CN102499094A - Method for increasing efficiency of tissue culture of Medicago sativa L. - Google Patents
Method for increasing efficiency of tissue culture of Medicago sativa L. Download PDFInfo
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- CN102499094A CN102499094A CN2011103775342A CN201110377534A CN102499094A CN 102499094 A CN102499094 A CN 102499094A CN 2011103775342 A CN2011103775342 A CN 2011103775342A CN 201110377534 A CN201110377534 A CN 201110377534A CN 102499094 A CN102499094 A CN 102499094A
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Abstract
The invention belongs to the technical field of plant biology, in particular relates to a method of tissue culture of Medicago sativa L. By way of developing a novel tissue culture technical method, the invention focuses on improvement and innovation of two technical nodes (seed disinfection and callus formation and differentiation) in culture steps. With adoption of a mercury bichloride and hydrogen peroxide compound disinfection method, the invention realizes the purposes of thorough disinfection effect and low pollution rate and minimum damage to seeds. Finally, the invention solves the problem of severe pollution to current explants. Meanwhile, a method of TDZ and 6-BA is adopted, thereby, the callus induction rate and the formation rate are remarkably increased, the callus quality is improved, the differentiation capacity of the callus is reserved to a maximum extent, the formation of embryoid sprouts is promoted so that the novel technical method of the tissue culture of the Medicago sativa L. which is efficient, saves time, and has low toxicity and low browning, is obtained.
Description
Technical field
The invention belongs to plant biotechnology field, specifically, relate to the method for tissue culture of a kind of alfalfa (Medicago sativa L.).The present invention has been through having developed new tissue culture technology method, and emphasis forms seed disinfection in the incubation step and callus and improves with differentiation two big technology nodes and innovate, thereby the clover group that obtains efficient, low toxicity, low brownization is trained novel technical method.
Background technology
Alfalfa (Medicago sativa L.) is important leguminous forage, is of high nutritive value because of it has, advantage such as good palatability, is described as " king of herbage ".The alfalfa tissue culture mainly is meant utilizes clover isolated organ (cotyledon, radicle etc.), through artificial culture, carries out the technical method of plant regeneration.This method is significant for the aspects such as breeding, preservation and the cultivation of clover genetically modified plants of the precious genetic stocks of clover.The clover tissue culture technology mainly comprises five steps: explant cultivation, callus induction, embryoid form and bud differentiation, root induction, regeneration plant.
In the clover tissue culture, callus inducing medium generally adopts single plant hormone or growth regulator at present, though this method is simple, the callus differentiation is slow, differentiation rate is low, callus quality is also relatively poor.For example, (Shu Wenhua, Geng Huazhu, Yan Lunxing etc. such as Shu Wenhua; Alfalfa plumular axis callus culture and plant regeneration, Practaculture Science, 1993,10 (3): 65-67) with the field clover be material; Through single 2,4-D (2,4 dichlorophenoxyacetic acid) hormone induction callus; Healing rate is 81%, and callus forms needs 7～8 days, and every callus is on average sprouted several 0.75～1.0;
In addition, and Liang Huimin etc. (Liang Huimin, Huang Jian, Xiayang etc., the foundation of clover tissue culture high-frequency regeneration system, Journal of Agricultural Biotechnology, 2003,11 (3): 321-322) discover, after optimizing, embryoid induction rate 58%;
In addition, Xiao Hexia (Xiao Hexia, the foundation of clover transformation tissue culture system and LEA-3 gene studies [D]; University Of Hebei, 2005) discover, be that material is cultivated with hypocotyl, stem, leaf; Wherein the hypocotyl effect is best, and best inducing culture is MS+2.0mg/L 2,4-D+0.5mg/L 6-BA; Embryoid induction rate 45%, brownization rate are 22%.
