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Efficient transgenic method for maize skeleton inbred lines

A backbone inbred line and transgenic technology, which is applied in the field of genetic engineering and crop breeding, can solve the problems of long transformation cycle, low positive rate, and low transformation efficiency, and achieve high positive rate, simple and convenient operation, and high transformation efficiency. Effect

Active Publication Date: 2015-07-01
先正达集团股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in actual operation, there are many shortcomings in the preparation of 'Xiang 249' transgenic varieties according to conventional transgenic methods, mainly in the aspects of low transformation efficiency, low positive rate, and long transformation cycle.

Method used

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  • Efficient transgenic method for maize skeleton inbred lines
  • Efficient transgenic method for maize skeleton inbred lines
  • Efficient transgenic method for maize skeleton inbred lines

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1 Preparation of Agrobacterium tumefaciens containing target gene

[0035] The Agrobacterium strains used in this specific embodiment are EHA105 and LBA4404. The herbicide-resistant gene Bar (gene sequence such as SEQ ID NO: 1) and the green fluorescent protein coding gene GFP (gene sequence such as SEQ ID NO: 2) were transformed into Agrobacterium by the Agrobacterium transformation method commonly used in the art. It should be understood that this specific embodiment uses the gene encoding green fluorescent protein as an example of the exogenous gene, but the present invention is not limited to a specific exogenous gene. The Agrobacterium-introduced gene used in this embodiment, the vector construction method and the plasmid used are only well known to those skilled in the art.

[0036] One week before infection, take 20 μL of glycerol bacteria from the -80°C refrigerator, and streak on the YEP medium plate with the corresponding antibiotics (EHA105 corres...

Embodiment 2

[0037] Example 2 Preparation of transgenic corn

[0038] The maize inbred line used in this example is 'Xiang 249', provided by China Seed Group Co., Ltd.

[0039] 1. Selection of ears and separation of immature embryos

[0040] (1) Select corn cobs whose young embryos have grown to 0.5-2.0 mm in 6-15 days after pollination, and the number of samples shall not be less than 500;

[0041] (2) Mix the sodium hypochlorite with a concentration of 6.15% with sterilized water at a concentration of 15% by volume, and add 1 drop (20ul) of Tween-20 to make a sterilant;

[0042] (3) Soak the cobs of corn in a sterilizing agent for 15 minutes, and then rinse them with sterile water for 3-5 times;

[0043] (4) Peel off the immature embryos, and put the immature embryos into the suspension.

[0044] 2. Infection and co-cultivation

[0045] (1) Put the stripped immature corn embryos into a 2ml EP tube containing 1.8mL of suspension, treat about 150 immature embryos within 30min, and then...

Embodiment 3

[0080] Example 3 Detection of transgenic corn

[0081] 1. Observation on the expression of exogenous gene GFP in tissues of transgenic maize

[0082] Take the 'Xiang 249' transformed immature embryos, callus and leaves of Example 2 to observe the expression of the exogenous gene (GFP) in the tissue. It turns green under fluorescence, indicating that the exogenous gene is successfully expressed, otherwise it is a negative material. Compared with the positive material, the negative material is darker under the fluorescence, the immature embryo and callus are yellow, and the leaves are red. see results Figure 1A , Figure 1B , Figure 1C . 2. PCR detection

[0083] (1) DNA extraction: Genomic DNA of the T0 transgenic maize obtained in Example 2 was extracted with a DNA extraction kit purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0084] (2) PCR detection

[0085] Thaw the following reagents from the -20°C refrigerator: 10×PCR reaction buffer (Takara), ...

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Abstract

The invention provides an efficient transgenic method for maize skeleton inbred lines. According to the method, an efficient transgenic effect for the maize skeleton inbred lines is realized by adding 0.01-1mg / L of cytokinin KT(6-glycosylaminopurine) in a co-culture medium and a rest culture medium, and determining the optimal screening agent concentration in a selective culture medium, i.e., the adding concentration of balaphos is 0.5-50mg / L. The method provided by the invention has the characteristics of being short in experiment period, high in positive rate, and simple and convenient to operate, used for preparing transgenic maize, and suitable for large-scale popularization.

Description

technical field [0001] The invention belongs to the field of genetic engineering and crop breeding, and in particular relates to a high-efficiency transgenic method for a corn backbone inbred line. Background technique [0002] GM corn is one of the most important GM crops in the world. In 2010, the global planting area of ​​genetically modified corn reached 46.8 million hectares, accounting for 26% of the total global corn area. In terms of corn transgenic technology, it was not until the late 1980s that a breakthrough was made in transformation. The first successful maize inbred line (A188) to produce regenerated plants was reported in 1975. Until 1988, Klein et al. used the gene gun method to transfer the cat gene to the embryoid body and non-embryoid body cells of maize, and found transient expression. At the same time, gene expression and labeling systems have also been developed. It was not until 1996 that the Agrobacterium co-cultivation method was successfully us...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H4/00A01H5/00
Inventor 许洁婷佘秋明周倩文琴黄磊胡燕琳卢涛章旺根
Owner 先正达集团股份有限公司
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