CN101643746B - Hotness-resistant Lucerne transgenic culturing method - Google Patents
Hotness-resistant Lucerne transgenic culturing method Download PDFInfo
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- CN101643746B CN101643746B CN2009101020711A CN200910102071A CN101643746B CN 101643746 B CN101643746 B CN 101643746B CN 2009101020711 A CN2009101020711 A CN 2009101020711A CN 200910102071 A CN200910102071 A CN 200910102071A CN 101643746 B CN101643746 B CN 101643746B
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- lucerne
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Abstract
The invention relates to a hotness-resistant Lucerne transgenic culturing method which is characterized by comprising the following steps of: selecting cotyledons of a main-bred Lucerne variety as explants; inducing calli; subculturing the calli till a fast proliferation period; pre-culturing the calli in a dark condition; carrying out agrobacterium co-culture transformation; carrying out 3 turns of selection by transferring the calli into a selection culture medium; selecting the resistant calli for induction and differentiation so as to obtain seedlings; carrying out histochemical staining and molecular detection on resistant plants; identifying the differentiated seedlings containing target genes; determining hotness resistance of selected transgenic plants; remaining the transgenic plants which are free from hotness damage and grow well; continuing to grow the transgenic plants; carrying out indoor and outdoor assessment and verification on the expression stability of the hotness resistance; carrying out seed proliferation on stable and fine plants; and culturing the hotness-resistant Lucerne which can normally go through the summer. The hotness-resistant Lucerne is more suitable for the growth in southern China and increases the supply of fine forage grass.
Description
Technical field
The present invention relates to a kind of transgenic culturing method of heat-proof lucerne.
Background technology
Clover is because of its high yield, high-quality, utilization year limit for length, well developed root system, and the structure of can improving the soil increases soil fertility, is described as " king of herbage ".In China, adjustment along with agricultural structure and livestock industry structure, grain-saving type livestock industry, phytophagous animal aquaculture particularly, present good development momentum, because clover conclusive effect of performance in the per unit area yield that improves milk cow, mutton sheep has become the first-selected fodder crop that China's " ternary " pattern of farming is adjusted, alfalfa industry exists wide development space as emerging sunrise industry.
Be difficult to plantation in China south owing to the reason of summer high temperature causes alfalfa, southern alfalfa hay is that transportation cost is high, has both improved feeding cost, and difficult quality guarantee, has greatly hindered the development of southern herbvore livestock and poultry from north allocation and transportation substantially.The CBF gene is a class gene of anti-retrocorrelation of finding several years ago, its coded activating transcription factor can with the DNA controlling element specific combination of CRT/DRE, promote to contain a plurality of heat and the drought-induced expression of gene of this controlling element in the promotor, thereby cause the enhancing of plant thermotolerance and resistance of reverse.
Under this background, the present invention proposes a kind of transgenic culturing method of heat-proof lucerne just, is intended to cultivate the heat-proof lucerne new variety that are more suitable for the south plantation, promotes the development of southern alfalfa alfalfa industry, increases the supply of high-quality forage grass.
Summary of the invention
The object of the present invention is to provide a kind of transgenic culturing method of heat-proof lucerne, to obtain to be fit to the heat-proof lucerne new variety of south plantation.
The transgenic culturing method of heat-proof lucerne is characterized in that may further comprise the steps:
1) getting main cotyledon of planting the alfalfa kind is explant, is seeded to inducing culture, induces the formation callus under 25 ℃ of optical condition;
2) get the callus of inducing 2 weeks of formation, be transferred to subculture medium, succeeding transfer culture to callus enters the fast breeding phase;
3) get the callus of fast breeding, carry out under 25 ℃ of dark condition that the acceptor callus is pre-to be cultivated 3 days;
4) get pre-incubated callus, in agrobacterium suspension, soaked 15 minutes, discard bacterium liquid afterwards, blot, be inoculated on the common substratum, under 23 ℃ of dark condition, cultivated altogether 3 days with aseptic filter paper;
5) get common cultured calli, after cleaning, drying, transfer to screening culture medium, under 25 ℃ of dark condition, cultivated for 2 weeks, collect the resistant calli that newly grows, be transferred on the new screening culture medium, carry out continuous 2 again and take turns the subculture screening, continue 1 time every 2 turnovers;
6) get 3 resistant callis of taking turns after the screening, be transferred to division culture medium, induce seedling differentiation, the antagonism plant carries out glucuronidase gene organization chemical staining and Molecular Detection, selects and remain to contain the transfer-gen plant of goal gene;
7) be taken into the transfer-gen plant of choosing, be transferred to heat-resisting 1 week of evaluation under 35 ℃ of temperature and the illumination 10000Lux, move to physical environment and grew for 1 week, place heat-resisting again 1 week of evaluation under 35 ℃ and the 10000Lux thereafter again, not withered and yellow, the well-grown transfer-gen plant of the blade of selecting and remain carries out seed growing;
8) continue the transgenic line that the plantation step 7) is screened, stable on heating expression stability is verified in the indoor and outdoor evaluation, and stable elite plant strain is carried out seminal propagation, and the heat-proof lucerne in summer can be normally got in cultivation.
