CN101869073B - Tartary buckwheat isolated regeneration culture method - Google Patents
Tartary buckwheat isolated regeneration culture method Download PDFInfo
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- CN101869073B CN101869073B CN2009100590262A CN200910059026A CN101869073B CN 101869073 B CN101869073 B CN 101869073B CN 2009100590262 A CN2009100590262 A CN 2009100590262A CN 200910059026 A CN200910059026 A CN 200910059026A CN 101869073 B CN101869073 B CN 101869073B
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- bitter buckwheat
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Abstract
The invention belongs to the technical field of biology, in particular to a tartary buckwheat isolated regeneration culture method. The invention cultures complete tartary buckwheat plants through four steps: aseptic seedling culture, callus inducement and subculture, bud division and root inducement. The invention has the advantages of high speed, economy, simplicity and convenience.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of isolated regeneration culture method of bitter buckwheat.
Background technology
Bitter buckwheat (F.tataricum Gaertn claims hull buckwheat again) is a polygonaceae (Polygonaceae), and the dicotyledonous crops of Fagopyrum (Fagopyrum) originates from China, is the cereal crops that a kind of drought resisting is cold-resistant, nutritious, medicinal health value is high.
Buckwheat in the production is divided into bitter buckwheat and sweet buckwheat, and the two exists reproduction to isolate.Wherein bitter buckwheat has higher alimentary health-care function, has higher international competitiveness in the international market, and the annual export volume of China accounts for 10% of international market demand amount.The flowering habit of bitter buckwheat and genetic characteristics cause its artificial hybridization pollination to be difficult to successfully, and this becomes bitter buckwheat crossbreeding and is difficult to the major reason that obtains to break through.Therefore utilize transgenic technology to import the new way that desirable genes might become bitter buckwheat genetic improvement, and the foundation of regeneration and transformation system is the basis of carrying out transgenic research.The tissue culture regeneration of sweet buckwheat and the existing report of research that transformation system is set up, but the research in this regard of bitter buckwheat does not appear in the newspapers as yet.The present invention is an experiment material with the hypocotyl of tartarian buckwheat, and its callus and Regeneration in Vitro are studied, and has set up the Regeneration in Vitro system of bitter buckwheat first, for the genetic improvement of bitter buckwheat provides new approach.
Summary of the invention
The present invention is directed to the problem that bitter buckwheat artificial hybridization pollination is difficult to success, fill up the blank of bitter buckwheat isolated regeneration culture method, a kind of quick, economic, easy bitter buckwheat isolated regeneration culture method is provided.
The present invention realizes through following mode:
The bitter buckwheat isolated regeneration culture method of the present invention may further comprise the steps:
(1), the cultivation of bitter buckwheat aseptic seedling;
(2), bitter buckwheat callus induces and subculture;
(3), the differentiation of bitter buckwheat bud;
(4), bitter buckwheat root induces.
The abbreviation of used medium of the present invention and plant hormone: 6-BA, 6-benzylaminopurine, 6-benzyl aminopurine; IBA, indole-3-butyric acid, indolebutyric acid; IAA, indol-3-acetic acid, heteroauxin; MS, Murashige and Skoog medium, Murashige and Skoog medium; NAA, α-naphthaleneacetic acid, naa; TDZ, thidiazuron, Thidiazuron; 2,4-D, 2,4-dichlorophenoxy, 2,4 dichlorophenoxyacetic acid; KT, 6-Furifuryl-aminopurine, 6-chaff aminopurine (kinetin)
One, the cultivation of bitter buckwheat aseptic seedling
Choose the ripe bitter buckwheat seed of full seed, clear water soaked 30-60 minute, peelled off kind of a skin, used the conventional sterilizing methods sterilization of seed, promptly in 75% ethanolic solution, soaked 1 minute, used 0.05%HgCl
2(conventional H gCl
2Surface sterilization concentration is 0.1%, and this research working concentration is 0.05% can reach sterilization effect, can reduce HgCl thus
2Toxicity.) solution surface sterilization 10 minutes, use sterile water wash again 4 times.Seed after the sterilization is blotted excessive moisture with aseptic paper, and being inoculated into additional 3% sucrose, 0.7% agar powder does not have on the MS solid culture medium of hormone and cultivates.Every day, illumination cultivation was 16 hours, secretly cultivated intensity of illumination 2000lx, 25 ℃ of temperature (medium that uses in this cultural method all passes through conventional autoclaving) 8 hours.