In addition, Zhao Jinmei (Zhao Jinmei, Li Fang, Zhou He etc., the foundation of alfalfa tissue culturing system, nuclear agricultural science newspaper, 2010,24 (3): 507-512) discover, after optimizing, form two kinds of methods.Its one alfalfa callus later stage of method differentiation rate reaches as high as 90%, but early stage callus induction difficulty, and yellow, water stain shape is second-rate, and brownization rate is up to being 25%.The early stage callus induction rate of its scheme two clovers is up to 100%, and is yellow green, and is loose, quality is high.But the callus later stage differentiation rate that this method forms is low, is merely 10%, and brownization also will be for seriously.
In addition, Xu Chunbo etc. (CN101946711A) discover that after optimizing, alfalfa callus later stage inductivity reaches 85%, but callus is yellow, water stain shape, and are second-rate.Brownization rate is up to being 33%.The later stage differentiation rate is also lower, is merely 63%.
Although it is ripe that the alfalfa regenerating system has been tending towards, but still have two big key technology difficult problems:
(1) the current seed disinfection method of seed disinfection, however Disinfection Effect is good, but big to the seed damage, influence seed germination; Otherwise little to the seed damage, but pollution appears in weak effect easily, causes group training failure;
(2) callus formation is slow with the differentiation early stage alfalfa callus formation of present technical system speed, embryo callus is few; Shortcomings such as middle and later periods embryo shape callus differentiation speed is slow, sprout less, browning is serious have a strong impact on clover organizational efficiency and even success or failure.Through after optimizing, though callus quality and differentiation rate increase, but still exist or be the high-quality callus, low callus breaks up; Be high callus differentiation rate, the key technology difficult problem of low callus induction efficient.Therefore, developing a kind of new high-efficient culture technical method, can guarantee high callus induction rate, can guarantee high callus differentiation rate again, is very necessary.
Therefore, it is very essential carrying out a kind of seed disinfection technical method efficient, low toxicity; Simultaneously, need develop a kind of technical method with differential period, can promote the formation of alfalfa callus in early days, can improve the differentiation capability of embryo callus subculture again, reduce brownization rate in callus formation.
Emphasis of the present invention has been developed new tissue culture technology method around this two big technical barrier, and emphasis improves these two key technology difficult problems and innovates, thus obtain efficient, save time, the clover group of low toxicity, low brownization trains novel technical method.
Summary of the invention
At present in the clover tissue culture, have mainly that explant is seriously polluted, callus forms and not enough this two big technical barrier of differentiation, this cause the clover tissue culture and inducement time long, efficient is low, of poor quality, problem such as brownization is serious.
Explant is seriously polluted mainly thoroughly not to be caused owing to seed disinfection.The seed disinfection method mainly is divided into mercuric chloride sterilization, clorox sterilization and three kinds of hydrogen peroxide solution sterilizations (referring to table 1) at present.Wherein mercuric chloride sterilization Disinfection Effect is best, but toxicity is big, and is serious to the seed damage, do not utilize the later stage explant to cultivate; The clorox Disinfection Effect is only second to mercuric chloride, but certain toxicity is also arranged.If disinfecting time is too short, then exist sterilization not thorough, the with serious pollution problem of seed; If disinfecting time is long, equally also cause the seed damage; Hydrogen peroxide solution is minimum to the seed damage, but Disinfection Effect is undesirable, and seed is seriously polluted.Through experiment repeatedly, the present invention realizes that through adopting mercuric chloride and hydrogen peroxide solution composite disinfecting method both Disinfection Effect was thorough, and pollution rate is low, to the minimum purpose of seed damage, finally solves the with serious pollution problem of current explant again.
