CN102124947B - Efficient transgene method for inducing caespitose shoots of soybean at high frequency - Google Patents
Efficient transgene method for inducing caespitose shoots of soybean at high frequency Download PDFInfo
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Abstract
The invention discloses an efficient transgene method for inducing caespitose shoots of soybean at a high frequency. The method comprises the following steps of: removing two cotyledons, primary leaves and redundant hypocotyls from an aseptic seedling along hypocotyls and growing point meristematic tissues of cotyledons to prepare an explant; analyzing pure tiny particles by adopting quartz sand; fully and uniformly mixing the pure tiny particles with engineering agrobacterium solution; performing intense vortex concussion on the explant on a vortex concussion instrument; and uniformly manufacturing a wound. According to the method, the dip dyeing cycle is shortened, the co-culture time is reduced, the toxic action on the plant explant is lightened and simultaneously the infected area of agrobacterium tumefaciens is increased; compared with cotylcdonary node transformation mediated by the agrobacterium tumefaciens, more caespitose shoot (mbt) tissues/tissue blocks of bud-leaf cushions are formed on a bud-induced culture medium, and transgene bud primordial can be detected about three weeks after the caespitose shoot (mbt) tissues/tissue blocks of the bud-leaf cushions are infected by the agrobacterium tumefaciens; besides, the regeneration frequency is high, so the genetic transformation efficiency of the soybean is greatly improved.
Description
Technical field
The present invention relates to biological technical field, exactly is the grow thickly high-efficient transgenic method of bud of a kind of high-frequency induction soybean.
Background technology
Soybean belongs to Papilionaceae, Glycine.Being one of the whole world most important high protein oil plant economic crops, is the main plant property dietary protein origin of human foods and animal-breeding.The soil of the whole world above 5,000 ten thousand hectares is used to planting soybean at present, and the quality and yield that therefore how to improve soybean has strategic meaning.The research and development of modern biotechnology is for through gene engineering improvement agricultural byproducts necessary precondition being provided, so people have invented a lot of transgene methods.Mainly contain particle gun conversion method, agrobacterium mediation converted method, pollen tube passage method, virus-mediated conversion (transient transfection), liposome-mediated protoplast transformation etc.These methods are applied to economic crops such as cereal crops such as soybean, corn, wheat and rape, have greatly promoted the genetic improvement of plant quality or crop varieties, for major contribution has been made in economic development with society.
Traditional agriculture bacillus mediated genetic transforming method, thus be to utilize infect the method that characteristic import foreign gene Plant Genome of Agrobacterium tumefaciems to plant.Because of its copy number is low, integrate complete, heredity more stable, receive the influence of genes of interest molecular weight less and easy and simple to handle, be widely used in the genetic transformation of plant.But no matter be important crops such as soybean, corn or wheat, its genetically modified transformation efficiency and regeneration efficiency are not high, only 2%-5%.Cause great waste so just for human resources, financial cost, also prolonged the time and the space of achievement output and release.
Therefore the object of the invention is that the shoot regeneration system of growing thickly of setting up the soybean high frequency reaches Agrobacterium genetic conversion system efficiently, improves regeneration efficiency and the transformation efficiency of soybean simultaneously.
Summary of the invention
The present invention seeks to the high-efficient transgenic method that provides a kind of high-frequency induction soybean to grow thickly bud.
This method may further comprise the steps:
1) with the soybean seedling of aseptic culture growing point, removes hypocotyl and part primary leaf, keep the primary leaf base portion, the preparation explant along hypocotyl and cotyledon;
2) explant is put into centrifuge tube; The agrobacterium liquid that adding contains genes of interest to be transformed is to 50ml; The pure quartz sand 2.0-2.3g of 20-200 goal analysis, in whirlpool concussion appearance, the concussion of 2000-3000rpm whirlpool mixed after 5-10 minute; Blot unnecessary bacterium liquid, be inoculated in solid and be total in the culture medium;
3) explant is transferred in the SIM medium induction of resistance grow thickly bud and/or MBT regeneration;
4) continue to cultivate by conventional method.
