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Gene transformation method for summer black grapes

A gene transformation and grape technology, applied in the field of genetic engineering, can solve the problems of Agrobacterium being unable to inhibit, cumbersome, and unsuccessful transgenesis, and achieve the effects of improving transgenic efficiency, simplifying the operation process, and avoiding blindness

Active Publication Date: 2016-08-17
JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY
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AI Technical Summary

Problems solved by technology

Although the genetic transformation technology route of grapes is mature, the transformation efficiency is very low, and too few transgenic plants are obtained, which brings obstacles to the later identification of transgenic plants
The operation process of the traditional Agrobacterium-mediated method is cumbersome, and many problems are prone to occur during the transgenic process, such as the selection of suitable vectors, the selection of suitable strains, the contamination of miscellaneous bacteria, the inability of Agrobacterium to be inhibited, and the concentration of bacteria solution is too high or too high. Low results in unsuccessful transgenics, control of the duration of infection, etc. These problems will affect the efficiency and speed of grape transgenics. The transgenic efficiency of grape plants obtained by traditional methods is less than 1%.

Method used

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  • Gene transformation method for summer black grapes
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Experimental program
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Embodiment

[0027] Example: First, prepare the main solution and the preparation of the culture medium:

[0028] Grape subculture medium (S1): MS 2.2g / L, sucrose 30g / L, agar 5.5g / L, IBA 0.4mg / L, pH 5.8, autoclaved, set aside.

[0029] Grape co-cultivation medium (S2): MS2.2g / L, sucrose 30g / L, agar 5.5g / L, TDZ 1mg / L, ZT1mg / L, IBA 0.4mg / L, pH5.4, autoclaved, cooled At about 60°C, divide into flat plates for later use.

[0030] Grape screening medium (S3): MS2.2g / L, sucrose 30g / L, agar 5.5g / L, TDZ 1mg / L, ZT 1mg / L, IBA 0.4mg / L, pH5.4, autoclaved, cooled Add kanamycin 20mg / L and carbenicillin 400mg / L to 60°C and put into culture flasks for later use.

[0031] Grape rooting screening medium (S4): MS2.2g / L, sucrose 30g / L, agar 5.5g / L, IBA 0.4mg / L, pH5.8, autoclaved, cooled to 60°C, added kanamycin 20mg / L, carbenicillin 300mg / L subpackage culture bottle for standby.

[0032] Secondly, the activation of the bacterial species in the bacterial liquid is carried out:

[0033] ① Streak the Agrob...

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Abstract

The invention discloses a gene transformation method for summer black grapes. The method comprises the steps of material selection from tissue culture seedling leaves, leaf stalks and stem fragments, infection, co-culture, screening culture and rooting culture. Material selection is optimized, growth portions and growth time of explant leaves are specifically regulated, and the phenomenon that the working efficiency is low due to blindness of material selection is avoided; meanwhile, the portions, including grape leaf stalks and stem fragments, except the leaves, of the explants are selected, better operation can be performed by using the leaf stalks and the stem fragments, the gene transformation efficiency can be improved, and the transformation efficiency can reach 20% or above; the problem of pollution of agrobacterium and infectious microbes in an agrobacterium-mediated method is solved; the problem that the grape conversion efficiency is low is solved; the problem that grape transgene transgenosis, transgenic test and farm transplanting period is long is solved, and the period is shortened; the problems that the transgenic test workload is large and workloads are large are solved, the detection method is simplified, and the detection speed is increased.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a grape gene transformation method. Background technique [0002] Grape is a crop with high economic value and rich nutritional value. In recent years, grape industry has developed rapidly in many areas of our country and has become an important part of agricultural industrialization in this area. In recent years, Chinese scientific researchers have also invested a lot of energy in the research of grapes. Especially with the rapid development of molecular biology and genetic engineering, the research focus has turned to the deep mining of excellent genes in grape germplasm resources, and the molecular markers and Identification and determination of gene expression sequences. At the same time, research results on genetic improvement such as stress resistance, storage and transportation resistance, and flavor quality have been reported one after another, and grape bree...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H4/00A01H5/00
CPCC12N15/8205
Inventor 王媛花颜志明郭正兵王全智杨宝林董慧
Owner JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY
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