Table 1: different sterilization methods are to the influence of alfalfa seed sprouting and seedling early growth
| Sterilization method | Seed number | Germination rate | Pollution rate | Aberration rate | Germinating time |
| The mercuric chloride sterilization | 100 | 53％ | 5％ | 15％ | 10-15 days |
| The clorox sterilization | 100 | 85％ | 45％ | 2％ | 7-10 days |
| The hydrogen peroxide solution sterilization | 100 | 80％ | 10％ | 5％ | 7-10 days |
Simultaneously, in alfalfa callus induction and the incubation, exist callus to form slowly; The embryoid induction rate is low, and the number that sprouts is few, and differentiation rate is low; Brownization is serious, and general callus appearance needs 5～7 day time, final embryoid induction rate 50%; Brownization of callus is serious, and brownization rate is up to 20～60%.What clover group training callus of induce mainly used at present is 2,4-D or KT.Use 2,4-D, callus induction rate is low, formation speed is slow; If the increasing usage amount, because of 2,4-D is a kind of growth inhibitor, is unfavorable for the formation of embryoid differentiation and bud, but also occurs browning easily, causes group training failure.
The present invention is through optimizing hormone combination and tissue culture technology flow process; Form new technical method; The method that present technique adopts TDZ+6-BA to be used first not only can significantly improve the callus of induce rate, form speed, improves the callus quality; But also kept the differentiation of calli ability to greatest extent, promote the formation of embryoid bud.This technology goes out sooner, callus quality is good, brownization rate is low, and the time that goes out to heal that can solve current callus cultivation existence is long, of poor quality, the technical barrier that brownization is serious.Application should technology after, the callus of induce time is 3 days, shortens 40%; Callus induction rate reaches 100%, and callus is yellow green, and is loose, quality is higher; Later stage callus differentiation rate reaches 75%, and brownization situation disappears basically.
The method for tissue culture that the purpose of this invention is to provide a kind of alfalfa, its acquisition by seed sterilization and aseptic seedling, explant are cultivated, induce and the inducing of successive transfer culture, embryoid, the differentiation of embryo callus subculture, the culture of rootage stage of callus form, and it is following specifically to make each step: choose healthy and strong seedling; The intercepting hypocotyl; Be cut into the 1cm segment, place the callus medium culture, cultivated for 4 weeks; Change callus over to subculture medium, cultivated 5-8 days; Change callus the formation of embryoid induction medium inducing embryoid body over to, cultivated 7-10 days, every at a distance from 24h, change a subculture.Change the embryoid that induces over to differential medium and carry out differentiation culture, 4 weeks of continuous culture.The plant that differentiates is changed in the root media, carry out culture of rootage, continuous culture 4 week and getting, the callus culture medium prescription is SH+2,4-D (2.0mg/L)+6-BA (0.5mg/L)+agar (7.5g/L)+sucrose (30g/L); The successive transfer culture based formulas is SH+2,4-D (2.0mg/L)+6-BA (0.25mg/L)+TDZ (0.25mg/L)+7.5g/L agar+30g/L sucrose; Embryoid culture medium prescription: MSO+NAA (1.0mg/L)+6-BA (0.5mg/L)+caseinhydrolysate (4g/L)+7.5g/L agar+30g/L sucrose; Differential medium MSO+NAA (1.0mg/L)+6-BA (0.5mg/L)+agar (7.5g/L)+sucrose (30g/L); The culture of rootage based formulas is 1/2MS+ agar (7.5g/L)+sucrose (30g/L).
Another object of the present invention just provides a kind of sterilizing methods of alfalfa seed, and concrete steps are following: choose the seed of full seed, behind the 75% alcohol-pickled 1min; 1% mercuric chloride soaks 5min; Then 30% hydrogen peroxide soaks 30min, uses distilled water flushing 3-4 time then, the seed of rinsing well is placed on blots water on the filter paper; Be inoculated in (pH=6.0) in the solid culture medium solid culture based formulas: MS+7.5g/L agar+30g/L sucrose; Condition of culture: 25 ℃, intensity of illumination 2500lk is divided into light dark period 16/8h, cultivates 5-7 days.
In sum, the present invention not only optimizes the seed disinfection process, adopts mercuric chloride and hydrogen peroxide to mix sterilization, and this method not only Disinfection Effect is good, and the seed pollution rate is low; And little to the alfalfa seed damage, handle the back seed and can sprout in 2-3 days; The method that also adopts TDZ+6-BA to be used first not only can significantly improve the callus of induce rate, form speed, improves the callus quality, but also has kept the differentiation of calli ability to greatest extent, promotes the formation of embryoid bud.