Two, bitter buckwheat callus induces and subculture
Get the seedling age of turning out in the step 1 and be 6-8 days bitter buckwheat aseptic seedling; With the segment that its hypocotyl is cut into about 0.5cm, be placed on the MS+2.0mg/L 2 behind the conventional high-temperature sterilization, evoked callus in the medium of 4-D+1.5mg/L 6-BA; The MS medium adds has 3% sucrose, 0.7% agar powder; PH5.8, incubation time are 7-10 days, and condition of culture is with the cultivation of the bitter buckwheat aseptic seedling of step 1.
Three, the differentiation of bitter buckwheat bud
The callus that the growth conditions of turning out in the step 2 is good goes in the differential medium of MS+0.1mg/L IAA+2.0mg/L 6-BA+1.0mg/L KT+0.5mg/L TDZ, incubation time 15 days, and other supplementary elements of medium and condition of culture are with step 2.
Four, bitter buckwheat root induces
The seedling of the robust growth of turning out in the step 3 is downcut from callus; Go to root induction on the 1/2MS medium that contains 0.5mg/L NAA; Condition of culture is with the cultivation of the bitter buckwheat aseptic seedling of step 1, and through 15 days cultivation, complete bitter buckwheat plant was just cultivated success.
The purposes of bitter buckwheat isolated regeneration culture method of the present invention:
1., can be used for carrying out the research of bitter buckwheat genetic transformation.
2., can be used for the screening of the good transgenic line of bitter buckwheat.
3., can be used for fast numerous preservation of bitter buckwheat germ plasm resource.
4., can be used for the screening of bitter buckwheat cell mutant.
5., can be used for batch production and from the bitter buckwheat of Regeneration in Vitro, extract medicinal ingredients such as rutin on a large scale.
6., can be used for the bitter buckwheat seedling of production high-quality detoxification.
The present invention has following advantage:
1., the hypocotyl that utilizes bitter buckwheat is as experiment material, can be fast, economical, carry out the regenerating and culturing of tartarian buckwheat easily;
2., utilize this method to realize that the cycle of bitter buckwheat Regeneration in Vitro is shorter, only need about 6 weeks from cultivating aseptic seedling to the regrowth overall process;
3., do not need complicated experiment condition and high-accuracy experimental instrument.This method only needs high-pressure sterilizing pot, sterile working platform and greenhouse to get final product, and this condition can be satisfied in domestic most laboratories, is easy to promote;
4., this method sets up the Regeneration in Vitro system of tartarian buckwheat first, for the further Study on Genetic Transformation of tartarian buckwheat lays the foundation;
5., the present invention proposes bitter buckwheat isolated regeneration culture method, operates simple and easy, with low cost.
6., (Thidiazuron TDZ) is applied in the differential medium of bitter buckwheat cultured in vitro process, and demonstrates tangible promotion differentiation with the new plant growth regulator Thidiazuron first.
Embodiment
Below in conjunction with specific embodiment, the present invention is described further.
Embodiment one: the Regeneration in Vitro of the bitter buckwheat in bitter buckwheat landrace Heisui River is cultivated:
(1), choose the mature seed of full seed, clear water soaked 30-60 minute, peelled off kind of a skin, used the conventional sterilizing methods sterilization of seed, promptly in 75% ethanolic solution, soaked 1 minute, used 0.05%HgCl
2Solution surface sterilization 10 minutes is used sterile water wash 4 times again.Seed after the sterilization is blotted excessive moisture with aseptic paper, and being inoculated into additional 3% sucrose, 0.7% agar powder does not have on the MS solid culture medium of hormone and cultivates.Every day, illumination cultivation was 16 hours, secretly cultivated intensity of illumination 2000lx, 25 ℃ of temperature (medium that uses in this cultural method all passes through conventional autoclaving) 8 hours;
(2), callus induces and subculture
Get seedling age and be 6-8 days the bitter buckwheat aseptic seedling in Heisui River,, be placed on the MS+2.0mg/L 2 behind the conventional high-temperature sterilization, evoked callus in the medium of 4-D+1.5mg/L 6-BA the segment that hypocotyl is cut into about 0.5cm.The MS medium is additional to have 3% sucrose, 0.7% agar powder, pH5.8, and condition of culture is the same;
(3), the differentiation of bud
The callus that growth conditions is good goes in the differential medium of MS+0.1mg/L IAA+2.0mg/L 6-BA+1.0mg/L KT+0.5mg/L TDZ, and other supplementary elements of medium and condition of culture are the same;
(4), root induces
The seedling of robust growth is downcut from callus, go to root induction on the 1/2MS medium that contains 0.5mg/L NAA.Plant regeneration frequency reaches about 10%.