Described step 1) is that aseptic seedling is removed two cotyledons, primary leaf and unnecessary following embryo by, preparation explant along the growing point meristematic tissue of hypocotyl and cotyledon;
Described Agrobacterium is Agrobacterium tumefaciems EHA105 or Agrobacterium tumefaciems AGL1;
Be preferably and add quartz sand 2.3 gram, the concussion of 3000rpm whirlpool mixed 10 minutes, 25 ℃ of dark culturing 24 hours.
The soybean high frequency that adopts method of the present invention to realize the inducing and transforming of bud tissue of growing thickly, more traditional agriculture bacillus mediated genetic transforming method has following advantage:
(1) the present invention takes the lead in adopting quartz sand molecule (analyzing pure level) and the abundant mixing of engineering agrobacterium liquid to go up the method for violent vortex concussion explant at vortex-shaker (QL866) first; Evenly make wound; Reduce incubation time altogether; Both alleviated toxic action, and strengthened the area that infects of Agrobacterium again simultaneously, thereby improved the genetic transformation efficiency of soybean greatly plant explants.
(2) explant that uses removes cotyledon and primary leaf (seeing accompanying drawing 1) from nascent leaf segment base portion (growing point); To explant not injury basically; Compare the piece of tissue that forms the bud of more growing thickly (MBT) tissue/bud-leaf pillow at the bud inducing culture with traditional agriculture bacillus mediated cotyledonary node conversion; They detect genetically modified bud original hase at about 3 Zhou Houke of agroinfection; And regeneration frequency is very high, can reach more than the 80%-90%, can improve the grow thickly regeneration efficiency of bud of soybean greatly from this point.
(3) explant that adopts the inventive method to obtain is easy to expand in a large number numerous resistance bud tissue (MBT) of growing thickly, more traditional method more low cost, more save time, obtain more effortlessly a large amount of transfer-gen plants.
(4) the dip-dye cycle shortens, and the present invention makes common incubation time be reduced to 2 days, and the common incubation time of traditional method is 3 days, has both alleviated the toxic action to plant explants, is unlikely to influence its transformation efficiency again.
(5) in addition the present invention also has a notable feature: save more medium and culture space, for traditional leaf segment method for transformation, only can cultivate 5 explants at a culture dish, yet the present invention can place up to 15 explants at single culture dish.
(6) in all soybean culture kinds, has similar regeneration capacity because of the growing point meristematic cell; So the method is not limited by genotype, applicable to most soybean culture kinds, and operation is very easy; Cost is very cheap; Transformation efficiency improves, and can obtain the transfer-gen plant that necessary for human is wanted in a large number fast, has accelerated the research and development process of the rearing new variety of soybean greatly.
Description of drawings
Fig. 1 is the grow thickly preparation sketch map of high-efficient transgenic method explant of bud of high-frequency induction soybean.
Fig. 2 is the preparation sketch map of traditional cotyledonary node transgenic method explant.
Fig. 3 is the pBI121 plasmid map.
Fig. 4 is the grow thickly high-efficient transgenic method flow chart of bud of high-frequency induction soybean.
Fig. 5 is traditional cotyledonary node transgenic method flow chart.
Fig. 6 is the expression of genes of interest in transfer-gen plant.
Embodiment
Further specify content of the present invention and effect through the practical implementation method below, this implementation method is merely elaboration relevant details of the present invention and embodiment preferred further, and embodiment does not limit range of application of the present invention in any form.
1. explant preparation
Through the disinfection by chlorine method soya seeds is carried out surface sterilization.200 soya seeds are on average put into two opening culture dishes, place a closed container, in the beaker that fills the 100ml70% clorox, add the 3ml12mol/L concentrated hydrochloric acid, be along walls of beaker adding drop by drop, sealing is noted in the fumigation of spending the night.Seed after the sterilization is inoculated in seed germination medium GM (seeing accompanying drawing 4A) (B5/B5+2% sucrose+3mM MES+TDZ 0.1mg/L+0.8% agar; PH5.8) cultivate after 7 days (seeing accompanying drawing 4B) in advance for 25 ℃; Prepare explant with the present invention and traditional method respectively; The inventive method: the aseptic seedling of 100 sproutings is removed two cotyledons, primary leaf and unnecessary hypocotyl along the growing point meristematic tissue of hypocotyl and cotyledon, obtain 100 explants (seeing accompanying drawing 1) that are used to transform; Conventional method: the aseptic seedling of 100 sproutings is removed unnecessary hypocotyl, primary leaf along the growing point meristematic tissue of cotyledon, keep two cotyledons, obtain 100 explants (seeing accompanying drawing 2) that are used to transform.
