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High-efficiency genetic transformation method using soybean Jilin No.35 embryonic tip as explant

A genetic transformation method and explant technology, applied in botany equipment and methods, using vectors to introduce foreign genetic material, horticultural methods, etc., can solve the problems of low transformation rate and strong genotype dependence, and achieve low regeneration rate Larger, shorter regeneration cycle, and the effect of solving the difficulty of soybean genetic transformation

Inactive Publication Date: 2012-07-04
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the embryo tip regeneration system also has some fatal shortcomings, such as strong genotype dependence and low transformation rate.

Method used

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  • High-efficiency genetic transformation method using soybean Jilin No.35 embryonic tip as explant
  • High-efficiency genetic transformation method using soybean Jilin No.35 embryonic tip as explant
  • High-efficiency genetic transformation method using soybean Jilin No.35 embryonic tip as explant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: The germination rate of clustered buds of soybean embryo tips of different genotypes

[0037] Using Jilin 35, Jilin 47, Ping'andou 8, Dongnong 42, American flat stem, Jidou No. 1, Jidou No. 2, Jidou No. 3, Hefeng 43 and Suinong 10 as materials, respectively, on the induction medium Add different concentrations of 6-BA (0, 0.2, 0.5, 1.0, 3.0 and 5.0 mg·L -1 ) (purchased from Beijing Dingguo), induced by light for 5 days, and transferred to MSB medium (MS macro, MS trace, MS iron salt and B5 organic), 30 explants per treatment, 3 repetitions. 14d investigation and statistics on the induction rate of adventitious buds at the embryonic tip of soybean genotypes, the results are as follows figure 1 .

[0038] From figure 1 It can be seen that the overall germination induction rate of the tested variety Zhongji soybean No. 2 is the highest, followed by Jilin 35. Overall, the appropriate concentration of 6-BA is mainly concentrated in the range of 0.2-3.0 mg. ...

Embodiment 2

[0039] Example 2: Detection results of different genotypes of soybean embryo tip GUS activity

[0040] The embryo tip explants of the above ten varieties were respectively immersed in the suspension of Agrobacterium EHA105 (purchased from Biovector) transformed into the pCAMBIA1301 (purchased from CAMBIA) plasmid vector, vacuum-infected for 15 min, and the attached explants were washed away. Bacterial solution, transfer the explants into the co-culture medium (1 / 2 MS large + MS trace + MS iron salt + B5 organic + 30 g / L sucrose + 0.8g / L agar + 100uMAS + 5mg / L6-BA PH5 .4), cultivated for 3 days.

[0041] Select about 100mg of unbrown embryo tip explants, add liquid nitrogen, grind into powder, put the powder into a 1.5m1 centrifuge tube, add 600ul GUS extraction buffer, mix well, 4°C, 12,000rpm, centrifuge for 10min, take The supernatant was divided into 2 new centrifuge tubes and stored at 4°C for later use. The supernatant was the crude extract of GUS protein, a part of the...

Embodiment 3

[0043] Embodiment 3: The impact of different disinfection methods on the embryo tip germination rate

[0044] Mature Jilin No. 35 soybean seeds with complete seed coats, no disease spots, no insect damage, and no mold were selected, and three seed disinfection methods were used. (1) Chlorine gas disinfection method: soybean seeds were placed in a closed container in a fume hood, mixed with 96 ml 5% Naclo and 4 ml HCl (both purchased from Beijing Dingguo), and sterilized for 6-10 h. (2) Mercury chloride disinfection method: rinse soybean seeds with tap water, treat with 75% alcohol for 35 s, then disinfect with 0.1% Hgcl2 for 10 min, and rinse with sterile water 3 to 5 times. (3) Alcohol disinfection method: rinse soybean seeds with tap water, treat with 75% alcohol for 50 s, and rinse with sterile water 3 to 5 times. Each disinfection method treated 100 seeds and repeated 3 times.

[0045] The results are as follows: three methods were used to disinfect the mature seeds of J...

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Abstract

The invention relates to a high-efficiency genetic transformation method using a soybean Jilin No.35 embryonic tip as an explant and belongs to the technical field of plant transgenosis. The method comprises the following steps of: selecting Jilin No.35 soybean seeds with integrated seed coat and without diseases; performing ultrasonic treatment on the explant; transferring the infected embryonic tip explant into a co-culture medium; co-cultivating; inoculating into a rooting medium; and transplanting to a nutrition pot after the root grows strong. According to the method, the regeneration cycle is shortened. Through the adoption of a strong and effect infection method, browning degree and necrosis degree during co-cultivation are reduced, and conversion rate is increased.

Description

technical field [0001] The invention belongs to the technical field of plant transgenics, and specifically relates to using soybean embryo tips as explants, screening genotypes with high regeneration rate and high sensitivity to Agrobacterium, and improving the transformation efficiency of Agrobacterium in combination with an efficient infection method. Background technique [0002] Soybean is an important oil crop and feed crop. Using transgenic technology to improve soybean quality is an important direction of breeding today. Although transgenic soybean has been planted on a large scale around the world, its genetic transformation is still one of the difficulties. Therefore, it is necessary to establish an efficient and stable The regeneration system is the cornerstone to solve the low genetic transformation rate of soybean. The research on soybean tissue culture can be traced back to the 1960s, and it was not until the 1980s that a breakthrough was made. Through explants ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00
Inventor 王庆钰闫帆王英李景文翟莹孙昕刘雅婧陈虹地赵健如张鑫生程浩杨旭光
Owner JILIN UNIV
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