Embodiment two: the bitter buckwheat Regeneration in Vitro in bitter buckwheat landrace Zhaotong is cultivated:
Its method is with embodiment one, and the bitter buckwheat plant regeneration frequency in Zhaotong reaches about 7%.
Embodiment three: the Regeneration in Vitro that No., bitter buckwheat kind west buckwheat is cultivated:
Its method is with embodiment one, and plant regeneration frequency of western buckwheat reaches about 5%.
Embodiment four: the bitter buckwheat Regeneration in Vitro in bitter buckwheat kind Jiujiang is cultivated:
Its method is with embodiment one, and the bitter buckwheat plant regeneration frequency in Jiujiang reaches about 3%.
Embodiment five: buckwheat Regeneration in Vitro in bitter buckwheat kind river is cultivated:
Its method is with embodiment one, and buckwheat plant regeneration frequency in river reaches about 5%.
Claims (1)
1. the isolated regeneration culture method of a bitter buckwheat is characterized in that: contain following steps:
(1), the cultural method of aseptic seedling is: bitter buckwheat seed was soaked 30-60 minute with clear water, peel off kind of a skin, use the conventional sterilizing methods sterilization of seed, promptly in 75% ethanolic solution, soaked 1 minute, use 0.05%HgCl
2Solution surface sterilization 10 minutes; Use sterile water wash again 4 times, the seed after the sterilization is blotted excessive moisture with aseptic paper, cultivate on the MS solid culture medium that be inoculated into the conventional autoclaving of process, add 3% sucrose, 0.7% agar powder does not have hormone; Every day, illumination cultivation was 16 hours; Dark cultivation 8 hours, intensity of illumination 2000lx, 25 ℃ of temperature;
(2), inducing and subculture method of callus: the bitter buckwheat aseptic seedling of getting the seedling age of turning out in the step 1 and be 6-8 days; With the segment that its hypocotyl is cut into 0.5cm, be placed on the MS+2.0mg/L 2 behind the conventional high-temperature sterilization, evoked callus in the medium of 4-D+1.5mg/L 6-BA; The MS medium adds has 3% sucrose, 0.7% agar powder; PH5.8, incubation time are 7-10 days, the cultivation of the bitter buckwheat aseptic seedling of the same step of condition of culture (1);
(3), the differentiation method of bud: the callus of turning out in the step (2) is gone in the differential medium of MS+0.1mg/LIAA+2.0mg/L 6-BA+1.0mg/L KT+0.5mg/L TDZ Thidiazuron; Incubation time 15 days, other supplementary elements of medium and the same step of condition of culture (2);
(4), the abductive approach of root is: the seedling of turning out in the step (3) is downcut from callus; Go to root induction on the 1/2MS medium that contains 0.5mg/L NAA; The cultivation of the bitter buckwheat aseptic seedling of the same step of condition of culture (1), through 15 days cultivation, complete bitter buckwheat plant was just cultivated success.
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| CN101869073B true CN101869073B (en) | 2012-07-04 |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102228006B (en) * | 2011-06-15 | 2012-12-19 | 中国医学科学院药用植物研究所 | Method for quickly growing seedling of wild buckwheat rhizome by tissue culture |
| CN102405833B (en) * | 2011-08-19 | 2012-10-10 | 西北大学 | Method for improving adventitious bud differentiation rate of tartary buck wheat through suspension culture |
| CN102960246B (en) * | 2012-11-20 | 2015-03-04 | 成都大学 | Tissue culturing method for effectively improving general flavone content of tartary buckwheat |
| CN105230495B (en) * | 2015-11-11 | 2017-12-19 | 四川农业大学 | A kind of rapid regeneration method for tissue culture of bitter buckwheat |
| CN106212491A (en) * | 2016-04-02 | 2016-12-14 | 江苏辉丰农化股份有限公司 | A kind of plant growth regualting composition |
| CN106386490B (en) * | 2016-09-13 | 2018-07-10 | 成都大学 | A kind of fast breeding method of Ledum palustre seedling |
| CN109042305B (en) * | 2018-09-11 | 2021-09-10 | 宝鸡市农业科学研究院 | Breeding method of tartary buckwheat variety |
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Non-Patent Citations (4)
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| JP平1-148134A 1989.06.09 |
| JP特开2008-228609A 2008.10.02 |
| 刘拥海等.苦荞种子萌发条件和愈伤组织的诱导.《湖南农业大学学报(自然科学版)》.2006,第32卷(第1期),第12-14页. * |
| 李占旗等.苦荞胚性愈伤组织诱导与植株再生研究.《西北植物学报》.2006,第26卷(第6期),第1150-1158页. * |
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