The description of more than summarizing the present invention, can further understand the present invention through some specific embodiment that provides with reference to this paper, these embodiment only are in order to explain rather than limit the present invention.Medium commonly used of some this areas wherein and composition thereof have been carried out explaination as follows:
The SOC medium: every liter of medium contains bacterial culture with tryptone 209 grams, yeast extract 59 grams, NaCl 0.59 gram, 250mmol/L KCL solution 10ml, pH7.0.Face with before adding people 2mol/L MgCl
2Solution 5ml and 1mol/L glucose solution 20ml.
SH medium: potassium nitrate (2830mg/L), ammonium nitrate (463mg/L), bitter salt (185mg/L); Two hydration calcium chloride (166mg/L), potassium dihydrogen phosphate (400mg/L), Zinc vitriol (8.6mg/L); Two molybdic acid hydrate sodium (0.25mg/L), Salzburg vitriol (0.025mg/L), cobalt chloride hexahydrate (0.025mg/L); Puridoxine hydrochloride (9.5mg/L), thiamine hydrochloride (9.9mg/L), nicotinic acid (4.5mg/L); Disodium ethylene diamine tetraacetate (37.3mg/L), green vitriol (27.8mg/L).
MSO medium: potassium nitrate (1900mg/L), ammonium nitrate (1650mg/L), bitter salt (370mg/L), two hydration calcium chloride (440mg/L); Potassium dihydrogen phosphate (170mg/L), four hydrated manganese sulfates (22.3mg/L), Zinc vitriol (8.6mg/L), boric acid (6.2mg/L); KI (0.83mg/L), two molybdic acid hydrate sodium (0.25mg/L), Salzburg vitriol (0.025mg/L), cobalt chloride hexahydrate (0.025mg/L); Inositol (100mg/L), puridoxine hydrochloride (1.0mg/L), thiamine hydrochloride (10.0mg/L); Nicotinic acid (1.0mg/L), disodium ethylene diamine tetraacetate (37.3mg/L), green vitriol (27.8mg/L).
MS medium: potassium nitrate (1900mg/L), ammonium nitrate (1650mg/L), bitter salt (370mg/L), two hydration calcium chloride (440mg/L); Potassium dihydrogen phosphate (170mg/L), four hydrated manganese sulfates (22.3mg/L), Zinc vitriol (8.6mg/L), boric acid (6.2mg/L); KI (0.83mg/L), two molybdic acid hydrate sodium (0.25mg/L), Salzburg vitriol (0.025mg/L), cobalt chloride hexahydrate (0.025mg/L); Inositol (100mg/L), glycine (2.0mg/L), puridoxine hydrochloride (0.5mg/L), thiamine hydrochloride (0.1mg/L); Nicotinic acid (0.5mg/L), disodium ethylene diamine tetraacetate (37.3mg/L), green vitriol (27.8mg/L).
Phenyl thiadiazolyl group urea (TDZ): a kind of emerging plant growth regulator.
(2,4-D): a plant growth regulators is to be used for evoked callus to form to 2,4 dichlorophenoxyacetic acid.
6-benzyl aminoadenine (6-BA): a plant growth regulators is to be used for evoked callus to form.
A-methyl (NAA): a plant growth regulators is to be used for evoked callus to form.
N6-furans methylamino purine (KT): have another name called kinetin, a plant growth regulators is to be used for evoked callus to form.
Description of drawings
Fig. 1: the tissue culture effect of alfalfa contrasts, and left figure (L) is callus before using, and right figure (R) is the callus after this technology of use;
Fig. 2: the tissue culture effect figure of alfalfa, A: aseptic seedling; B: callus induction; C: embryoid induction; D: indefinite bud forms; E: differentiation plant; F: regeneration plant.
