced embryonic callus of cotton leaf to cultivate cotton leaf, obtain embryo callus;
2) the embryo callus succeeding transfer culture in the embryoid germination medium that step 1) is obtained obtains embryoid, obtains cotton regenerated seedling.
Above-mentioned embryoid germination medium is in the MSB1 basic culture solution, adds the substratum that KT, 6-BA, sucrose and Gel rite obtain, and said MSB1 basic culture solution solvent is a water, and solute is seen table 1.
The final concentration of KT is 0.001-0.15mg/L in the said embryoid germination medium, and the final concentration of 6-BA is 0.005-0.15mg/L, sucrose 25-35g/L, and the final concentration of Gel rite is 2.0-2.5g/L.
Further, the final concentration of KT is 0.001-0.1mg/L in the above-mentioned embryoid germination medium, and the final concentration of 6-BA is 0.005-0.15mg/L, and the final concentration of sucrose is 28-32g/L, and the final concentration of Gel rite is 2.0-2.3g/L.
Optimally, the final concentration of KT is 0.01mg/L in the above-mentioned embryoid germination medium, and the final concentration of 6-BA is 0.01mg/L, and the final concentration of sucrose is 30g/L, and the final concentration of Gel rite is 2.2g/L.
Above-mentioned steps 1) cotton leaf in is through pretreated, and said pre-treatment is with 0.1% HgCl with cotton leaf
2Carry out disinfection, then at least once with aseptic water washing.
Above-mentioned cotton leaf can be the true leaf of the different varieties of upland cotton, is not suitable for parts such as cotyledon, bract.Be particularly suitable for middle cotton institute 24, middle cotton institute 27, Ji close 713 or the Ji close 321, this method also is fit to cotton 312, the jade-like stone word 201 of jade-like stone word simultaneously, and in 394, in 091 etc. be easy to tissue culture cotton material.
Method of the present invention is to utilize the true leaf of land for growing field crops cotton plant to set up tissue culturing system as explant, finally sets up blade tissue culturing system directly differentiation, stability and high efficiency.This system is directly induced by blade and is differentiated embryo callus; And without callus; Effectively shortened the cotton tissue culture cycle, can be used for the quick genetic transformation of foreign gene, also can be used for the differentiation rate genetic research, be easy to the aspects such as material selective breeding of tissue culture.
Embodiment
Below in conjunction with specific embodiment the present invention is described further, but the present invention is not limited to following examples.
Among the following embodiment,, be ordinary method like no specified otherwise.
The acquisition of embodiment 1, cotton regenerated seedling and statistical observation thereof
1, the preparation of embryo callus
1) pre-treatment of explant
The true leaf of getting the land for growing field crops cotton plants is as explant.After cotton leaf fetched, prepare earlier 8 through high-temperature sterilization (121 ℃, petridish 14min) adds an amount of aqua sterilisa (121 ℃ 14min), add mass percentage concentration and be 0.1% HgCl in the 2nd, 3,4 petridish in the 1st, 6,7, No. 8 petridish
2Solution.
Wherein cotton is provided with 3 kinds, is respectively:
Middle cotton institute 24, science and technology trading company of The Chinese Academy of Agriculture Science and Technologys Cotton Research Institute sells;
The Ji closes 321, and science and technology trading company of The Chinese Academy of Agriculture Science and Technologys Cotton Research Institute sells;
Middle cotton institute 27, science and technology trading company of The Chinese Academy of Agriculture Science and Technologys Cotton Research Institute sells.
The pre-treatment concrete operations: the blade that will pluck from the land for growing field crops earlier washes one time the 1st petridish, moves on to successively then to soak sterilization in the 2nd, 3,4 petridish, in each petridish, soaks 1-2min, is 0.1%HgCl in mass percentage concentration
2Middle immersion sterilization total time is proper at 4-5min, because vacuum side of blade leaf hair is more, will fully stir during sterilization, in order to avoid form small bubbles at blade surface, makes sterilization not thorough, is prone to pollute; In the 6th, 7, No. 8 petridish, respectively wash 1 time, cleaning down to fall residual HgCl
2, residual HgCl
2Also can cause blade death, embryonic callus induction, difficult growth etc.Blade after the flushing is cut into the explant subsequent use (noting not removing vein) about 0.5cm * 0.5cm in main lobe arteries and veins zone.
2) acquisition of embryo callus
The subsequent use explant that step 1) is obtained; Under aseptic condition, receiving substratum through the direct induced embryonic callus of cotton leaf of high-temperature sterilization (being called for short the EC1 substratum) goes up (after substratum prepares; Pour in the 100ml triangular flask, every bottle adds about 60ml, subsequent use behind high-temperature sterilization.Sterilising conditions is 121 ℃, 14min), notices that face of blade upwards, contacts substratum with vacuum side of blade.After 25 days, can be observed callus growth, after 35-60 days, can obtain embryo callus.
