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Method for transgenosis of ovules in ovaries of young pomegranate fruits by using agrobacterium injection technology and application of method

A technology of Agrobacterium injection and transgene, which is applied in the field of pomegranate ovary injection, can solve the problems of difficulty, high cost, immaturity, etc., and achieve the effect of solving breeding difficulties and low cost

Inactive Publication Date: 2020-12-11
ZAOZHUANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is costly and difficult, and the positive rate of pomegranate transgenic seedlings obtained is low. Moreover, this method is not mature in China.
As a woody plant, pomegranate has no direct genetic transformation reports on live pomegranate plants

Method used

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  • Method for transgenosis of ovules in ovaries of young pomegranate fruits by using agrobacterium injection technology and application of method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] (1) Preparation of Agrobacterium injection

[0049] The PBI121 vector carries the β-glucosidase gene (GUS) and the kanamycin resistance gene (KaNa), and the PBI121 vector is transformed into GV3101 Agrobacterium tumefaciens , to obtain the vector containing PBI121 GV3101 Agrobacterium tumefaciens , the bacterial species was expanded by shaking in the LB liquid medium added with double antibodies, and then the bacterial cells were collected by centrifugation, and the bacterial cells were added to the invasion solution. The bacterial concentration in the infection solution is OD 600 =1.0, use after standing for 3h.

[0050] The formulation of the infection solution is: add MgCl to every 300 mL of deionized water 2 0.2856 g, MES 0.63 g, then sealed with a parafilm, put into a high-temperature and high-pressure steam sterilizer for sterilization, and the sterilization condition is 121 ° C for 20 minutes. Cool to room temperature after sterilization, add AS 11.772mg on...

Embodiment 2

[0062] (2) Preparation of Agrobacterium Injection

[0063] The CRISPR cas9 BGK01 vector carries the hygromycin resistance gene (HYG) and the kanamycin resistance gene (KaNa), and part of the pomegranate glucuronic acid decarboxylase gene (UDP-glucuronic acid decarboxylase 5) The exon sequence is inserted into the multiple cloning site of the BGK01 vector, thereby constructing the pomegranate glucuronide decarboxylase gene BGK01 vector. Transfer the constructed vector into GV3101 Agrobacterium tumefaciens Competent cells to obtain the target strain.

[0064] The bacterial species was expanded by shaking in LB liquid medium with double antibodies, and then the bacterial cells were collected by centrifugation, and the bacterial cells were added to the invasion solution. The bacterial concentration in the infection solution is OD600 =1.0, use after standing for 3h.

[0065] The formulation of the infection solution is: add MgCl to every 300 mL of deionized water 2 0.2856 g, M...

Embodiment 3

[0073] (1) Preparation of Agrobacterium injection

[0074] The pK7GWIWG2D(II) vector carries spectinomycin (Spec) resistance gene and GFP marker gene, and GFP protein can show green fluorescence under blue fluorescence excitation. Using the kit method, the partially sequenced correct cDNA sequence of the pomegranate glucuronic acid decarboxylase gene (UDP-glucuronic acid decarboxylase 2) was inserted into the multiple cloning site of the pK7GWIWG2D(II) vector to construct the pomegranate glucuronic acid decarboxylase gene pK7GWIWG2D (II)-RNAi vector. Transfer the constructed vector into GV3101 Agrobacterium tumefaciens Competent cells to obtain the target strain.

[0075] The strain was expanded by shaking in LB liquid medium with spectinomycin (Spec) and rifampicin (Rif) double antibodies, and then the bacteria were collected by centrifugation, and the bacteria were added to the invasion solution. The bacterial concentration in the infection solution is OD 600 =1.0, use ...

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Abstract

The invention provides a method for transgenosis of ovules in ovaries of young pomegranate fruits by using an agrobacterium injection technology and application of the method. According to the method,during the period of the young pomegranate fruits, agrobacterium is injected into the ovaries of the young pomegranate fruits, the agrobacterium is in full contact with the ovules of the pomegranatefruits, so that exogenous genes are effectively introduced into young pomegranate embryo cells and further developed into seeds, and transgenic plants of the pomegranate fruits are obtained through screening and detection. The method is low in cost, and the problem of difficulty in transgenic breeding of the pomegranate fruits can be effectively solved. The young pomegranate embryos are directly infected, the transformed young embryos are developed into the seeds in the fruits, the number of the ovules in each pomegranate ovary is about 200, and even if the transformation efficiency is low, the number of the obtained transformed plants is also very considerable; and the obtained positive plants are F1 generations, and the breeding cycle is shortened.

Description

technical field [0001] The method belongs to the field of genetic engineering breeding, and uses the Agrobacterium tumefaciens mediated technology to transfer foreign genes into wild-type pomegranates and successfully expresses related traits of the foreign genes, and specifically relates to a transgenic method of pomegranate ovary injection. Background technique [0002] Pomegranate( Punica granatum ) is a plant of the pomegranate family Pomegranate, which is planted in temperate and tropical regions all over the world. Lansky and others have found that the leaves, flowers, fruits, roots and other parts of pomegranate have special pharmacological effects. In recent years, the cultivation, development and utilization of pomegranate have attracted attention. The current pomegranate breeding method is still based on traditional hybrid breeding, which is low in efficiency and slow in speed, and it is difficult to meet the demand. How to change the horticultural traits of pom...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/65A01H4/00A01H5/10A01H6/00
CPCC12N15/8205C12N15/65A01H4/00
Inventor 张立华任宏伟
Owner ZAOZHUANG UNIV
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