Embodiment
Embodiment
1. the acquisition of seed sterilization and aseptic seedling
Choose the seed of full seed, behind the 75% alcohol-pickled 1min, 1% mercuric chloride soaks 5min; Then 30% hydrogen peroxide soaks 30min; Use distilled water flushing 3-4 time then, the seed of rinsing well is placed on blots water on the filter paper, be inoculated in (pH=6.0) in the solid culture medium.Culture medium prescription: MS+ agar (7.5g/L)+sucrose (30g/L).Condition of culture, 25 ℃, intensity of illumination 2500lk is divided into light dark period 16/8h, cultivates 5-7 days.
Through adopting mercuric chloride and hydrogen peroxide solution composite disinfecting method, realize that both Disinfection Effect was thorough, pollution rate is low, to the minimum purpose of seed damage, finally solves the with serious pollution problem of current explant again.
2. callus induces
Choose healthy and strong seedling, intercepting hypocotyl (root top stem is with the lower part).Be cut into the 1cm segment, place the callus medium culture.Callus culture medium prescription: SH+2,4-D (2.0mg/L)+6-BA (0.5mg/L)+7.5g/L agar+30g/L sucrose.Around the cultivation.(referring to Fig. 1).
3. the subculture of callus
Changing callus over to subculture medium cultivates.After callus produces, transfer to subculture medium and continue to cultivate.Callus culture medium prescription: SH+2,4-D (2.0mg/L)+6-BA (0.25mg/L)+TDZ (0.25mg/L)+7.5g/L agar+30g/L sucrose.Condition of culture, 25 ℃, intensity of illumination 2500lk is divided into light dark period 16/8h, cultivates 5-8 days.
4. embryoid induces
Callus is changed over to the formation of embryoid induction medium inducing embryoid body.Embryoid culture medium prescription: MSO+NAA (1.0mg/L)+6-BA (0.5mg/L)+caseinhydrolysate (4g/L)+7.5g/L agar+30g/L sucrose.Cultivated 7-10 days, every at a distance from 24h, change a subculture.
5. the differentiation of plant tissue
Change the embryoid that induces over to differential medium and carry out differentiation culture.Differential medium MSO+NAA (1.0mg/L)+6-BA (0.5mg/L)+7.5g/L agar+30g/L sucrose.4 weeks of continuous culture.
6. culture of rootage
The plant that differentiates is changed in the root media, carry out culture of rootage.Root media 1/2MS+7.5g/L agar+30g/L sucrose.In continuous culture 4-5 week, the incubation of whole alfalfa is referring to Fig. 2.
Application should technology after, the callus of induce time is 3 days, shortens 40%; The embryoid induction rate reaches 90%, increases by 40%; Brownization situation disappears basically, and brownization rate reduces by 20%.And the increase of the formation quantity of callus indefinite bud, increasing nearly 150% (number that sprouts is increased to 2.1/every callus from 1.0/every callus), the time of sprouting shortens 30%.
Claims (2)
1. the method for tissue culture of an alfalfa is characterized in that its acquisition by seed sterilization and aseptic seedling, explant are cultivated, induce and the inducing of successive transfer culture, embryoid, the differentiation of embryo callus subculture, the culture of rootage stage of callus form, and specifically preparation process is following: choose healthy and strong seedling; The intercepting hypocotyl; Be cut into the 1cm segment, place the callus medium culture, cultivated for 4 weeks; Change callus over to subculture medium, cultivated 5-8 days; Change callus the formation of embryoid induction medium inducing embryoid body over to, cultivated 7-10 days, every at a distance from 24h, change a subculture.Change the embryoid that induces over to differential medium and carry out differentiation culture, 4 weeks of continuous culture.The plant that differentiates is changed in the root media, carry out culture of rootage, continuous culture 4 week and getting, the callus culture medium prescription is SH+2,4-D (2.0mg/L)+6-BA (0.5mg/L)+agar (7.5g/L)+sucrose (30g/L); The successive transfer culture based formulas is SH+2,4-D (2.0mg/L)+6-BA (0.25mg/L)+TDZ (0.25mg/L)+7.5g/L agar+30g/L sucrose; Embryoid culture medium prescription: MSO+NAA (1.0mg/L)+6-BA (0.5mg/L)+caseinhydrolysate (4g/L)+7.5g/L agar+30g/L sucrose; Differential medium MSO+NAA (1.0mg/L)+6-BA (0.5mg/L)+agar (7.5g/L)+sucrose (30g/L); The culture of rootage based formulas is 1/2MS+ agar (7.5g/L)+sucrose (30g/L).
