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An efficient, rapid and stable gene transformation method for blueberry

A technology of gene transformation and blueberry, applied in biochemical equipment and methods, genetic engineering, botanical equipment and methods, etc., can solve the problems of low efficiency, long dipping time, low success rate, etc., and achieve the reduction of polyphenols and the effect of pigment interference

Active Publication Date: 2021-03-23
FRUIT TREE INST OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the gene transformation method mediated by Agrobacterium has been widely used in plants, the success rate of gene transformation is only less than 1% due to its cumbersome operation steps and long dipping time, and the efficiency is not high.

Method used

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  • An efficient, rapid and stable gene transformation method for blueberry
  • An efficient, rapid and stable gene transformation method for blueberry

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] A method for efficient, rapid and stable gene transformation of blueberries, comprising the following steps:

[0025] S1, preparation of explants: cut the young and tender stems of blueberry tissues, cut them into small sections of 0.3 cm, soak them in disinfectant solution for sterilization, wash them with sterile water, drain the water, obtain explants, and set aside; The liquid is 1g / 100ml sodium hypochlorite solution, soaking time is 20min;

[0026] S2, the preparation of Agrobacterium: the Agrobacterium containing the target gene was propagated and cultured to the cell concentration OD 600 =0.4, culture temperature is 28 ± 1 ℃, 200rpm shaking culture, obtains the Agrobacterium bacterium liquid, for subsequent use; What adopted in embodiment 1 is the Agrobacterium LBA4404 that contains pCAMBIA 2301 plasmid, wherein contains the Agrobacterium LBA4404 of pCAMBIA 2301 plasmid according to routine Method preparation, here does not involve the point of invention, so the...

Embodiment 2

[0035] A method for efficient, rapid and stable gene transformation of blueberries, comprising the following steps:

[0036] S1, preparation of explants: cut the young and tender stems of blueberry tissue, cut them into small sections of 0.5 cm, soak them in disinfectant solution for sterilization, wash them with sterile water, drain the water, obtain explants, and set aside; The liquid is a 70% ethanol solution by volume fraction, and the soaking time is 20 minutes;

[0037] S2, the preparation of Agrobacterium: the Agrobacterium containing the target gene was propagated and cultured to the cell concentration OD 600 =0.8, culture temperature is 28 ± 1 ℃, 200rpm shaking culture, obtains the Agrobacterium bacterium liquid, for subsequent use; What adopt in embodiment 2 is the Agrobacterium EHA105 that contains pCAMBIA 2301 plasmid, wherein contains the Agrobacterium EHA105 of pCAMBIA 2301 plasmid according to routine Method preparation, here does not involve the point of inven...

Embodiment 3

[0046] A method for efficient, rapid and stable gene transformation of blueberries, comprising the following steps:

[0047] S1, preparation of explants: cut the young leaves of blueberry tissue, cut them into small pieces of 0.5 cm, soak them in disinfectant solution for sterilization, wash them with sterile water, drain the water, obtain explants, and set aside; the disinfectant solution 0.5g / 100ml sodium hypochlorite solution, soaking time is 20min;

[0048] S2, the preparation of Agrobacterium: the Agrobacterium containing the target gene was propagated and cultured to the cell concentration OD 600 =0.6, culture temperature is 28 ± 1 ℃, 200rpm shaking culture, obtains Agrobacterium bacterium liquid, for subsequent use; What adopted in embodiment 3 is the Agrobacterium GV3101 that contains pK7WG2D plasmid, wherein the Agrobacterium GV3101 that contains pK7WG2D plasmid is prepared according to conventional methods , the invention point is not involved here, so the preparati...

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Abstract

The invention belongs to the technical field of gene transformation methods, and concretely relates to an efficient and rapid stable gene transformation method for blueberries. The method comprises the following steps of preparing an explant, preparing an agrobacterium tumefaciens, carrying out primary coculture transformation on the explant, induced culturing, carrying out secondary coculture transformation on a callus, culturing and screening a transgenic plant and the like. According to the efficient and rapid stable gene transformation method for the blueberries provided by the invention,the influence of a gene transformation process of cellular constituents such as cellulose in blueberry tissues is comprehensively considered, an existing agrobacterium tumefaciens mediated gene transformation method is improved, the coculture transformation of the agrobacterium tumefaciens is carried out in a tender tissue period and a callus period, the gene transformation success rate can achieve 25.7 percent or more, the gene transformed blueberry seedling differentiation survival rate achieves 99 percent or more, and the efficient, rapid and stable gene transformation method is developed.

Description

technical field [0001] The invention belongs to the technical field of gene transformation methods, and in particular relates to a high-efficiency, rapid and stable gene transformation method for blueberries. Background technique [0002] Blueberry belongs to Rhododendronaceae, Vaccinium genus, dark blue fruit, spherical or oblate, about 1cm in diameter, thin peel, blue-purple ripe fruit, sweet and sour taste, high edible. Blueberry fruit is rich in polysaccharides, pigments, pectin, and DNA that retains genetic information. Due to the differences in the DNA of different blueberry varieties, the relationship between DNA and blueberry phenotypes was studied. Genetic modification breaks the boundaries between different plant species, recombines the genetic material of different species of organisms at the molecular level, and can completely transform organisms according to human will or purpose. Therefore, breeding transgenic blueberry varieties with special traits or physio...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/36
CPCC12N15/8205
Inventor 宋杨刘凤之张红军王海波刘红弟
Owner FRUIT TREE INST OF CHINESE ACAD OF AGRI SCI