Agrobacterium tumefaciens-mediated peanut efficient transgenic method

An Agrobacterium tumefaciens, transgenic technology, applied in the fields of biochemical equipment and methods, genetic engineering, plant genetic improvement, etc., can solve the problems of strong genotype dependence and cumbersome operation, and achieve the effect of convenient and rapid acquisition.

Active Publication Date: 2011-09-28
SHANDONG PEANUT RES INST
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Problems solved by technology

[0005] The technical effects of the present invention can overcome the above-mentioned defects, and provide a high-efficiency peanut transgenic method mediated by Agrobacterium tumefaciens, which makes up for the shortcomings of the current peanut transgenic technology, such as generally low conversion rate, strong genotype dependence, and cumbersome operation

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  • Agrobacterium tumefaciens-mediated peanut efficient transgenic method
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  • Agrobacterium tumefaciens-mediated peanut efficient transgenic method

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Embodiment Construction

[0017] The specific application method of the present invention comprises the following steps:

[0018] (1) Transform the constructed Ti plasmid vector carrying the gene of interest into Agrobacterium tumefaciens using conventional methods;

[0019] (2) Shake the transformed bacteria in YEP medium at 25°C-30°C until OD 600 The value is at 0.1-0.6;

[0020] (3) Take 2-6 μl of resuspended bacterial solution and inject it into the aerial part of the peanut plant 30-60 days after the peanut is sown, so that Agrobacterium can infect and transform the peanut plant;

[0021] (4) After harvesting the seeds, take thin slices of peanut cotyledon tissue, and quickly extract the DNA of the sample to be tested as a PCR template; (see "Easy and Quick DNA Extraction Method for Peanut Healthy and Diseased Tissues", Patent Application No.: 200910255786.0)

[0022] (5) Use specific primers designed according to the target gene sequence for PCR identification, and electrophoresis detection res...

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Abstract

The invention relates to a plant transgenic technology and in particular relates to an Agrobacterium tumefaciens-mediated peanut transgenic method. The method comprises the following steps: (1) adopting the conventional method to deliver the constructed Ti plasmid vector carrying the target gene into Agrobacterium tumefaciens; (2) using the transformed bacteria to perform shake culture in YEP culture medium; (3) taking 2-6mu l of weight drop bacterial solution to inject in a peanut plant in the suitable period of the growth of peanut to ensure the infection and convertion of Agrobacterium tumefaciens in the peanut plant; (4) taking the sheets of the peanut cotyledon tissue after harvesting seeds, rapidly extracting the DNA of the sample to be tested to use the DNA as the PCR template; and(5) using a specific primer designed according to the target gene sequence to perform PCR identification, and performing electrophoresis detection. In the method, an Agrobacterium tumefaciens-mediated transgenic technology is utilized, thus viable offspring can be obtained conveniently and rapidly and the single tissue culture regeneration stage is not required.

Description

technical field [0001] The invention relates to plant transgenic technology, in particular to a peanut transgenic method mediated by Agrobacterium tumefaciens. Background technique [0002] Peanut is one of the most important oil crops in the world. Most of the peanut transgenic technologies established so far require the procedure of plant tissue culture regeneration, which shows strong genotype dependence and low transformation rate. Parental material of economic importance may not necessarily be easily transformed. The lack of a set of universal transgenic technology has become a bottleneck restricting the basic research of peanut molecular biology and the applied research of breeding. [0003] The International Crops Research Institute for the Semi-arid Tropics (ICRISAT) has obtained transgenic peanuts without selectable markers through direct PCR screening. The conversion rate of the Spanish-type variety JL24 is above 75%, but the steps of tissue culture are still req...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 王传堂王秀贞李贵杰于洪涛唐月异杨伟强张建成崔凤高禹山林
Owner SHANDONG PEANUT RES INST
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