Vacuum-infiltration assisted exogenous gene transforming method of soybean germinating embryo

A technology of vacuum infiltration and exogenous genes, which is applied in the field of plant genetic engineering, can solve the problems of far from reaching the model level, low frequency of soybean transformation, poor repeatability, etc., to overcome the limitations of genetic transformation, short transformation cycle, regeneration fast effect

Inactive Publication Date: 2010-11-10
SHANDONG UNIV
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  • Abstract
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AI Technical Summary

Problems solved by technology

In 2000, Donaldson et al. used Agrobacterium to transform the cotyledon nodes of 12 short-season soybean varieties, and the infection rate could reach 92%. However, only genetically stable transgenic plants were obtained from the variety Accolibri, and the transformation rate was extremely low.
Recently, some new transformation methods have been discovered: ultrasonic-assisted Agrobacterium transformation (SAAT), vacuum filtration-assisted Agrobacterium transformation of whole plants, particle bombardment and Agrobacterium comprehensive method, etc., but the frequency of soybean transformation is still very low. , far from reaching the level of modeling, and there are still shortcomings such as poor repeatability and being limited by soybean genotypes. Therefore, in-depth research is still needed

Method used

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  • Vacuum-infiltration assisted exogenous gene transforming method of soybean germinating embryo
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  • Vacuum-infiltration assisted exogenous gene transforming method of soybean germinating embryo

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Experimental program
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Effect test

Embodiment 1

[0048] Jidou 12 (purchased from Weifang Academy of Agricultural Sciences) was used as the acceptor material, and the transformed strain was Agrobacterium strain AGL104 carrying the psnac1::K7WG2D vector (the vector was purchased from Invitrogen).

[0049] (1) Seed disinfection and pre-cultivation treatment

[0050] Soybean seeds were sterilized with 70% ethanol for 5 minutes, and then cleaned with 0.1% HgCl 2 Disinfect for 10 minutes, rinse with sterile water 5-6 times, and soak the seeds in sterile water for 12 hours at 25°C-28°C.

[0051] Soybeans whose embryos swelled and germinated after soaking were put into the pre-cultivation medium, and cultured in light at 28°C for 1 day, wherein the formula of the pre-culture medium was: MS+3.0mg / L 6-BA (6-benzylaminoadenine) +20g / L sucrose+7g / L agar, pH5.8.

[0052] (2) After the seed embryo swells and germinates, it is cut to expose or damage the germinated embryo part

[0053] When the whole pre-cultivated soybean embryo is enl...

Embodiment 2

[0071] Shengdou No. 9 (purchased from Weifang Academy of Agricultural Sciences) was used as the recipient material, and the transformed strain was Agrobacterium AGL104 carrying psnac6::K7WG2D (p2K7GW7 vector was purchased from Invitrogen).

[0072] (1) Seed disinfection and pre-cultivation treatment

[0073] Soybean seeds were sterilized with 70% ethanol for 5 minutes, and then cleaned with 0.1% HgCl 2 Disinfect for 10 minutes, rinse with sterile water 5-6 times, and soak the seeds in sterile water for 12 hours at 25°C-28°C.

[0074] Soybeans whose embryos swelled and germinated after soaking were put into the pre-cultivation medium, and cultured in light at 28°C for 1 day, wherein the formula of the pre-culture medium was: MS+3.2mg / L 6-BA (6-benzylaminoadenine) +20g / L sucrose+7g / L agar, pH5.8.

[0075] (2) After the seed embryo swells and germinates, it is cut to expose or damage the germinated embryo part

[0076] When the whole pre-cultivated soybean embryo is enlarged a...

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Abstract

The invention discloses a vacuum-infiltration assisted exogenous gene transforming method of a soybean germinating embryo, which comprises the following steps of: 1. carrying out disinfection and pre-culture processing on seeds; 2. after the expansion and the germination of seed embryos, cutting, exposing or damaging germinating embryo parts; 3. infecting the germinating embryos by agrobacteria containing target genes; 4. carrying out co-culture; 5. regenerating and screening clustered buds as well as rooting and screening plantlets; and 6. regenerating and screening the clustered buds as well as rooting and screening the plantlets. The method is applicable to multiple genotypes, has high regenerating speed and is a reliable system for soybean transgenic breeding.

Description

technical field [0001] The invention relates to a method for exogenous gene transformation assisted by vacuum infiltration of germinated soybean embryos, belonging to the field of plant genetic engineering. Background technique [0002] Soybean is an important economic crop, so improving and optimizing soybean quality has become a focus of attention. Establishing a good tissue culture regeneration system is the prerequisite for successful genetic transformation of soybean. Although genetically modified soybeans have been planted on a large scale around the world, their genetic transformation is still one of the difficulties in the field of genetic engineering. Soybean genetic transformation system is limited to cotyledon node transformation system and germ transformation system mediated by Agrobacterium. These transformation systems all have problems such as low transformation frequency, difficulty in reproducibility, and dependence on specific genotypes. Advances in soyb...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00
Inventor 向凤宁姬丹丹王鹏
Owner SHANDONG UNIV
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