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Method for increasing elongation of soybean tissue culture cluster buds and improving genetic transformation efficiency

A technology of genetic transformation efficiency and bud elongation, which is applied in the field of significantly improving the elongation rate of soybean tissue culture clustered buds and genetic transformation efficiency, and can solve the problems of low soybean genetic transformation efficiency

Inactive Publication Date: 2018-07-10
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, compared with the transformation efficiency of other crops, such as rice 23% (Ge et al.2006; Lin and Zhang 2005), maize 30%-40% (Ishida et al.1996; Yang et al.2006), the genetic The conversion efficiency is still low
[0005] Many factors, such as soybean recipient species, Agrobacterium strains and infection activity, type of screening agent, medium composition and hormone ratio, etc. all affect soybean transformation efficiency. After regeneration of , only a few clustered shoots can elongate (Song et al.2013), therefore, the elongation rate of clustered shoots is the bottleneck to improve the efficiency of soybean genetic transformation

Method used

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  • Method for increasing elongation of soybean tissue culture cluster buds and improving genetic transformation efficiency
  • Method for increasing elongation of soybean tissue culture cluster buds and improving genetic transformation efficiency
  • Method for increasing elongation of soybean tissue culture cluster buds and improving genetic transformation efficiency

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Experimental program
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Embodiment 1

[0050] A kind of bud elongation culture medium, described culture medium comprises: the gibberellin of 1mg / L and the auxin of 0.1mg / L, and a kind of preferred specific proportioning scheme is: MS large amount 50ml / L, MS trace amount 5ml / L L, Fe salt 5ml / L, B5 organic 10ml / L, sucrose 30g / L, MES 0.58g / L, add gel 3.5g / L after adjusting the pH of the solution to 5.6, add gibberellin (GA 3 ) 1mg / L, auxin (IAA) 0.1mg / L, zeatin (ZR) 1mg / L, L-asparaginase (Asp) 50mg / L, L-pyroglutamine (L-pyro) 100mg / L L, cephalosporin (Cef) 75mg / L, carbenicillin (Carb) 500mg / L, screening agent 3mg / L; Wherein the formula of basal medium is as shown in table 1, dilutes when using:

[0051] Formulation of table 1 basal medium

[0052]

[0053]

[0054] Comparative experiment 1

[0055] Soybean varieties Jack Purple and Tianlong No. 1 were selected for experimentation. Experimental design: Soybean varieties JackPurple and Tianlong No. 1 were selected for experimentation. Experimental design: GA 3...

Embodiment 2

[0067] Embodiment 2 soybean genetic transformation method

[0068] The present embodiment provides a soybean genetic transformation method, which specifically includes the following steps:

[0069] 1) Disinfection of soybean seeds: select mature soybean seeds with large grains, plumpness, no spots, and intact seed coats, wipe the surface of the seeds with 75% alcohol, put them in a vacuum sterilizer, and perform chlorine gas sterilization for 6 hours;

[0070] 2) Imbibition of soybean seeds: soak the sterilized seeds in step 1) in the dark with sterile water for 16-18 hours, and the soaking temperature is 23°C;

[0071] 3) Collection and resuspension of Agrobacterium: Melt the transformed bacteria solution stored at -80°C on ice and mix well. Dip the bacteria solution with a sterile inoculation loop and draw a line on the YEB solid medium added with antibiotics. Cultivate on a shaker at 28°C for two days; the first activation is to pick a single clone and inoculate it into a ...

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Abstract

The invention provides a novel bud elongation culture medium and a soybean genetic transformation method for increasing elongation of cluster buds and improving genetic transformation efficiency. Theculture medium comprises 0.9-1.1mg / L of gibberellins and 0.08-0.12mg / L of auxin. By the culture medium and the method, the elongation of the soybean tissue culture cluster buds can be increased whilesoybean genetic transformation efficiency can be improved, and technical support is provided for soybean gene function research and soybean molecular breeding in the future.

Description

technical field [0001] The invention relates to the field of genetic transformation of crops, in particular to a method capable of significantly improving the elongation rate of clustered buds of soybean tissue culture and the efficiency of genetic transformation. Background technique [0002] Soybean is recognized as one of the most difficult crops for genetic transformation in the world. The reported soybean genetic transformation systems include Agrobacterium-mediated method, pollen tube method, particle gun method, electric shock method, PEG method, microinjection method, etc., among which Agrobacterium-mediated method is a commonly used method for soybean. 85% of transgenic plants are obtained through Agrobacterium-mediated method (Yu et al.2011). [0003] According to different explants, the Agrobacterium-mediated soybean genetic transformation system can be mainly divided into soybean cotyledonary node method, embryo tip method, hypocotyl method, etc., among which th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H4/00A01H6/54
CPCA01H4/00C12N15/8205C12N15/8267
Inventor 李艳李姝璇丛亚辉王婷婷盖钧镒
Owner NANJING AGRICULTURAL UNIVERSITY
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