Establishment method of tobacco hairy root genetic transformation system

A technology for a genetic transformation system and a method for establishing a genetic transformation system is applied in the field of establishing a genetic transformation system of tobacco hairy roots, which can solve the problems of shortening the research period, long research period, and accelerating the research process of tobacco functional genes, so as to shorten the research period and shorten the period. , the effect of accelerating the research process

Pending Publication Date: 2022-03-04
YUNNAN ACAD OF TOBACCO AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the functional research of tobacco genes is generally realized through transgenic tobacco plants, and the research cycle is relatively long, and the research cycle can be greatly shortened by using hairy roots. The research process of functional genes

Method used

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  • Establishment method of tobacco hairy root genetic transformation system
  • Establishment method of tobacco hairy root genetic transformation system
  • Establishment method of tobacco hairy root genetic transformation system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] 1.1. Obtaining sterile tobacco vaccines: including tobacco seed disinfection and seed germination.

[0036] 1.1.1 Disinfection of tobacco seeds: soak the seeds with 75% alcohol for 60s~70s, discard the alcohol, then sterilize with 50% (v / v) sodium hypochlorite shock for 9~11min, and finally rinse with sterile water 5~6 times to obtain aseptic Tobacco seeds.

[0037] 1.1.2 Seed Germination: Inoculate sterile tobacco seeds in MS medium (2 / 3MS 2.95g / L, Sucrose 20g / L, Agrose 8g / L, pH: 5.8), culture in the dark for 2 days and place in the light incubator (Under the condition of 25°C, cultivate in light for 6 hours per day and in dark for 18 hours per day). After 1 week of germination, the seedlings were transplanted into sterile culture bottles to continue culturing; cultured in sterile culture bottles for 2 weeks to obtain 2-week-old tobacco aseptic seedlings, and 2-week-old tobacco aseptic seedlings were as follows: figure 1 Shown in a.

[0038] 1.2. Genetic transformat...

Embodiment 2

[0045] The acquisition of tobacco sterile seedlings in this embodiment: including tobacco seed disinfection and seed germination.

[0046] 2.1.1 Disinfection of tobacco seeds: soak the seeds with 75% alcohol for 60s-70s, discard the alcohol, then sterilize with 50% (v / v) sodium hypochlorite for 9-11 minutes, and finally rinse with sterile water for 5-6 times to obtain aseptic Tobacco seeds.

[0047] 2.1.2 Seed germination: Inoculate sterile tobacco seeds in MS medium (2 / 3MS 2.95g / L, Sucrose 20g / L, Agrose 8g / L, pH: 5.8), culture in dark for 2 days and then place in light incubator (Under the condition of 25°C, cultivate in light for 6 hours per day and in dark for 18 hours per day). After 1 week of germination, the seedlings were transplanted into sterile culture bottles to continue culturing; cultivated in sterile culture bottles for 2 weeks to obtain 2-week-old tobacco aseptic seedlings, and 2-week-old tobacco aseptic seedlings were as follows: figure 1 Shown in a.

[0048...

Embodiment 3

[0055] The acquisition of tobacco sterile seedlings in this embodiment: including tobacco seed disinfection and seed germination.

[0056] 3.1.1 Disinfection of tobacco seeds: soak the seeds with 75% alcohol for 60s-70s, discard the alcohol, then sterilize with 50% (v / v) sodium hypochlorite shock for 9-11 minutes, and finally rinse with sterile water 5-6 times to obtain aseptic Tobacco seeds.

[0057] 3.1.2 Seed Germination: Inoculate sterile tobacco seeds in MS medium (2 / 3MS 2.95g / L, Sucrose 20g / L, Agrose 8g / L, pH: 5.8), culture in dark for 2 days and then place in light incubator (Under the condition of 25°C, cultivate in light for 6 hours per day and in dark for 18 hours per day). After 1 week of germination, the seedlings were transplanted into sterile culture bottles to continue culturing; cultivated in sterile culture bottles for 2 weeks to obtain 2-week-old tobacco aseptic seedlings, and 2-week-old tobacco aseptic seedlings were as follows: figure 1 Shown in a.

[00...

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Abstract

The invention discloses an establishment method of a tobacco hairy root genetic transformation system. Comprising the steps of sterilizing tobacco seeds to obtain sterile tobacco seeds, germinating the tobacco seeds to obtain tobacco sterile seedlings, preparing agrobacterium, preparing agrobacterium infection liquid, infecting, co-culturing, sterilizing and culturing, screening and culturing to obtain tobacco hairy roots capable of being used for gene function detection, identifying the hairy roots and the like. The method is simple and easy to implement and short in period, enough hairy roots can be obtained in 8 weeks, and the obtained hairy roots can be applied to functional identification of tobacco genes (including detection of expression quantity of transformed genes; detection of the expression quantity of other target genes, detection of the content of nicotine, nornicotine, neonicotine and other tobacco substances); the technical problem that the traditional stable tobacco plant transformation from genetic transformation to T1-generation plants capable of being used for gene function identification takes a long time of about one year is solved; compared with the prior art, the tobacco hairy root genetic transformation system obviously shortens the research period.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for establishing a tobacco hairy root genetic transformation system. Background technique [0002] Hairy root is widely used as a tool in the production of plant secondary metabolism, especially in the production of resource plant secondary metabolites, which has unique advantages and prospects. For example, the content and accumulation efficiency of diosgenin in hairy roots of Baiying were 4.5 and 10 times that of wild-type Baiying. The content of total flavonoids in the hairy root of Astragalus mongolica was significantly higher than that of the control, and the content of various low-polarity saponins in the hairy root tissue of Jintiesuo was significantly higher than that of Jintiesuo. In addition, hairy roots can also be used as an effective tool for gene function research, for example, using the hairy root system to study the gene functions of chalcone...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/06A01H6/82C12Q1/6895
CPCC12N15/8205C12Q1/6895C12Q2600/13
Inventor 王丙武赵璐高玉龙宋中邦孔光辉王亚辉隋学艺张谊寒焦芳婵吴兴富李永平贺晓辉
Owner YUNNAN ACAD OF TOBACCO AGRI SCI
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