Wherein, the EC1 substratum is on the basis of MSB1 basic culture solution, adds kinetin (KT), 6-BA, 2,4-D, IAA, glucose, Gel rite (Sigma, article No.: G1910); Wherein, The final concentration of KT is that the final concentration of 0.05mg/L, 6-BA is 0.1mg/L, 2; The final concentration of 4-D is that the final concentration of 0.005mg/L, IAA is that the final concentration of 0.005mg/L, glucose is that the final concentration of 25g/L, Gelrite is 2.2g/L; Regulate pH to 5.8, the solvent of wherein said MSB1 basic culture solution is a water, and solute is seen table 1.
Culture condition is, triangular flask is placed under the intensity of illumination of 2000-4000lx to cultivate, and is better with the 3000lx light intensity; Every day, light was cultivated 14 hours, secretly cultivated 10 hours; Culturing room's temperature is controlled at 20-30 ℃.
3) statistical observation
Experiment is provided with three kinds, repeats 3 times, whenever is treated to one liter of substratum, is divided in the triangular flask of 18 100ml 5 explants of every bottle graft.Experimental result sees the following form 2.Can find out that from table 2 middle cotton institute 24 inductivities are the highest, the Ji is closed 321 and is taken second place, and middle cotton institute 27 is minimum.Explain equally with aseptic cotton hypocotyl, there is genotypic difference in inducing of embryo callus.
Table 2. differing materials blade differentiation rate statistics
2, embryo callus seedling differentiation
1) preparation of embryoid germination medium
Embryoid germination medium (called after SE1 substratum) is on the basis of MSB1 basic culture solution, to add KT, 6-BA, sucrose and Gel rite.Wherein: the final concentration of KT is 0.01mg/L, and the final concentration of 6-BA is 0.01mg/L, and the final concentration of sucrose is 30g/L, the final concentration 2.2g/L of Gel rite, and regulating pH is 6.5, and the solvent of wherein said MSB1 basic culture solution is a water, and solute is seen table 1.
2) acquisition of differentiation seedling
The embryo callus embryoid germination medium that places the step 1) of above-mentioned steps 2 to obtain of preparation in the above-mentioned steps 1 is cultivated, and culture condition is, triangular flask is placed under the intensity of illumination of 2000-4000lx to cultivate, and is better with the 3000lx light intensity.Every day, light was cultivated 14 hours, secretly cultivated 10 hours.Per 40 days subcultures once behind succeeding transfer culture 2-3 time, just can obtain regrowth.
3) statistical observation
For the embryo callus in clear and definite differing materials source to becoming the reaction of seedling substratum; Select for use middle cotton institute 24, middle cotton institute 27, Ji to close 321 3 cotton varieties; Every kind is chosen from 20 of the embryo callus of blade, and every is switched to 3 bottles and becomes on the seedling substratum.Add up into the seedling number next time during subculture.During succeeding transfer culture, one bottle of every bottle of subculture does not expand numerous.Can find out that from table 3 result middle cotton institute 24, middle cotton institute 27 close 321 with the Ji and compare, and become the seedling number less.Be illustrated as the seedling cultivation stage and also have genotypic difference.
The one-tenth seedling number of table 3. differing materials embryo callus
The acquisition of embodiment 2, cotton regenerated seedling and statistical observation thereof
The difference of present embodiment and embodiment 1 is following 3 points, and all the other steps are identical:
1, used starting material are merely middle cotton institute 24, are cultivated by precocious breeding seminar of The Chinese Academy of Agriculture Science and Technologys Cotton Research Institute, are sold by science and technology trading company of The Chinese Academy of Agriculture Science and Technologys Cotton Research Institute.
2, EC1 substratum: on the basis of MSB1 basic culture solution, added kinetin (KT), 6-BA, 2,4-D, IAA, glucose, Gel rite; Wherein, The final concentration of KT is that the final concentration of 0.005mg/L, 6-BA is 0.005mg/L, 2; The final concentration of 4-D is that the final concentration of 0.0001mg/L, IAA is that the final concentration of 0.001mg/L, glucose is that the final concentration of 25g/L, Gel rite is 1.8g/L, regulates pH to 5.8.
3, SE1 substratum: be on the basis of MSB1 basic culture solution, to add KT, 6-BA, sucrose and Gel rite; Wherein: the final concentration of KT is 0.001mg/L, and the final concentration of 6-BA is 0.005mg/L, and the final concentration of sucrose is 25g/L, and the final concentration of Gel rite is 2.0g/L, and regulating pH is 6.2.
Experimental result is seen table 4.Can find out from table 2, table 4, the reduction of hormonal readiness, less to the frequency of embryonic callus induction influence.Find out that from the embryonic callus induction experimental observation embryo callus occurs later, just observes embryo callus after about 30 days.In the succeeding transfer culture stage, because the embryo callus poor growth causes sprouting the growth period limited speed at embryoid, the time that obtains regeneration plant postpones, and quantity reduces.