2. explant infects
(1) engineering Agrobacterium bacterium liquid preparation: the Agrobacterium tumefaciems bacterial classification (EHA105/AGL1 is preserved by this laboratory respectively) that will contain pBI121 plasmid vector (plasmid map is seen accompanying drawing 3) joins (dusty yeast 10g/L in 50 milliliters of YEP liquid nutrient mediums that contain kanamycin; Peptone 10g/L; Sodium chloride 5g/L; PH7.0), 28 ℃, the 180-200rpm incubated overnight is to OD
602Between 0.5-0.7.Collected thalline in centrifugal 10 minutes at Sigma HermleZ36HK centrifuge 5000rpm, and the common culture fluid of usefulness (1/10B5/B5+3% sucrose+200 μ M acetosyringone+20mM MES+0.03%Silwet77, pH5.4) resuspended to OD
600=0.5-0.7.
(2) above-mentioned resuspended Agrobacterium being infected liquid adds with 100 explants through the inventive method preparation and fills 2.3g quartz sand molecule (Beijing Chemical Plant produces; Analyze pure level 20-200 order) aseptic 50ml centrifuge tube in fully mixing go up the violent vortex concussion of 2000-3000rpm explant at vortex-shaker (QL866), carry out Agrobacterium and infect.Time of infection is 5-10min; Add in 100 ml flasks in the full temperature shaken cultivation of HZQ-F160 case with 100 explants through conventional method preparation and above-mentioned resuspended Agrobacterium is infected liquid, 25 ℃, 3000rpm carries out Agrobacterium and infects.Time of infection is 15-30min.
3. explant is cultivated altogether
Respectively above-mentioned explant after two kinds of methods infect is blotted unnecessary bacterium liquid with aseptic filter paper, be inoculated in the solid that is covered with aseptic filter paper and be total in the culture medium, wherein, 25 ℃ of the inventive method, dark culturing 24 hours (seeing accompanying drawing 4C); And 25 ℃ of conventional methods, dark culturing 72 hours (seeing accompanying drawing 5C).
4. bud/MBT the tissue of growing thickly is induced
The explant of respectively above-mentioned two kinds of methods being cultivated is altogether transferred to SIM medium (B5/B5+3% sucrose+BA0.5mg/L+250mg/L Cef+100mg/L Kan+3mM MES+0.8% agar; PH5.8) the induction of resistance bud/MBT regeneration of growing thickly in; The explant of handling through the inventive method can produce the MBT tissue earlier; Further differentiate a large amount of buds of growing thickly again and (see accompanying drawing 4D, 4E); Conventional method then directly produce the bud of growing thickly (see accompanying drawing 5D, 5E, 5F).Equal 25 ℃, cultivated 30 days in 18/6 hour, whenever shift an explant in fresh SIM medium at a distance from two weeks.Each transfer all will be done a new otch in the bases of explant.
5.MBT hyperblastosis
The above-mentioned resistance MBT tissue that obtains through the inventive method is transferred in the subculture medium, breeds MBT tissue (seeing accompanying drawing 4F) in a large number.(B5/B5+3% sucrose+TDZ 0.1mg/L+250mg/L Cef+100mg/L Kan+3mM MES+0.8% agar, pH5.8)
6. grow thickly bud elongation of resistance
Respectively with the resistance of two kinds of methods of above-mentioned warp differentiation grow thickly bud be transferred among the elongation medium SEM (1/2MS/B5+2% sucrose+1mg/L zeatin ribonucleotide+0.1mg/L heteroauxin+0.5mg/L gibberellin+100mg/L pyroglutamic acid+50mg/L asparagine+250mg/L Cef+3mM MES+0.8% agar, pH5.8).Be stretched to 3-4 centimetre seedling and (see accompanying drawing 4G, 5G) until the resistance bud of growing thickly.
