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Efficient agrobacterium transgene method of zoysia japonica

A technology of Agrobacterium and transgene, applied in the field of genetic engineering, can solve the problems of not being resistant to temperature, not being resistant to cold, restricting further development, etc., and achieve the effect of short time consumption and easy implementation

Inactive Publication Date: 2015-11-11
SICHUAN AGRI UNIV
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Problems solved by technology

[0005] Although zoysia has the advantages of trampling and drought resistance because of its advantages such as resistance to trampling and drought, it also has shortcomings such as temperature resistance and cold resistance. It is one of the research priorities in this field to carry out the breeding of excellent varieties, and the above-mentioned zoysia transgenic The problems faced by the work greatly limit the in-depth development of work in this field

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  • Efficient agrobacterium transgene method of zoysia japonica
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  • Efficient agrobacterium transgene method of zoysia japonica

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Embodiment 1

[0033] 1) 1) Embryos were stripped from the mature seeds of Zoysia grass, divided into 5 groups, and cultured in callus induction medium MT4 medium; among them, the first group was cultured for 3 days, and the second group was cultured for 4 days , the third group was cultivated for 5 days, the fourth group was cultivated for 6 days, and the fifth group was cultivated for 7 days; two control groups were set up, the control group 1 was cultivated by the above method for 1 day, and the control group 2 was cultivated by the above method for 2 days; plant resistance selection gene hpt gene and β-glucuronidase gene gus The pMDC99IG plasmid of the reporter gene was transformed into the Agrobacterium strain EHA105 by the freeze-thaw method; at 200rpm and 28°C, the Agrobacterium containing the above-mentioned exogenous plasmid was cultured, and the medium contained 5gL -1 Yeast extract, 10gL -1 Bacto-peptone, 5gL -1 Sodium chloride, 50mgL -1 Karamycin, pH 7.0, grown to OD 600 =0...

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Abstract

The invention provides an efficient agrobacterium transgene method of zoysia japonica. The method is characterized by comprising the following steps: 1, stripping embryos out of mature seeds of the zoysia japonica, and placing the embryos in a calli induction medium MT4 medium for three days at the minimum; 2, conducting excitation on the agrobacterium with foreign plasmid, conducting suspension on the agrobacterium by utilizing a liquid calli subculture medium MT4-S liquid medium, and obtaining agrobacterium suspension liquid; 3, placing plant tissue obtained in the step 1 into the agrobacterium suspension liquid prepared in the step 2, processing the plant tissue through a vacuum dryer for 30 minutes, and conducting shake cultivation slowly for 1 hour; 4, taking out the plant tissue, placing the plant tissue into an MT4-S solid medium containing acetosyringone, and conducting cultivation away from light for three days at the minimum, wherein the concentration of the acetosyringone is 20 mg / L. According to the method, the transgene operation process takes short time, and it takes less than 10 days; no complex operation is needed, and the method is easy to implement.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a high-efficiency Agrobacterium transgene method for zoysia weed. Background technique [0002] Since the introduction of exogenous DNA into plant cells, plant genetic engineering has developed rapidly. The success of introducing exogenous DNA into plants is related to the plant species and gene introduction technology. The genetic transformation method of Zoysia can be divided into protoplast transformation method and transformation not dependent on protoplast. [0003] Due to the complex operation of protoplast culture and the difficulty in regenerating plants, this approach was gradually abandoned. Transformation methods that do not rely on protoplasts mainly include biolistic transformation and Agrobacterium-mediated transformation. [0004] However, at present, there are still problems in the transformation of Zoysia grass, such as complex operation, long-ti...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00
Inventor 王迅马啸汪志辉张新全付刚赖云松梁东林立金唐懿夏惠
Owner SICHUAN AGRI UNIV
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