Above-mentioned stability is meant that the thermotolerance of selected alfalfa can express consistently (2 seasons and more than) between different year between (2 years and more than), season and intersite (2 places and more than), and characteristic is not subjected to the remarkably influenced in time, season and place.
Among the present invention, said inducing culture adds NAA5mg, BA0.5mg, sucrose 30g, agar powder 6.8g for the MS minimum medium, pH sterilization preceding 5.8.
Among the present invention, said subculture medium adds NAA0.5mg, BA2mg, sucrose 30g, agar powder 6.8g for the MS minimum medium, pH sterilization preceding 5.8.
Among the present invention, said substratum altogether adds NAA0.5mg, BA2mg, sucrose 30g, agar powder 6.8g for the MS minimum medium, pH sterilization preceding 5.8.
Among the present invention, said screening culture medium is added Totomycin 100mg, NAA0.5mg, BA2mg, sucrose 30g, agar powder 6.8g for the MS minimum medium, pH sterilization preceding 5.8.
Among the present invention, said division culture medium adds KT1mg, sucrose 30g, agar powder 6.8g for the MS minimum medium, pH sterilization preceding 5.8.
Above-mentioned MS minimum medium is macroelement (NH
4NO
3650mg, KNO
3900mg, CaCl
22H
2O 440mg, MgSO
47H
2O 370mg, KH
2PO
4700mg), trace element (KI0.83mg, H
3BO
36.2mg, MnSO
44H
2O 22.3mg, ZnSO
47H
2O 8.6mg, Na
2MnO
42H2O0.25mg, CuSO
45H
2O 0.025mg, CoCl
26H
2O 0.025mg, FeSO
47H
2O 27.8mgNa
2-EDTA2H
2O 37.3mg) and organic composition (inositol 100mg, nicotinic acid 0.5mg, vitamin B6 0.5mg, vitaminB10 .5mg, glycine 2mg).
Among the present invention, the bacterial strain of said Agrobacterium is super virulence type EHA105, includes plasmid pCMD.The T-DNA district of pCMD carries the Arabidopis thaliana CBF1 of anti-retrocorrelation gene, hygromycin phosphotransferase gene and glucuronidase gene.CBF1 gene length 642bp constitutes the expression framework jointly with CaMV35S promotor and rouge alkali synthetase terminator.Bacterial strain EHA105 can conveniently buy or give acquisition, in general plant genetic engineering laboratory preservation is arranged all, referring to document Wu Guan front yard etc., and Scientia Agricultura Sinica, 2005,38 (12): 2395-240.
Among the present invention, said glucuronidase gene organization chemical staining method is referring to Rueb and Hensgens.Rice Genetics Newsletter, 1989, (6): 168-169, the hygromycin gene molecular detecting method is referring to Wu Guanting etc., Scientia Agricultura Sinica, 2005,38 (12): 2395-240.
The present invention has the beneficial effect of following several respects:
The heat-proof lucerne new variety of cultivating can normally be got over the summer, be more suitable for China's south plantation, promote the alfalfa clover at the southern industry development of China, increase the supply of high-quality forage grass, promote the development of China's phytophagous animal aquaculture, be particularly advantageous in the rural economic development and the farmers' income of promoting the southern hills mountain area.
