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Method for high-throughput genetic transformation of tobacco

A genetic transformation and high-throughput technology, applied in biochemical equipment and methods, botany equipment and methods, and the use of vectors to introduce foreign genetic materials, etc., can solve the problems of long time, various types of culture media, and labor-intensive, etc., to achieve Reduced mechanical damage, reduced repeated explant transfer, and reduced mechanical loss

Inactive Publication Date: 2019-02-22
CHINA TOBACCO YUNNAN IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After decades of technology accumulation, in general, tobacco has formed a set including aseptic seedling culture, Agrobacterium streaking, Agrobacterium monoclonal liquid activation culture, leaf disk pre-culture, infection, co-cultivation, and co-cultivation of leaves. There are nine steps of stable genetic transformation system including plate cleaning, selection and differentiation culture, and rooting culture. However, due to the small scale of past research, there are many shortcomings in the process of tobacco genetic transformation, such as a large variety of operation steps and media, labor-intensive, and long time. It has always existed, and there is an increasing need for a tobacco genetic transformation technology that is simple, efficient, low in human resources, and suitable for large-scale operations

Method used

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  • Method for high-throughput genetic transformation of tobacco
  • Method for high-throughput genetic transformation of tobacco
  • Method for high-throughput genetic transformation of tobacco

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] The high-throughput genetic transformation method of the tobacco variety Honghua Dajinyuan of the present embodiment comprises the following steps:

[0034] Step (1) Cultivation of aseptic seedlings of the tobacco variety Honghua Dajinyuan: Take plump tobacco seeds, soak them in 75% alcohol for 30s in an ultra-clean workbench, rinse them twice with sterile water, and then sterilize them with 10% sodium hypochlorite 10 min, wash 5 times with sterile water, inoculate into MS1 medium after absorbing the water on the surface of the seeds with high-pressure sterilized filter paper, and cultivate them for about 35 days at 25°C to obtain sterile seedling plants, such as figure 1 shown.

[0035] The specific composition of MS1 medium is: MS basic medium 4.43g / L, sucrose 30g / L, agar 8g / L, pH=5.75.

[0036] Step (2) Preparation of infecting Agrobacterium: Take out the LBA4404 engineered Agrobacterium containing the pbi121 plasmid that has been frozen in advance from the -80°C re...

Embodiment 2

[0045] The high-throughput genetic transformation method of the K326 variety of the present embodiment comprises the following steps:

[0046] Step (1) Cultivation of aseptic seedlings of K326 variety: take plump tobacco seeds, soak them in 75% alcohol for 30 seconds in an ultra-clean workbench, rinse them twice with sterile water, then disinfect them with 10% sodium hypochlorite for 10 minutes, and then sterilize them with sterile water Wash 5 times, dry the water on the surface of the seeds with high-pressure sterilized filter paper, inoculate them into MS1 medium, and cultivate them for about 35 days at 25°C to obtain sterile seedling plants.

[0047] Step (2) Preparation of infecting Agrobacterium: Take out the LBA4404 engineered Agrobacterium containing the pbi121 plasmid that has been frozen in advance from the -80°C refrigerator, absorb 50 μL of pbi121 engineered Agrobacterium and spread evenly until it contains 50 mg / L rifampicin, 50 mg / L In the YEB resistance medium o...

Embodiment 3

[0053] The high-throughput genetic transformation method of the tobacco variety Honghua Dajinyuan of the present embodiment comprises the following steps:

[0054] Step (1) Cultivation of aseptic seedlings of the tobacco variety Honghua Dajinyuan: Take plump tobacco seeds, soak them in 75% alcohol for 30s in an ultra-clean workbench, rinse them twice with sterile water, and then sterilize them with 10% sodium hypochlorite After 10 minutes, wash 5 times with sterile water, dry the water on the surface of the seeds with high-pressure sterilized filter paper, inoculate them into MS1 medium, and cultivate them at 20°C for 30 days to obtain sterile seedling plants.

[0055] Step (2) Preparation of infecting Agrobacterium: Take out the LBA4404 engineered Agrobacterium containing the pbi121 plasmid that has been frozen in advance from the -80°C refrigerator, absorb 50 μL of pbi121 engineered Agrobacterium and spread evenly until it contains 50 mg / L rifampicin, 50 mg / L In the YEB resi...

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Abstract

The invention relates to a method for high-throughput genetic transformation of tobacco, belonging to the field of plant genetic transformation. The method comprises the following steps of: cultivating sterile seedlings of the tobacco, preparing infected agrobacterium, infecting, carrying out co-cultivation, selecting, and carrying out differentiation culture and rooting culture. The method has the beneficial effects that the time-consuming and laborious steps of pre-cultivating a tobacco leaf disc, activating the infected agrobacterium, cleaning after co-cultivation and the like are eliminated, the human cost and the time cost are saved, the pollution risk caused by the complicated manual operation in the conversion process is reduced, and meanwhile, the genetic transformation efficiencyis further improved; and the method is suitable for the large-scale genetic transformation of tobacco.

Description

technical field [0001] The invention relates to a method for genetic transformation, in particular to a method for high-throughput genetic transformation of tobacco, belonging to the field of plant genetic transformation. Background technique [0002] Common tobacco (Nicotiana tabacum) is a model organism of Solanaceae, which plays an important role in basic research of model organisms and national economic production. At the same time, tobacco is also the earliest plant for tissue culture. The MS basic medium developed according to tobacco (Murashige and Skoog, 1962) has been widely used in tissue culture of hundreds of plants. Although it has a first-mover advantage, tobacco genetic research has always been limited by its weak foundation and small scale. [0003] In recent years, CRISPR / Cas9-based gene editing technology (hereinafter referred to as gene editing technology) has emerged. It has simple principles, efficient mutations, low cost, and wide application. It has s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/06A01H6/82
CPCC12N15/8205
Inventor 张建铎李雪梅陈章玉杨光宇向海英曾婉俐张涛高茜宋春满许力蒋佳芮邓乐乐杨文武贾凌夏庆友
Owner CHINA TOBACCO YUNNAN IND
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