2. the method for tissue culture of the alfalfa described in the claim 1 is characterized in that, the concrete steps of seed sterilization are: the seed of choosing full seed; Behind the 75% alcohol-pickled 1min, 1% mercuric chloride soaks 5min, and then 30% hydrogen peroxide soaks 30min; Use distilled water flushing 3-4 time then; The seed of rinsing well is placed on blots water on the filter paper, be inoculated in (pH=6.0) in the solid culture medium solid culture based formulas: MS+7.5g/L agar+30g/L sucrose; Condition of culture: 25 ℃, intensity of illumination 2500lk is divided into light dark period 16/8h, cultivates 5-7 days.
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| CN102907326A (en) * | 2012-11-07 | 2013-02-06 | 云南农业大学 | Tissue culture propagation method for Medicagao Sativa L. |
| CN103843663A (en) * | 2014-03-14 | 2014-06-11 | 中国农业科学院北京畜牧兽医研究所 | Method for promoting rooting of alfalfa tissue culture seedlings |
| CN105918123A (en) * | 2016-05-03 | 2016-09-07 | 北京林业大学 | Method for tissue culture of medicago sativa L.cv.Baoding for ecological restoration |
| CN107691220A (en) * | 2017-10-26 | 2018-02-16 | 中国农业科学院兰州畜牧与兽药研究所 | A kind of breeding method of alfalfa tissue-cultured seedling by space mutagenesis |
| CN113061170A (en) * | 2019-12-16 | 2021-07-02 | 青海师范大学 | Alfalfa antioxidant glycoprotein and preparation method and application thereof |
| CN114158479A (en) * | 2021-11-10 | 2022-03-11 | 内蒙古蒙草生态环境（集团）股份有限公司 | Method for culturing tissue culture seedlings of wild alfalfa in Alaska region |
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| CN102907326A (en) * | 2012-11-07 | 2013-02-06 | 云南农业大学 | Tissue culture propagation method for Medicagao Sativa L. |
| CN102907326B (en) * | 2012-11-07 | 2013-09-25 | 云南农业大学 | Tissue culture propagation method for Medicagao Sativa L. |
| CN103843663A (en) * | 2014-03-14 | 2014-06-11 | 中国农业科学院北京畜牧兽医研究所 | Method for promoting rooting of alfalfa tissue culture seedlings |
| CN103843663B (en) * | 2014-03-14 | 2015-07-15 | 中国农业科学院北京畜牧兽医研究所 | Method for promoting rooting of alfalfa tissue culture seedlings |
| CN105918123A (en) * | 2016-05-03 | 2016-09-07 | 北京林业大学 | Method for tissue culture of medicago sativa L.cv.Baoding for ecological restoration |
| CN107691220A (en) * | 2017-10-26 | 2018-02-16 | 中国农业科学院兰州畜牧与兽药研究所 | A kind of breeding method of alfalfa tissue-cultured seedling by space mutagenesis |
| CN113061170A (en) * | 2019-12-16 | 2021-07-02 | 青海师范大学 | Alfalfa antioxidant glycoprotein and preparation method and application thereof |
| CN113061170B (en) * | 2019-12-16 | 2023-04-07 | 青海师范大学 | Alfalfa antioxidant glycoprotein and preparation method and application thereof |
| CN114158479A (en) * | 2021-11-10 | 2022-03-11 | 内蒙古蒙草生态环境（集团）股份有限公司 | Method for culturing tissue culture seedlings of wild alfalfa in Alaska region |
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