Embodiment
Below further specify the present invention by specific examples.
Embodiment 1:
Cotyledon with alfalfa kind " Victoria " is an explant, is seeded to inducing culture, induces the formation callus; Get and induce the callus that formed for 2 weeks, be transferred to subculture medium, succeeding transfer culture to callus enters the fast breeding phase; Get the callus of fast breeding, under 25 ℃ of dark condition, carry out the pre-cultivation of acceptor callus 3 days; Get pre-incubated callus (about 3000 culture dish contain the callus more than 30000), in agrobacterium suspension, soaked 15 minutes, discard bacterium liquid afterwards, blot, be inoculated on the common substratum, under 23 ℃ of dark condition, cultivated altogether 3 days with aseptic filter paper; Get common cultured calli, after cleaning, drying, transfer to screening culture medium, under 25 ℃ of dark condition, cultivated for 4 weeks, collect the resistant calli (about 200) that grows, be transferred on the new screening culture medium, carry out continuous 2 again and take turns the subculture screening, continue 1 time every 4 turnovers; Get 3 resistant callis (53) of taking turns after the screening, be transferred to division culture medium, induce seedling differentiation 49 strains, the antagonism plant carries out glucuronidase gene organization chemical staining and hygromycin gene Molecular Detection, choose differentiation seedling 27 strains that contain goal gene, be transferred to root media and grow up to normal seedling 19 strains; Be taken into the transfer-gen plant of choosing, be transferred to and carry out 1 week of resistance to cold evaluation, 2 rounds, evil, well-grown transfer-gen plant 5 strains of not being heated of selecting and remain under 35 ℃ of temperature and the illumination 10000Lux; Continue the selected transgenosis alfalfa strain system of plantation, estimate the heat-stable expression stability of Simulation evaluation in the 2007-2008 indoor and outdoor, the transgenosis heat-proof lucerne strain that obtains stably express is 1, i.e. THT-Alfa1-1.The THT-Alfa1-1 that cultivates of plantation can normally get over the summer, and promptly hot season in midsummer, blade kept green, can normal growth, can provide the stable feed that high-quality nutrition is provided, and common alfalfa material is because thermo-labile, and production is stagnated in dormancy, and blade is withered and yellow.
Claims (7)
1. the transgenic culturing method of a heat-proof lucerne is characterized in that may further comprise the steps:
1) getting main cotyledon of planting the alfalfa kind is explant, is seeded to inducing culture, induces the formation callus under 25 ℃ of optical condition;
2) get the callus of inducing 2 weeks of formation, be transferred to subculture medium, succeeding transfer culture to callus enters the fast breeding phase;
3) get the callus of fast breeding, carry out under 25 ℃ of dark condition that the acceptor callus is pre-to be cultivated 3 days;
4) get pre-incubated callus, soaked 15 minutes in agrobacterium suspension, discard bacterium liquid afterwards, blot with aseptic filter paper, be inoculated on the common substratum, cultivated altogether 3 days under 23 ℃ of dark condition, the bacterial strain of said Agrobacterium is EHA105, includes plasmid pCMD;
5) get common cultured calli, after cleaning, drying, transfer to screening culture medium, under 25 ℃ of dark condition, cultivated for 2 weeks, collect the resistant calli that newly grows, be transferred on the new screening culture medium, carry out continuous 2 again and take turns the subculture screening, continue 1 time every 2 turnovers;
6) get 3 resistant callis of taking turns after the screening, be transferred to division culture medium, induce seedling differentiation, the antagonism plant carries out glucuronidase gene organization chemical staining and Molecular Detection, selects and remain to contain the transfer-gen plant of goal gene;
7) be taken into the transfer-gen plant of choosing, be transferred to heat-resisting 1 week of evaluation under 35 ℃ of temperature and the illumination 10000Lux, move to physical environment and grew for 1 week, place heat-resisting again 1 week of evaluation under 35 ℃ and the 10000Lux thereafter again, not withered and yellow, the well-grown transfer-gen plant of the blade of selecting and remain carries out seed growing;
8) continue the transgenic line that the plantation step 7) is screened, stable on heating expression stability is verified in the indoor and outdoor evaluation, and stable elite plant strain is carried out seminal propagation, and the heat-proof lucerne in summer can be normally got in cultivation.