7. resistant plant is taken root
Respectively the seedling of two kinds of methods of above-mentioned warp elongation is transferred among the root media RM (1/2MS/MS+2% sucrose+0.5mg/LNAA+100mg/L pyroglutamic acid+50mg/L asparagine+250mg/L Cef+3mM MES+0.8% agar, pH5.8).Grow 2-3 bar root system until resistant plant and (see accompanying drawing 4H, 5H).
8. transfer-gen plant is identified
When the blade of treating two kinds of method regeneration plants of above-mentioned warp grew to suitable size, clip transgenosis and non-transgenic soybean leaves detected respectively.Adopt the fluophotometer method to measure its gus gene active (with reference to the fluorescent quantitation method of Jefferson (1987)) in the present embodiment, three repeated tests are averaged, and carry out the analysis of gus gene expression activity.
The result sees that table 1 is with shown in the accompanying drawing 6:
The expression activity analysis of table 1 genes of interest in transfer-gen plant and non-transgenic plant
Testing result shows: 15 times of the specific activity non-transgenic plant mean heights of GUS in the rotaring gene plant blade that above-mentioned two kinds of methods obtain.Illustration purpose gene GUS expresses in transfer-gen plant as reporter gene simultaneously.
9. transfer-gen plant refines seedling
Choose above-mentionedly through identifying the transfer-gen plant refining seedling be positive, bottleneck is crack and in medium, water suitable moisture content with cultivating, and prevents the dry hardening of medium; Cultivated in 18/6 hour; Domestication in the controlled environment chamber, the domestication time is about 48 hours and (sees accompanying drawing 4I, 5I).
10. transfer-gen plant is transplanted
Transfer-gen plant after the above-mentioned domestication is taken out from medium; Thoroughly remove the remaining medium of root; Seedling replanting to filling vermiculite: in the flowerpot of the composite soil of soil=1: 1; Hide seedling with preservative film and preserve moisture, place the indoor cultivation of artificial climate to remove preservative film after 2 days, normal management.Obtain transfer-gen plant, can be used for follow-up genetic analysis and (see accompanying drawing 4J, 5J).
In conjunction with content of the present invention following two embodiment are provided:
The grow thickly high-efficient transgenic method of bud of A Agrobacterium tumefaciems EHA105 mediation high-frequency induction soybean
The experiment kind is that Ji is educated 72,100;
Choose smooth surface and do not have 100 of the unabroken soybean mature seeds of assorted spot; It is carried out the chlorine surface sterilization described in implementation method 1; Seed after the sterilization is inoculated in seed germination medium GM (seeing accompanying drawing 4A) and cultivates after 7 days (seeing accompanying drawing 4B) in advance for 25 ℃; To remove two cotyledons, primary leaf and unnecessary hypocotyl along the growing point meristematic tissue of hypocotyl and cotyledon by the aseptic seedling of 100 seed germinations, obtain 100 explants (seeing accompanying drawing 1) that are used to transform;
100 explants of method for preparing are infected liquid with resuspended Agrobacterium add in the aseptic 50ml centrifuge tube that fills 2.3g quartz sand molecule (analyzing pure level) fully mixing and go up the violent vortex concussion of 3000rpm explant, carry out Agrobacterium and infect at vortex-shaker (QL866).Time of infection is 5-10min; Be inoculated in the solid that is covered with aseptic filter paper then and be total in the culture medium, 25 ℃, dark culturing 24 hours (seeing accompanying drawing 4C);
Above-mentioned 100 explants after cultivating are altogether transferred to induction of resistance MBT regeneration in the SIM medium, and each explant can produce MBT tissue earlier, further differentiate again a large amount of buds of growing thickly (see accompanying drawing 4D, 4E, 4F); 25 ℃, cultivated 30 days in 18/6 hour, whenever shift an explant in fresh SIM medium at a distance from two weeks.Each transfer all will be done a new otch in the bases of explant.The bud number of growing thickly (seeing attached list 2) that breaks up on each explant of back statistics all around.
