Method for efficiently and rapidly stabilizing gene transformation for tomatoes

A gene transformation and tomato technology, applied in the field of genetic engineering, can solve the problems of unsuccessful transgenesis, inability to suppress Agrobacterium, and long time consumption, so as to improve the survival rate and regeneration rate, increase the efficiency of transgenic, and avoid tedious work.

Active Publication Date: 2015-12-30
JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY
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  • Abstract
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  • Application Information

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Problems solved by technology

Although the genetic transformation technology route of tomato is mature, the traditional method is laborious, time-consuming, cumbersome and inefficient, and too few transgenic plants are obtained, which brings obstacles to the later identification of transgenic plants
The operation process of the traditional Agrobacterium-mediated method is cumbersome, and many problems are prone to occur during the transgenic process, such as the selection of suitable

Method used

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1 The preparation of culture medium, bacterial strain and plant expression vector of the present invention

[0028] 1. Materials

[0029] (1) Plant material

[0030] Tomato Micro-Tom Seeds and Tissue Culture Plantlets.

[0031] (2) Preparation of main solution and culture medium

[0032] The medium preparation method is as follows:

[0033] Tomato seed medium: S1

[0034] MS2.2g / L, sucrose 30g / L, agar 5.5g / L, pH5.8, autoclaved, ready for use.

[0035] Medium for tomato transgenic seedlings: S2

[0036] MS2.2g / L, sucrose 30g / L, agar 5.5g / L, ZT1mg / L, NAA0.4mg / L, kanamycin 20mg / L, carbenicillin 400mg / L, pH5.8, autoclaved, Let cool and set aside.

[0037] (3) Strains and plant expression vectors

[0038] The bacterial strain used in the present invention is Agrobacterium strain EHA105, which is a commonly used bacterial strain in tomato transgenic and has strong infection ability. The strain was purchased from ATCC (American type culture collection).

...

Embodiment 2

[0040] Embodiment 2 A kind of high-efficiency, rapid and stable gene transformation method of tomato

[0041] (1) Activation of strains

[0042] ① Streak Agrobacterium EHA105 containing plasmid pK7WG2D on LB solid medium (containing 50mg / LSPR (spectinomycin) and 50mg / LRif (rifampicin)) and culture at 28°C for 2 days.

[0043] ②Pick 2-3 good single colonies that grow on the plate, inoculate them into 50mL LB liquid medium (containing 50mg / LSPR (spectinomycin) and 50mg / LRif (rifampin)), shake at 28°C and 220rpm Shake the culture in the bed until the OD600 value is 0.4 (about 12-16h).

[0044] ③ Centrifuge at 5000rmp for 8 minutes at room temperature, remove the supernatant, turn the centrifuge tube upside down on sterile filter paper, remove the supernatant as much as possible, resuspend the bacteria in 100mL MS liquid, and culture in a shaker for about 1 hour to measure OD600 The value of 0.2 is the best (this value is very important, it cannot be lower than 0.2, it cannot be...

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Abstract

The invention discloses a method for efficiently and rapidly stabilizing gene transformation for tomatoes. The method comprises the steps that 1, activation of stains is conducted; 2, preparation of plant materials is conducted; 3, infection is conducted; 4, screening and culturing are conducted. According to the method, the tomato transgenic efficiency is increased, and the transformation rate can reach over 30 percent. The concentration of bacterial liquid is reduced, the infection time is prolonged, and on one hand, agrobacterium pollution is avoided; on the other hand, the infection efficiency is increased. The infection bacterial liquid is not added with drugs for assisting in infection, so that damage to tomato leaves is reduced, the survival rate and the regeneration rate of leaf culture are increased; in a traditional method, drugs such as acetosyringone which assists in infection are usually added, damage is caused to leaves, and the regeneration capacity of the leaves is reduced. By means of carriers carrying strong expressed reporter genes, transgenic plants can be obtained directly by screening through observation, and tedious work in traditional detection is avoided. The cycle of transgenes is shortened.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a method for efficient, rapid and stable gene transformation of tomato. Background technique [0002] Tomato (Lycopersicone sculentum Mill.) is an important food crop in the world. It has many varieties, high yield, rich nutrition and wide application. Micro-Tom is a tomato mutant, which retains the basic characteristics of common tomato as a model plant, but the plant is short and has a shorter life cycle, which has the great advantage of saving research space and shortening research time. With the rapid development of molecular biology and genetic engineering in recent years, the research focus has turned to the deep mining of excellent genes in tomato germplasm resources, and the identification and determination of molecular markers and gene expression sequences. At the same time, research results on genetic improvement such as stress resistance, storage and tran...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00
Inventor 王媛花张立伟史红林
Owner JIANGSU POLYTECHNIC COLLEGE OF AGRI & FORESTRY
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