Method for quickly building genetic transformation regeneration system of grapes

A genetic transformation, grape technology, applied in plant regeneration, horticultural methods, botanical equipment and methods, etc., can solve the problems of difficult expression of transgenic plants, complex genetic characteristics of transgenic plants, and difficult planting

Inactive Publication Date: 2013-12-18
TOBACCO RES INST HENAN ACADEMY OF AGRI SCI
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Problems solved by technology

The gene gun method has the advantage of transforming many cells in one treatment, but it is difficult to obtain single-copy integrated plants, the genetic...

Method used

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  • Method for quickly building genetic transformation regeneration system of grapes
  • Method for quickly building genetic transformation regeneration system of grapes
  • Method for quickly building genetic transformation regeneration system of grapes

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Embodiment Construction

[0026] 1. Activation of Agrobacterium tumefaciens: Agrobacterium tumefaciens strains containing the green fluorescent protein gene (GFP) were cultured on LB solid medium, single colonies were picked for PCR verification, and positively expressed plaques were added to the resistance-inducing medium Medium (resistance induction medium formula is: 5g L -1 Beef extract, 1g L -1 Yeast paste, 5g L -1 Peptone, 5gL -1 Sucrose, 4gL -1 MgSO 4 ·7H 2 O, 100 μmol L -1 Acetosyringone, 50mg L -1 Kanamycin, 50mg L -1 Rifampicin, adjusted to pH 5.2), 28°C, 250rpm culture to OD 600 = 1.0. Centrifuge the bacterial solution at 5000rpm for 10 minutes, discard the supernatant, and add liquid co-cultivation medium to resuspend (the formula of liquid co-cultivation medium is MS macroelement, MS trace element, MS iron salt, 0.5mgL -1 Thiamine Hydrochloride, 0.2mg L -1 Pyridoxine hydrochloride, 0.5mg L -1 Niacin, 150mg L -1 Inositol, 50mg L -1Hydrolyzed casein, 10g L -1 Coconut milk, 5% ...

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Abstract

The invention provides a method for quickly building a genetic transformation regeneration system of wine grape Riesling, and belongs to the technical field of transgenic breeding of grapes. The method is characterized by comprising the following steps: performing tissue culture to induce grape anther to obtain callus; subculturing and screening to obtain embryonic callus; with embryonic callus as a receptor, screening through a selective medium by an agrobacterium tumefaciens-mediated method to obtain resistant callus; and then regenerating a grape plant. Whether the gene gfp is expressed can be observed and reported by PCR (Polymerase Chain Reaction) verification and through a stereo fluorescence microscope, and a transformant also can be identified quickly, therefore, the grape transgenosis efficiency can be improved. Compared with the prior art, the method has the advantages that the culture medium has a good effect on culturing and screening, and achieves quick regeneration of a Riesling transgenic grape plant, thus the grape transgenosis efficiency is greatly improved, and the regeneration rate reaches 81%; the transformation cycle is short, the transgenic plant can be obtained within seven months; the transformant is greatly increased; the transformation rate can reach 63%.

Description

technical field [0001] The invention belongs to the technical field of new plants, in particular to the technical field of grape transgenic breeding. Background technique [0002] Cultivating new grape varieties, improving fruit quality, and increasing plant resistance are issues that grape researchers are very concerned about. However, the number of grape genes is large and most of them are in a heterozygous state, and the genetic background is complex. Using traditional methods for cross-breeding, the cultivation process is cumbersome. , the cycle is long, the cost is high, and the efficiency is low. The method of obtaining excellent varieties through hybridization is easily limited by parental materials. The hybridization process often brings in bad economic traits and is restricted to a large extent. The rapid development of tissue culture technology, molecular biology and genetic engineering technology has opened up new ways for the research of new grape varieties and q...

Claims

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Application Information

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IPC IPC(8): A01H4/00
Inventor 赵凤霞高相彬王正平宋学立朱景伟
Owner TOBACCO RES INST HENAN ACADEMY OF AGRI SCI
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