B Agrobacterium tumefaciems EHA105 mediates traditional cotyledonary node transgenic method
The experiment kind is that Ji is educated 72,100;
Choose smooth surface and do not have 100 of the unabroken soybean mature seeds of assorted spot; It is carried out the chlorine surface sterilization described in implementation method 1; Seed after the sterilization is inoculated in seed germination medium GM (seeing accompanying drawing 5A) and cultivates after 7 days (seeing accompanying drawing 5B) in advance for 25 ℃; To remove unnecessary hypocotyl, primary leaf and keep two cotyledons by the aseptic seedling of 100 seed germinations growing point meristematic tissue, obtain 100 explants (seeing accompanying drawing 2) that are used to transform along cotyledon;
100 explants of method for preparing are infected liquid with resuspended Agrobacterium add in 100 ml flasks in the full temperature shaken cultivation of HZQ-F160 case, 25 ℃, 3000rpm carries out Agrobacterium and infects.Time of infection is 15-30min. Be inoculated in the solid that is covered with aseptic filter paper then and be total in the culture medium, 25 ℃, dark culturing 72 hours (seeing accompanying drawing 5C);
Above-mentioned 100 explants after cultivating are altogether transferred in the SIM medium induction of resistance shoot regeneration of growing thickly, each explant can differentiate some indefinite buds (see accompanying drawing 5D, 5E, 5F); 25 ℃, cultivated 30 days in 18/6 hour, whenever shift an explant in fresh SIM medium at a distance from two weeks.Each transfer all will be done a new otch in the bases of explant.The bud number of growing thickly (seeing attached list 2) that breaks up on each explant of back statistics all around.
Result such as following table:
The inventive method of table 2 Agrobacterium tumefaciems EHA105 mediation and the comparison of conventional method
(the bud number of always growing thickly of the average transgenosis bud number of average genetic transformation efficiency=per 100 explants differentiation/explant differentiation)
Embodiment 3
The grow thickly high-efficient transgenic method of bud of A Agrobacterium tumefaciems AGL1 mediation high-frequency induction soybean
The experiment kind is that Ji is educated 72,100;
Choose smooth surface and do not have 100 of the unabroken soybean mature seeds of assorted spot; It is carried out the chlorine surface sterilization described in implementation method 1; Seed after the sterilization is inoculated in seed germination medium GM (seeing accompanying drawing 4A) and cultivates after 7 days (seeing accompanying drawing 4B) in advance for 25 ℃; To remove unnecessary hypocotyl, primary leaf along the growing point meristematic tissue of cotyledon by the aseptic seedling of 100 seed germinations, keep two cotyledons, obtain 100 explants (seeing accompanying drawing 2) that are used to transform;
100 explants of method for preparing are infected liquid with resuspended Agrobacterium add in the aseptic 50ml centrifuge tube that fills 2.0g quartz sand molecule (analyzing pure level) fully mixing and go up the violent vortex concussion of 2000rpm explant, carry out Agrobacterium and infect at vortex-shaker (QL866).Time of infection is 5-10min; Be inoculated in the solid that is covered with aseptic filter paper then and be total in the culture medium, 25 ℃, dark culturing 24 hours (seeing accompanying drawing 4C);
Above-mentioned 100 explants after cultivating are altogether transferred to induction of resistance MBT regeneration in the SIM medium, and each explant can produce MBT tissue earlier, further differentiate again a large amount of buds of growing thickly (see accompanying drawing 4D, 4E, 4F); 25 ℃, cultivated 30 days in 18/6 hour, whenever shift an explant in fresh SIM medium at a distance from two weeks.Each transfer all will be done a new otch in the bases of explant.The bud number of growing thickly (seeing attached list 3) that breaks up on each explant of back statistics all around.