Table 4 middle cotton the tissue culture effect of 24 blades
The acquisition of embodiment 3, cotton regenerated seedling and statistical observation thereof
The difference of present embodiment and embodiment 1 is following 3 points, and all the other steps are identical:
1, used starting material are merely middle cotton institute 24, are cultivated by precocious breeding seminar of The Chinese Academy of Agriculture Science and Technologys Cotton Research Institute, are sold by science and technology trading company of The Chinese Academy of Agriculture Science and Technologys Cotton Research Institute.；
2, EC1 substratum: on the basis of MSB1 basic culture solution, added kinetin (KT), 6-BA, 2,4-D, IAA, glucose, Gel rite; Wherein, The final concentration of KT is that the final concentration of 0.15mg/L, 6-BA is 0.15mg/L, 2; The final concentration of 4-D is that the final concentration of 0.001mg/L, IAA is that the final concentration of 0.02mg/L, glucose is that the final concentration of 35g/L, Gel rite is 2.5g/L, regulates pH to 5.8.
3, SE1 substratum: be on the basis of MSB1 basic culture solution, to add KT, 6-BA, sucrose and Gel rite; Wherein: the final concentration of KT is that the final concentration of 0.1mg/L, 6-BA is that the final concentration of 0.15mg/L sucrose is that the final concentration of 35g/L, Gel rite is 2.5g/L, regulates pH to 6.5.
Experimental result is seen table 5.Can find out from table 2, table 5, because the rising of hormonal readiness has considerable influence to frequency of embryonic callus induction.Find out that from the embryonic callus induction experimental observation because the increase of hormonal readiness, the non-embryonic callus tissue growth is vigorous, generally after 20 days, promptly can be observed callus.The raised growth of callus has influenced the acquisition and the growth of embryo callus.Just observe the part embryo callus after about 30 days in callus subregion on every side.In the succeeding transfer culture stage, because callus growth is very fast, disturbed the growth of embryo callus, make embryoid sprout the growth period limited speed, the time that obtains regeneration plant postpones, and quantity reduces.
Table 5 middle cotton the tissue culture effect of 24 blades
Claims (10)
1. the substratum of the direct induced embryonic callus of cotton leaf is in the MSB1 basic culture solution, to add the substratum that following material obtains: KT, 6-BA, 2,4-D, IAA, carbon source and gelifying agent;
The solvent of said MSB1 basic culture solution is that water, solute are as follows:
The solute of MSB1 basic culture solution
The final concentration of KT is that 0.005-0.15mg/L, 6-BA final concentration are 0.005-0.15mg/L, 2 in the said substratum, and the final concentration of 4-D is that the final concentration of 0.0001-0.001mg/L, IAA is 0.001-0.02mg/L.
2. substratum according to claim 1 is characterized in that: said gelifying agent is agar powder, carrageenin or Gelrite; Said carbon source is glucose or sucrose.
3. substratum according to claim 2 is characterized in that: KT, 6-BA, 2 in the said substratum, and the final concentration of 4-D, IAA, glucose and Gelrite is following:
KT is that 0.005-0.15mg/L, 6-BA are 0.005-0.15mg/L, 2, and 4-D is 0.0001-0.001mg/L, and IAA is 0.001-0.02mg/L, and glucose is that 25-35g/L and Gelrite are 1.8-2.5g/L.
4. method of producing cotton regenerated seedling may further comprise the steps:
1) places the arbitrary described substratum of claim 1-3 to cultivate cotton leaf, obtain embryo callus;
2) the embryo callus succeeding transfer culture in the embryoid germination medium that step 1) is obtained obtains embryoid, obtains cotton regenerated seedling.
5. method as claimed in claim 4 is characterized in that: said embryoid germination medium is in the MSB1 basic culture solution, adds the substratum that KT, 6-BA, sucrose and Gelrite obtain; The final concentration of KT is 0.001-0.1mg/L in the said embryoid germination medium, and the final concentration of 6-BA is 0.005-0.15mg/L, sucrose 25-35g/L, and the final concentration of Gelrite is 2.0-2.5g/L; The solvent of said MSB1 basic culture solution is that water, solute are as follows:
The solute of MSB1 basic culture solution
6. method as claimed in claim 5 is characterized in that: the final concentration of KT is 0.001-0.1mg/L in the said embryoid germination medium, and the final concentration of 6-BA is 0.005-0.15mg/L, and the final concentration of sucrose is 30g/L, and the final concentration of Gelrite is 2.2g/L.
7. method as claimed in claim 6 is characterized in that: the final concentration of KT is 0.01mg/L in the said embryoid germination medium, and the final concentration of 6-BA is 0.01mg/L, and the final concentration of sucrose is 30g/L, and the final concentration of Gelrite is 2.2g/L.
8. like the arbitrary described method of claim 4-7, it is characterized in that: said cotton leaf is through pretreated, and said pre-treatment is with 0.1% HgCl with cotton leaf
2Carry out disinfection, then at least once with aseptic water washing.
9. method as claimed in claim 8 is characterized in that: said cotton leaf is a true leaf.
10. method as claimed in claim 9 is characterized in that: said cotton is that middle cotton institute 24, middle cotton institute 27 or Ji close 321.
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| Title |
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| 冷春旭,等.亚洲棉再生体系研究进展.《棉花学报》.2005,第17卷(第3期), * |
| 张宝红,等.棉花优良品种中棉所19高频体细胞胚胎发生细胞系筛选与植.《作物学报》.2000,第26卷(第2期), * |
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