Agrobacterium-mediated tomato genetic transformation method

A genetic transformation method and technology mediated by Agrobacterium, applied in the field of tomato genetic transformation mediated by Agrobacterium, can solve the problems of genetic instability of transgenic offspring, affecting the efficiency of genetic transformation, silencing of foreign genes, etc. The effect of genetic stability and expression level improvement

Inactive Publication Date: 2020-10-30
HAINAN UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The sensitivity of different genotypes of tomato varieties to growth regulators is quite different, resulting in the specificity of different genotypes of tomato varieties to growth regulators, which affects the efficiency of genetic transformation
[0005] As one of the main model plants for plant transgenic research, tomato's genetic transformation system has been reported a lot, but there are still many problems, such as low transformation rate, genetic instability of transgenic offspring, exogenous gene silencing, complicated operation, etc.

Method used

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  • Agrobacterium-mediated tomato genetic transformation method
  • Agrobacterium-mediated tomato genetic transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1: a kind of tomato genetic transformation method mediated by Agrobacterium, specifically comprising:

[0065] Step 1: Planting of sterile seedlings

[0066] 11) In the ultra-clean bench, put a certain amount of Micro-Tom seeds into a sterile plate, pour in 75% absolute ethanol, soak for 5 minutes, discard the solution, add a 4% sodium hypochlorite solution, and soak 5min, discard the solution, wash 5 times with sterile water, and wash the sodium hypochlorite solution to prevent it from affecting the germination rate of the seeds;

[0067] 12) Use sterile tweezers to evenly place the seeds into culture bottles containing T0 medium, 30 seeds in each culture bottle;

[0068] 13) Place it in a dark box and cultivate it for 3 days at a temperature of 25°C until the rooted seeds germinate and the buds grow to 2cm;

[0069] 14) Place the culture bottle of germinated seeds in the cultivation room, and cultivate under light for 2 days at a temperature of 25°C until...

Embodiment 2

[0102] Embodiment 2: Different from Embodiment 1:

[0103] A method for tomato genetic transformation mediated by Agrobacterium, specifically comprising:

[0104] Step 1: Planting of sterile seedlings

[0105] 11) In the ultra-clean bench, put a certain amount of Micro-Tom seeds into a sterile plate, pour in 75% absolute ethanol, soak for 5 minutes, discard the solution, add a 4% sodium hypochlorite solution, and soak 5min, discard the solution, wash 5 times with sterile water, and wash the sodium hypochlorite solution to prevent it from affecting the germination rate of the seeds;

[0106] 12) Use sterile tweezers to evenly place the seeds into culture bottles containing T0 medium, 30 seeds in each culture bottle;

[0107] 13) Place it in a dark box and cultivate it for 4 days at a temperature of 25°C until the rooted seeds germinate and the buds grow to 2.5cm;

[0108] 14) Place the culture bottle of germinated seeds in the cultivation room, and cultivate under light for ...

Embodiment 3

[0136] Embodiment 3: Different from Embodiment 1:

[0137] A method for tomato genetic transformation mediated by Agrobacterium, specifically comprising:

[0138] Step 1: Planting of sterile seedlings

[0139] 11) In the ultra-clean bench, put a certain amount of Micro-Tom seeds into a sterile plate, pour in 75% absolute ethanol, soak for 5 minutes, discard the solution, add a 4% sodium hypochlorite solution, and soak 5min, discard the solution, wash 5 times with sterile water, and wash the sodium hypochlorite solution to prevent it from affecting the germination rate of the seeds;

[0140] 12) Use sterile tweezers to evenly place the seeds into culture bottles containing T0 medium, 35 grains in each culture bottle;

[0141] 13) Place it in a dark box and cultivate it for 5 days at a temperature of 25°C until the rooted seeds germinate and the buds grow to 3cm;

[0142] 14) Place the culture bottle of germinated seeds in the cultivation room, and cultivate under light for 4...

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Abstract

The invention relates to the technical field of plant genetic engineering, and provides an agrobacterium-mediated tomato genetic transformation method. The method specifically comprises the followingsteps: obtaining a sterile tomato leaf explant; infecting the leaf explant with agrobacterium tumefaciens; screening the resistance of the explant; carrying out subculture on the explant; rooting culture of the explant; carrying out soil moving culture on the transgenic plant; according to the method, the procedure of genetic transformation is simplified, the whole process is simple, and the transformation period is effectively shortened; ; the heredity of the transgenic offspring tends to be stable; the operation is simple, the expression level of the exogenous gene is obviously improved, andthe conversion efficiency is greatly improved.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a tomato genetic transformation method mediated by Agrobacterium. Background technique [0002] Tomato belongs to the Solanaceae family. As a vegetable and economic crop widely grown in the world, it has the advantages of small genome (950Mb), short growth cycle (45-100d), self-pollination, etc. It is also used to cultivate transgenic plants and plant biopharmaceutical products. pattern plants. In recent years, the total tomato production in the world has also increased significantly, and it has become one of the important sources of minerals and vitamins in many countries. [0003] In 1986, McCormick et al. proposed for the first time to use the Agrobacterium-mediated leaf disc method to transform tomato. This method uses tomato cotyledons and hypocotyls as explants, and uses single sequences and multi-copy sequences as exogenous genes to obtain More than 300...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00A01H6/82
CPCA01H4/00C12N15/8205
Inventor 孟兰环王宇史学群
Owner HAINAN UNIVERSITY
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