Tomato transformation method of agrobacterium tumefaciens mediation

A technology of Agrobacterium tumefaciens and tomato, which is applied in the field of genetic engineering, can solve the problems of long transformation cycle, cumbersome operation process, and low efficiency of tomato genetic transformation, and achieve the effect of strong repeatability and high transformation efficiency

Inactive Publication Date: 2016-11-23
FUJIAN AGRI & FORESTRY UNIV
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a tomato transformation method mediated by Agrobacterium tumefaciens, overcome the difficulties of low genetic transformation efficiency of existing tomato, long transformation cycle and complicated operation process, and provide a method for overall infection of tomato seeds, thereby Improve the transfection efficiency of tomato

Method used

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  • Tomato transformation method of agrobacterium tumefaciens mediation
  • Tomato transformation method of agrobacterium tumefaciens mediation
  • Tomato transformation method of agrobacterium tumefaciens mediation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Take the transformation of tomato with lycopene β-cyclase gene (β-LCY) as an example, including the following:

[0037] (1) Seed disinfection and cultivation

[0038] Select fresh tomato seeds that are plump, of the same size, and soak in water for 2 hours, rinse with sterile water for 3 times after disinfection, and inoculate the seeds into medium A for cultivation;

[0039] (2) Obtaining cotyledon explants

[0040] Tomato cotyledons with a seedling age of 8 days were selected, inoculated on medium B, and used for transformation after dark culture for 1 day;

[0041] (3) Conversion

[0042] Insert the explants into the EHA105 Agrobacterium suspension, and dip for 20 minutes to make the Agrobacterium adsorb to the explants, and the concentration of the suspension is OD 600 =0.2; move the infected explants to sterilized filter paper, absorb the surface liquid, transfer to solid co-cultivation medium C, carry out co-cultivation at 28°C for 3 days, take out the explant...

Embodiment 2

[0055] A tomato transformation method mediated by Agrobacterium tumefaciens, comprising the following steps:

[0056] (1) Seed disinfection and cultivation

[0057] Select fresh tomato seeds that are plump, of the same size, and soak in water for 2 hours, rinse with sterile water for 3-5 times after disinfection, and inoculate the seeds into medium A for cultivation;

[0058] (2) Obtaining cotyledon explants

[0059] Tomato cotyledons with a seedling age of 8 days were selected, inoculated on medium B, and used for transformation after dark culture for 1 day;

[0060] (3) Conversion

[0061] Insert the explants into the EHA105 Agrobacterium suspension, and dip for 20 minutes to make the Agrobacterium adsorb to the explants, and the concentration of the suspension is OD 600 =0.6; move the infected explants to sterilized filter paper, absorb the surface liquid, transfer to solid co-cultivation medium C, carry out co-cultivation at 28°C for 3 days, take out the explants after...

Embodiment 3

[0096] Taking the transformation of tomato with saffron zeaxanthin lyase gene (CsZCD) as an example, the details are as follows:

[0097] 1. Effect of feeder layer on tomato transformation efficiency

[0098] Table 2 shows that the transformation efficiency of tomato without feeder layer cells can reach more than 20%, but co-cultivation with feeder layer cells can effectively increase the transformation efficiency of tomato. Suspension cells of tomato, potato and petunia can be used as feeder cells, but their preparation is cumbersome, time-consuming and consumable.

[0099] Table 2 Effect of feeder cells on genetic transformation of tomato cotyledons

[0100]

[0101] 2. The effect of seedling age on genetic transformation efficiency of tomato

[0102] The transformation rate of tomato cotyledons increased with the increase of seedling age. When the seedling age was 2-3 days, the transformation rate of tomato cotyledons was the lowest at 5.5%, and when the seedling age w...

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Abstract

The invention provides a tomato transformation method of agrobacterium tumefaciens mediation. Cotyledon explants (the seedling age is 8 days) are precultured for one day in an MSB5 culture medium with 2.0 mg/L of zeatin and 0.1mg/L of IAA, and are then infected by EHA105 agrobacterium for 20 minutes; the concentration of OD600 is 0.2 to 0.6; after the culture is performed for 3 days on a co-culture medium, the co-culture is performed for one day on a culture medium of 500mg/L of cephalosporin; the screening is performed for 6 to 8 weeks on the culture medium containing 100mg/L of kanamycin and 500mg/L of carbenicillin. The method has the advantages that the conversion efficiency is high; feeder layer cells, acetosyringone and bleeding sap are not needed; the conversion efficiency reaches 20 percent or higher; simplicity and high repeatability are realized.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a tomato transformation method mediated by Agrobacterium tumefaciens. Background technique [0002] tomato( Lycopersicon esculentum Mill) is a worldwide vegetable and one of the first higher plants to conduct gene transformation research. Transgenic tomatoes such as anti-virus, insect-resistant, extended storage period, and improved quality have been obtained. The first commercially available transgenic plant, the shelf-stable tomato, has found its way into consumer households. Therefore, tomato has become one of the most popular model plants in vegetable genetic engineering research. At present, although methods such as pollen tube passage and gene gun can be used to introduce foreign genes into the tomato genome, the Agrobacterium-mediated method is the most used and most mature technology in genetic transformation of tomato. Agrobacterium including ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00
CPCC12N15/8205
Inventor 邱栋梁马杰刘婷婷陈文胜
Owner FUJIAN AGRI & FORESTRY UNIV
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