High-efficiency agrobacterium-mediated transformation method of soybean root hairs

An Agrobacterium-mediated, high-efficiency technology, applied in the field of plant genetic engineering, can solve the problems of easy contamination by Agrobacterium and other miscellaneous bacteria, complicated operation, etc., and achieve the effects of not easy contamination, simple operation, and high rooting rate.

Active Publication Date: 2019-02-26
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method is cumbersome to operate and is easily contaminated by Agrobacterium and other miscellaneous bacteria

Method used

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  • High-efficiency agrobacterium-mediated transformation method of soybean root hairs
  • High-efficiency agrobacterium-mediated transformation method of soybean root hairs
  • High-efficiency agrobacterium-mediated transformation method of soybean root hairs

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Experimental program
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Effect test

Embodiment 1

[0050] like figure 2 The shown Agrobacterium-mediated soybean root hair transformation method comprises the following steps:

[0051] 1. Soybean germination

[0052] The sterilized beans are planted in GM solid medium, and the bean hilum is inserted into the medium to 2 / 3 of the seeds. There are about 20 grains in one dish, and the petri dish is sealed with parafilm, and cultured in the dark at 23°C for 16 hours in an incubator.

[0053] 2. Agrobacterium Preparation

[0054] Aspirate 400-500 μl of the positive K599 strain bacterial liquid and spread it on a plate containing 25 mg / L rifampicin and 100 mg / L kanamycin, and culture at 28°C for 36-48 hours to grow a lawn.

[0055] 3. Preparation of Infection Solution

[0056] Add AS (weigh 0.008g and dissolve it in 20μl dimethyl sulfoxide), GA3 (add 100μl to 0.5mg / ml mother solution), DTT (154.2mg Add 200 μl / ml)), L-cysteine ​​(add 200 μl to mother solution 580 mg / ml), NaOH (add 600 μl to mother solution 1M), shake well and p...

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Abstract

The invention discloses a high-efficiency agrobacterium-mediated transformation method of soybean root hairs. The method includes the following steps: 1) obtaining an agrobacterium rhizogenes lawn carrying a target gene, performing lawn suspension by utilizing a liquid co-culture medium, and adjusting an OD600 value to 0.6-1.0; 2) splitting soybean seeds germinated in a germination medium along the hilum so that half seeds can be obtained, putting the half seeds into the liquid co-culture medium containing the suspension lawn prepared by the step 1), and performing infection for 15-30 min; 3)distributed the half seeds infected by the step 2) in an empty culture container, and performing co-culture in the dark for 3-5 days with a temperature of 23-25 DEG C; and 4) cutting the roots of thehalf seed co-cultured by the step 3), distributing the half seeds in a culture medium, performing dark culture with the temperature of 23-25 DEG C, and selecting positive roots with lengths of 4-6 cmto cut for individual culturing after the roots are grown out. The method is short in operation time, high in rooting rate and positive incidence, and simple and efficient; and explants are not easilypolluted by agrobacterium and other competitors.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a method for transforming soybean root hairs mediated by high-efficiency Agrobacterium. Background technique [0002] Agrobacteriam rhizogenes is a Gram-negative soil bacterium capable of infecting most dicotyledonous plants. Plants infected with Agrobacterium rhizogenes produce many rapidly growing adventitious roots called hairy or hairy roots [4] . Agrobacterium rhizogenes has important application value in plant transgenic research. It is infected through plant wounds, and its T-DNA is randomly inserted into the chromosome of the host cell, co-expressed with the gene of the host cell, and induces the plant to produce hairy roots [1] . [0003] As early as 1907, Smith and Townsend discovered that Agrobacterium rhizogenes could induce plants to form hairy roots. In 1934, Hildebrand explained this phenomenon again in his research on apple trees. [2] . Th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82
CPCC12N15/8205
Inventor 邢邯黄颜众轩慧冬郭娜黄鹭王海棠
Owner NANJING AGRICULTURAL UNIVERSITY
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