Efficient genetic transformation method for agrobacterium-mediated vacuum infection of pigeon peas

A genetic transformation method, Agrobacterium-mediated technology, applied in the fields of molecular biology and plant transgenics, can solve the problems of low efficiency and long period of pigeonpea genetic transformation

Active Publication Date: 2020-11-03
BEIJING FORESTRY UNIVERSITY
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Problems solved by technology

At the same time, the advantages of this method include simple operation steps, and the ability to obtain sterile transgenic materials, which solves the problems of long genetic transformation period and low efficiency of pigeonpea. This method can skip steps such as callus regeneration, and directly obtain transgenic materials from the stem. Roots, while aerial parts can be used for rapid validation of transient gene expression

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  • Efficient genetic transformation method for agrobacterium-mediated vacuum infection of pigeon peas
  • Efficient genetic transformation method for agrobacterium-mediated vacuum infection of pigeon peas
  • Efficient genetic transformation method for agrobacterium-mediated vacuum infection of pigeon peas

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Embodiment Construction

[0048] The following is attached Figure 1-4 The present invention is described in further detail.

[0049] We design specific examples based on the genetic transformation method established above. The application case this time is to construct an overexpression vector of the marker gene eGFP, obtain the root system of the overexpression eGFP gene by this method, combine PCR sequencing and laser confocal microscopic analysis to verify, and use trypan blue staining and fluorescence on the leaves Quantitative determination of gene expression efficiency after transient transformation. Below in conjunction with embodiment expand the detailed description to each step:

[0050] (1) Pigeon Pea Seed Selection, Disinfection and Aseptic Culture

[0051] Pigeon bean seeds with plump and well-proportioned particles and no insect eyes are selected for disinfection with sodium hypochlorite. The specific disinfection process is 75% alcohol disinfection for 30 seconds, followed by disinfec...

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Abstract

The invention relates to an efficient genetic transformation method of agrobacterium tumefaciens mediated vacuum infected pigeon peas, and belongs to the technical field of molecular biology and planttransgenic engineering. Experimental steps of the method mainly comprise four parts: explant screening; preparing an agrobacterium infection solution; and performing vacuum infiltration and transgenosis identification on whole pigeon pea plants. According to the invention, a stable transgenic root system induced by stems can be obtained, and leaves of the pigeon peas can be used for rapid identification of gene expression after transient transfection. The inventor finds that transgenic roots can grow from stems of woody plant seedlings through agrobacterium tumefaciens vacuum infiltration forthe first time. The method not only can be directly applied to genetic transformation of the pigeon peas, but also can provide a new thought for a genetic transformation system of woody plants.

Description

technical field [0001] The invention belongs to the field of molecular biology and plant transgenic technology, and specifically relates to a high-efficiency genetic transformation method using Agrobacterium-mediated vacuum infection of pigeonpea plants and its application, which can obtain stably transformed transgenic roots and instantaneously transformed leaves. Background technique [0002] Pigeon pea is native to India and is the sixth largest edible bean in the world. It is the main economic crop in semi-arid areas and has high nutritional and medicinal value. In recent years, as the medicinal value of pigeonpea has been continuously developed, the planting range has also been continuously expanded. Cultivating pigeonpea with traditional methods can no longer meet the market demand. How to cultivate high-yield and high-quality pigeonpea through molecular means has become a problem of concern, and related scientific research is also constantly being carried out. Howeve...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
CPCC12N15/8205
Inventor 孟冬曹红燕付玉杰杨清李航航刘腾跃杨婉珑
Owner BEIJING FORESTRY UNIVERSITY
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