B Agrobacterium tumefaciems AGL1 mediates traditional cotyledonary node transgenic method
The experiment kind is that Ji is educated 72,100;
Choose smooth surface and do not have 100 of the unabroken soybean mature seeds of assorted spot; It is carried out the chlorine surface sterilization described in implementation method 1; Seed after the sterilization is inoculated in seed germination medium GM (seeing accompanying drawing 5A) and cultivates after 7 days (seeing accompanying drawing 5B) in advance for 25 ℃; To remove unnecessary hypocotyl, primary leaf along the growing point meristematic tissue of cotyledon by the aseptic seedling of 100 seed germinations, keep two cotyledons, obtain 100 explants (seeing accompanying drawing 2) that are used to transform;
100 explants of method for preparing are infected liquid with resuspended Agrobacterium add in 100 ml flasks in the full temperature shaken cultivation of HZQ-F160 case, 25 ℃, 3000rpm carries out Agrobacterium and infects.Time of infection is 15-30min. Be inoculated in the solid that is covered with aseptic filter paper then and be total in the culture medium, 25 ℃, dark culturing 72 hours (seeing accompanying drawing 5C);
Above-mentioned 100 explants after cultivating are altogether transferred in the SIM medium induction of resistance shoot regeneration of growing thickly, each explant can differentiate some indefinite buds (see accompanying drawing 5D, 5E, 5F); 25 ℃, cultivated 30 days in 18/6 hour, whenever shift an explant in fresh SIM medium at a distance from two weeks.Each transfer all will be done a new otch in the bases of explant.The bud number of growing thickly (seeing attached list 3) that breaks up on each explant of back statistics all around.
The inventive method of table 3 Agrobacterium tumefaciems EHA105 mediation and the comparison of conventional method
Show from above result: infect the soybean varieties Ji with the Agrobacterium tumefaciems AGL1 that contains the pBI121 plasmid vector and educate 72, method of the present invention induces the soybean transgene that the differentiates bud of growing thickly to how on average 3-4 transgenosis bud at least than conventional method; And time of infection and incubation time altogether are still less, genetic transformation efficiency is higher, embodied the high-frequency induction soybean more and grown thickly that the high-efficient transgenic method of bud saves time, laborsaving and characteristics of high efficiency.
(the bud number of always growing thickly of the average resistant buds number of genetic transformation efficiency=per 10 explants differentiation/explant differentiation)
Claims (4)
1. the high-frequency induction soybean high-efficient transgenic method of bud of growing thickly may further comprise the steps:
1) with the soybean seedling of aseptic culture growing point, removes hypocotyl and part primary leaf, keep the primary leaf base portion, the preparation explant along hypocotyl and cotyledon;
2) explant is put into centrifuge tube; The agrobacterium liquid that adding contains genes of interest to be transformed is to 50ml, and the pure quartz sand 2.0-2.3 of 20-200 goal analysis g is in whirlpool concussion appearance; The concussion of 2000-3000rpm whirlpool mixed after 5-10 minute; Blot unnecessary bacterium liquid, be inoculated in solid and be total in the culture medium dark culturing 24 hours;
3) explant is transferred in the SIM medium induction of resistance shoot regeneration of growing thickly; Described SIM medium is B5+3% sucrose+BA 0.5mg/L+250mg/L Cef+100mg/L Kan+3mM MES+0.8% agar, pH5.8;
4) continue to cultivate by conventional method.
2. the grow thickly high-efficient transgenic method of bud of a kind of high-frequency induction soybean according to claim 1; It is characterized in that: described explant; Be that aseptic seedling is removed two cotyledons, primary leaf and unnecessary hypocotyl along the growing point meristematic tissue of hypocotyl and cotyledon, the preparation explant.
3. the grow thickly high-efficient transgenic method of bud of a kind of high-frequency induction soybean according to claim 1 and 2, it is characterized in that: described Agrobacterium is Agrobacterium tumefaciems EHA105 or Agrobacterium tumefaciems AGL1.
4. the grow thickly high-efficient transgenic method of bud of a kind of high-frequency induction soybean according to claim 3, it is characterized in that: add quartz sand 2.3 grams, the concussion of 3000rpm whirlpool mixed 10 minutes
, 25 ℃ of dark culturing 24 hours.
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| 李文霞等.农杆菌介导大豆子叶节转化系统的优化.《中国农业科学》.2008,第41卷(第4期),971-977. * |
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