Agrobacterium tumefaciens mediated explants genetic transformation method

Abstract

The invneiton discloses an agrobacterium tumefaciens mediated explants genetic transformation method, which comprises agrobacterium activation, agrobacterium infection explants, cocultivation of agrobacterium and explants, induction, screening differentiation of explants, wherein the operations of the cocultivation of the agrobacterium and the explants are that sterilization filter paper is placed on a solid co-culture medium, and a bacterial liquid which is arranged on the surfaces of the explants which are infected by the agrobacterium is placed on the sterilization filter paper to culture for 3-4 days after being sucked clean. A method for preparing the solid co-culture medium is that 0.7% (W / V) agar powder is added into distilled water, and is added with 200 uM acetosyringone after being sterilized in high voltage. The agrobacterium tumefaciens mediated explants genetic transformation method enables the agrobacterium to be capable of fully contacting with the explants not to grow overly when the agrobacterium and the explants are co-cultivated during the genetic transformation process, improves transformation efficiency, and can reduce pollution probability by simplifying culture mediums.

Description

technical field [0001] The invention belongs to the field of transgenic experiment methods, in particular to an agrobacterium-mediated explant genetic transformation method. Background technique [0002] Co-cultivation is the most critical part of the transformation process of plant transgenic experiments. The transfer and integration of T-DNA are completed at this time. After Agrobacterium attaches to the explants, it cannot transform immediately, and the tumor can be induced to go through the "cell regulation period" after 16 hours of attachment, so that T-DNA transfer occurs. Therefore, the co-cultivation time is longer. However, if the co-cultivation time is too long, it may also lead to the excessive proliferation of Agrobacterium, causing the plant cells to be poisoned and die. The proliferation speed of Agrobacterium in co-culture is directly related to the transformation, and the proliferation and growth status of Agrobacterium are related to its infection ability...

AI Technical Summary

Problems solved by technology

[0003] Most of the Agrobacterium cultures are directly cultured by explants on solid medium, because the commonly used medium is added with a large number of elements, trace elements, sucrose and other substances (commonly used co-culture medium components are: MS (mass, trace, organic, Inositol) + 3% (W / V) sucrose + 200uMAS + 0.7% (W / V) agar powder), first, it is easier to infect bacteria; second, due to the high water content of the medium, and there is no filter paper, it is easy to cause Agrobacterium Overgrowth, leading to browning and death of explants due to inability to inhibit the growth of excessive Agrobacterium

When Agrobacterium tumefaciens infects the explants, it is necessary to fully contact the bacterial liquid and the explants so that the T-DNA can be transferred to the plant cells, and the explants must not be too poisoned by Agrobacterium, which will cause the inability to inhibit the explants in the later stage. Excessive Agrobacterium growth browns and kills explants

In the cultivation process of the prior art, the conversion rate is low or even subsequent experiments cannot be carried out due to improper operation of the co-cultivation process.

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