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Method for cultivating tobacco mutants on basis of EMS (ethyl methanesulfonate) mutagenesis

A technology of mutants and tobacco, applied in botany equipment and methods, seed and rhizome treatment, plant genetic improvement, etc., can solve gene function disturbance, target gene function loss, and there is no very detailed mutagenesis of the main tobacco variety K326 Technology optimization system and other issues

Inactive Publication Date: 2018-02-09
YUNNAN ACAD OF TOBACCO AGRI SCI
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Problems solved by technology

But knockout mutations have two drawbacks: first, "gene knockout" can lead to severe disruption of gene function, and transposon insertion usually results in complete loss of target gene function
[0007] Tobacco K326 is the main variety of tobacco, which has the advantages of easy testing and high quality. At present, there is no very detailed optimization system for the mutagenesis technology of the main tobacco variety K326.

Method used

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  • Method for cultivating tobacco mutants on basis of EMS (ethyl methanesulfonate) mutagenesis
  • Method for cultivating tobacco mutants on basis of EMS (ethyl methanesulfonate) mutagenesis
  • Method for cultivating tobacco mutants on basis of EMS (ethyl methanesulfonate) mutagenesis

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Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1: Tobacco variety K326 seed pretreatment

[0036] At room temperature 25°C, take 61.5mL of 1 mol / L K 2 HPO 4 and 38.5mL of 1mol / L KH 2 PO 4 Mix well to prepare 100mL solution, then dilute 10 times to 1000mL to prepare 0.1mol / L potassium phosphate buffer. Potassium phosphate buffer solution is generally prepared and used immediately. About 40,000 seeds were collected in a 50 mL centrifuge tube, and 50 mL of 0.1 mol / L potassium phosphate buffer solution with pH 7.0 was added for pretreatment at 25°C for 6 hours. Put it into the mixer during pretreatment to ensure that the seeds are fully treated. After the pretreatment, the seeds were blotted dry with filter paper and then air-dried naturally. After weighing, they were divided into 19 parts on average (6 gradients × 3 times + 1 control), each with about 2000 grains, and set aside.

Embodiment 2

[0037] Embodiment 2: EMS concentration situation and seed treatment

[0038]The EMS buffer was prepared with 0.1mol / L phosphate buffer (pH 7.0), and the volume fractions of EMS were 0%, 0.30%, 0.50%, 0.70%, 0.70%, 1.10%, and 1.50%. Prepare 1% EMS mother solution first, and then prepare working solution according to Table 1. Among them, all pipette tips and centrifuge tubes that come into contact with the EMS solution should be placed in the sodium hypochlorite solution so that the toxicity of the EMS is oxidized and eliminated. Pour tobacco K326 seeds into a centrifuge tube filled with EMS solution, soak each concentration of EMS at 28°C for 8h, 12h and 16h respectively, and the treatment and time numbers of different volume fractions are A8-F16, in which potassium phosphate buffer The control solution was named CK (Table 2). All the centrifuge tubes were placed in a mixer at 28°C at 40 r / min, dipped and shaken in the dark.

[0039] Table 1 EMS solution preparation table

...

Embodiment 3

[0043] Example 3: Cleaning of EMS treated tobacco seeds and EMS waste liquid treatment

[0044] Rinse the seeds in the centrifuge tube with phosphate buffer solution 50 times, add to 50mL of the centrifuge tube each time, tighten the tube cap (to prevent leakage of EMS residue), and then shake it up and down about 30 times. Finally, pour it into a small sieve, mark it and rinse it with running water for 0.5 h. The cleaned seeds were blotted dry with filter paper and then dried naturally for later use.

[0045] After the treatment was completed, the EMS waste liquid obtained from the first 5 washes was poured into 1mol / L NaOH solution for digestion.

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Abstract

The invention belongs to the technical field of tobacco breeding and particularly relates to a large-scale efficient cultivation method for tobacco mutants on the basis of an EMS (ethyl methanesulfonate) mutagen. The method comprises steps of pretreatment of tobacco seeds, treatment with EMS, seed germination and transplanting, screening of variant plants as well as calculation of germination rateand survival rate. EMS treatment conditions are particularly optimized for main tobacco cultivar K326, the tobacco K326 is enabled to be sub-vital through probing, and the optimal EMS treatment conditions are obtained. The method can be applied to construction of a tobacco mutant library, can be directly applied to breeding of new varieties and has the advantage that advanced backcross is not needed, and genetic breeding resources of tobacco are enriched.

Description

technical field [0001] The invention belongs to the technical field of tobacco breeding, and in particular relates to a method for large-scale and high-efficiency cultivation of tobacco mutants by using EMS (ethyl methylsulfonate) mutagen. Background technique [0002] Traditional breed selection mainly includes systematic breeding and hybrid breeding. In the process of creating an excellent variety, the systematic breeding method forms a system (group) through repeated screening and comparison of an excellent mutant individual plant, and finally becomes a new variety. , regional test and production test, variety approval and promotion several steps). However, the introduction of high-quality genetic resources from wild tobacco resources to cultivated tobacco by cross-breeding is often accompanied by linkage burden, which will destroy the optimal genetic composition of excellent germplasm, and may bring unfavorable traits. Traditional tobacco breeding usually takes 5-8 yea...

Claims

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Application Information

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IPC IPC(8): A01H1/06A01C1/00
CPCA01H1/06A01C1/00
Inventor 杨大海姚恒谢贺白戈陈学军逄涛李勇黄昌军刘勇方敦煌肖炳光李永平
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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