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Agrobacterium-mediated peach genetic transformation method

A genetic transformation method, Agrobacterium-mediated technology, applied in the field of genetic engineering of horticultural crops, can solve the problems of not meeting current needs and low transformation efficiency

Inactive Publication Date: 2021-04-23
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the conversion efficiency of this method is low and cannot meet today's needs

Method used

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  • Agrobacterium-mediated peach genetic transformation method
  • Agrobacterium-mediated peach genetic transformation method
  • Agrobacterium-mediated peach genetic transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] A kind of Agrobacterium-mediated peach genetic transformation method of the present embodiment comprises the following steps:

[0038] (1) Establishment of genetically transformed receptors

[0039] From March to April, the hypocotyls of peach new shoots were cut from the field as explants, and inoculated on the surface of the callus induction medium; the induced callus was subcultured 4 times, such as figure 1 As shown, it can be used as a genetic transformation recipient.

[0040] Among them, the formula of callus induction medium is: MS+1.5mg / L 6-BA+0.4mg / L IBA+20g / L sucrose+7g / L agar+0.5g / L MES, pH 5.7; The conditions for tissue growth are: 16h light, 8h dark photoperiod, light intensity 1750lx, temperature 25°C, sterile.

[0041] (2) The cultivation of Agrobacterium and the configuration of the infection solution

[0042] The pSAK277-GUS plasmid was transformed with Agrobacterium GV3101 to obtain the Agrobacterium GV3101 carrying the pSAK277-GUS plasmid. Among ...

Embodiment 2

[0058] A kind of Agrobacterium-mediated peach genetic transformation method of the present embodiment comprises the following steps:

[0059] (1) Establishment of genetically transformed receptors

[0060] From March to April, the hypocotyls of peach new shoots were cut from the field as explants, and inoculated on the surface of callus induction medium; the induced callus can be used as a genetically transformed recipient after three subcultures. body.

[0061] Among them, the formula of callus induction medium is: MS+1.0mg / L 6-BA+0.2mg / L IBA+20g / L sucrose+6g / L agar+0.5g / L MES, pH 5.6; The conditions for tissue growth are: 16h light, 8h dark photoperiod, light intensity 1500lx, temperature 25°C, sterile.

[0062] (2) The cultivation of Agrobacterium and the configuration of the infection solution

[0063] The pSAK277-GUS plasmid was transformed with Agrobacterium GV3101 to obtain the Agrobacterium GV3101 carrying the pSAK277-GUS plasmid. Among them, the construction map o...

Embodiment 3

[0079] A kind of Agrobacterium-mediated peach genetic transformation method of the present embodiment comprises the following steps:

[0080] (1) Establishment of genetically transformed receptors

[0081] From March to April, the hypocotyls of peach new shoots were cut from the field as explants, and inoculated on the surface of callus induction medium; the induced callus can be used as a genetic transformation recipient after 5 subcultures. body.

[0082] Among them, the formula of callus induction medium is: MS+2.0mg / L 6-BA+0.6mg / L IBA+20g / L sucrose+8g / L agar+0.5g / L MES, pH 5.8; The conditions for tissue growth are: 16h light, 8h dark photoperiod, light intensity 2000lx, temperature 25°C, sterile.

[0083] (2) The cultivation of Agrobacterium and the configuration of the infection solution

[0084] The pSAK277-GUS plasmid was transformed with Agrobacterium GV3101 to obtain the Agrobacterium GV3101 carrying the pSAK277-GUS plasmid. Among them, the construction map of the...

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Abstract

The invention relates to the field of gardening crop genetic engineering, and provides an agrobacterium-mediated peach genetic transformation method. The method comprises the following steps: establishing a genetic transformation receptor, culturing agrobacterium, preparing an infection solution, conducting infection and co-culturing, conducting degerming, screening and culturing, and carrying out GUS staining identification on resistant calluses. According to the method, the calluses are used as receptors, GUS genes are used as reporter genes, genetic transformation of peaches is carried out with an agrobacterium GV3101 mediation method, the resistant calluses are obtained, and the resistant calluses are determined to be positive calluses through multiple times of subculture and GUS staining. According to the method, the success rate of agrobacterium-mediated transformation can be remarkably increased, and meanwhile, the method can be widely applied to peach genetic engineering, cell engineering, metabolic engineering and establishment of a peach genetic transformation system.

Description

technical field [0001] The invention relates to the field of genetic engineering of horticultural crops, in particular to an Agrobacterium-mediated peach genetic transformation method. Background technique [0002] Peach (Amygdalus persica L.): Rosaceae, peach plant, deciduous small tree. Peach trees are native to China and have been documented as early as the tenth century BC. Peach is mainly grown in Asian countries such as China and Japan. Its types include yellow peaches, juicy peaches, nectarines, and flat peaches. [0003] Peach has a long childhood, and it takes a long time to breed with traditional breeding techniques, which will greatly increase labor costs and reduce land utilization. The use of genetic engineering for germplasm improvement can solve this problem and greatly shorten the breeding time. [0004] The field of molecular biology continues to progress and develop, and the genetic transformation of fruit tree horticultural crops is also gradually perf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/29A01H5/00A01H6/74
CPCC07K14/415C12N15/8205C12N15/8212
Inventor 蔡永萍黄满畅周晖张金云潘海发邱可立盛玉陈红莉谢庆梅
Owner ANHUI AGRICULTURAL UNIVERSITY
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