2. the transgenic culturing method of heat-proof lucerne according to claim 1 is characterized in that said inducing culture adds NAA5mg, BA0.5mg, sucrose 30g, agar powder 6.8g for the MS minimum medium, pH sterilization preceding 5.8.
3. the transgenic culturing method of heat-proof lucerne according to claim 1 is characterized in that said subculture medium adds NAA0.5mg, BA2mg, sucrose 30g, agar powder 6.8g for the MS minimum medium, pH sterilization preceding 5.8.
4. the transgenic culturing method of heat-proof lucerne according to claim 1 is characterized in that said substratum altogether adds NAA0.5mg, BA2mg, sucrose 30g, agar powder 6.8g for the MS minimum medium, pH sterilization preceding 5.8.
5. the transgenic culturing method of heat-proof lucerne according to claim 1 is characterized in that said screening culture medium is MS minimum medium interpolation Totomycin 100mg, NAA0.5mg, BA2mg, sucrose 30g, agar powder 6.8g, pH sterilization preceding 5.8.
6. the transgenic culturing method of heat-proof lucerne according to claim 1 is characterized in that said division culture medium adds KT1mg, sucrose 30g, agar powder 6.8g for the MS minimum medium, pH sterilization preceding 5.8.
7. according to the transgenic culturing method of arbitrary described heat-proof lucerne among the claim 2-6, it is characterized in that said MS minimum medium is NH
4NO
3650mg, KNO
3900mg, CaCl
22H
2O 440mg, MgSO
47H
2O 370mg, KH
2PO
4700mg, KI 0.83mg, H
3BO
36.2mg, MnSO
44H
2O22.3mg, ZnSO
47H
2O 8.6mg, Na
2MnO
42H
2O 0.25mg, CuSO
45H
2O 0.025mg, CoCl
26H
2O 0.025mg, FeSO
47H
2O 27.8mg, Na
2-EDTA2H
2O 37.3mg, inositol 100mg, nicotinic acid 0.5mg, vitamin B6 0.5mg, VITMAIN B1 0.5mg and glycine 2mg.
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| CN105132457A (en) * | 2015-10-19 | 2015-12-09 | 宁夏农林科学院 | Method for conducting rapid genetic transformation of alfalfa |
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| WO2011116708A1 (en) * | 2010-03-26 | 2011-09-29 | 中国科学院上海生命科学研究院 | Method of changing plants' characters |
| CN102499094B (en) * | 2011-11-11 | 2013-08-28 | 中国农业科学院草原研究所 | Method for increasing efficiency of tissue culture of Medicago sativa L. |
| CN113040009A (en) * | 2021-04-07 | 2021-06-29 | 张家口市农业科学院(河北省高寒作物研究所) | Screening and planting method for grassland pasture germplasm resources |
| CN114711146A (en) * | 2022-05-19 | 2022-07-08 | 中国农业科学院北京畜牧兽医研究所 | Screening method of alfalfa tissue culture rapid differentiation plants |
| CN117502127B (en) * | 2024-01-03 | 2024-03-19 | 中国农业科学院草原研究所 | Directional cultivation method for grassland community structure based on plant priority effect |
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| CN101283673A (en) * | 2008-05-12 | 2008-10-15 | 浙江大学 | Heat-proof lucerne breeding process |
| US7449617B2 (en) * | 2004-01-05 | 2008-11-11 | The Regents Of The University Of California | Plants with elevated levels of gallic acid/polyphenol oxidase and methods of generating such plants |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7449617B2 (en) * | 2004-01-05 | 2008-11-11 | The Regents Of The University Of California | Plants with elevated levels of gallic acid/polyphenol oxidase and methods of generating such plants |
| CN101283673A (en) * | 2008-05-12 | 2008-10-15 | 浙江大学 | Heat-proof lucerne breeding process |
Non-Patent Citations (2)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105132457A (en) * | 2015-10-19 | 2015-12-09 | 宁夏农林科学院 | Method for conducting rapid genetic transformation of alfalfa |
| CN105132457B (en) * | 2015-10-19 | 2018-08-03 | 宁夏农林科学院 | A kind of method of fast genetic transformation clover |
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