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Agrobacterium mediated efficient corn backbone selfing line genetic transformation method

A genetic transformation method and technology of inbred lines, applied in the field of genetic transformation of high-efficiency corn backbone inbred lines

Inactive Publication Date: 2016-05-11
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the inbred lines X178 and Zong 31, no efficient genetic transformation method has been disclosed

Method used

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  • Agrobacterium mediated efficient corn backbone selfing line genetic transformation method
  • Agrobacterium mediated efficient corn backbone selfing line genetic transformation method
  • Agrobacterium mediated efficient corn backbone selfing line genetic transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1、X178

[0103] Embodiment 1, X178 field planting and immature embryo obtain

[0104] 1. Field planting of X178

[0105] Seed: X178

[0106] Sowing time (twice): May 10, 2014 and May 10, 2015.

[0107] Test location: Shangzhuang Experimental Station of China Agricultural University.

[0108] Sowing area (each time): 0.5 mu; the row length is 5 meters, the row spacing is 0.6 meters, and the plant spacing is 0.3 meters.

[0109] Apply 200 catties of compound fertilizer / mu and 4,000 catties / mu of organic matter before sowing; topdressing will not be carried out after sowing. Irrigate according to the water conditions in the field, watering 4 times in total in 2014 and 2 times in 2015.

[0110] Sow 2 seeds in each hole, grow to 5 leaves and start thinning, leaving one seedling in each hole.

[0111] Use normal weed and pest management during planting.

[0112] 2. Artificial pollination

[0113] Artificial pollination is adopted to ensure that the size of the immature embryos after p...

Embodiment 2、X178

[0124] Embodiment 2, establishment of X178 genetic transformation method

[0125] 1. Preparation of Agrobacterium infection solution

[0126] (1) Plasmid pCAMBIA3301 (vector schematic diagram see figure 1 ) to transform Agrobacterium tumefaciens EHA105, pick the recombinant Agrobacterium, and name the recombinant Agrobacterium EHA105 / pCAMBIA3301.

[0127] (2) Inoculate the EHA105 / pCAMBIA3301 obtained in step (1) on AB basic medium containing 50 mg / L kanamycin and 25 mg / L rifampicin, and culture at 28° C. for 3 days.

[0128] (3) After completing step (2), pick a single colony with an inoculation needle, inoculate it on a YEP solid plate containing 50 mg / L kanamycin and 25 mg / L rifampicin, and culture at 20° C. for 3 days.

[0129] (4) After completing step (3), scrape the colony with a bacterial inoculation loop (about 5 mm), inoculate it into 5-10 mL of infection medium, and cultivate it with shaking at 23 ° C and 100-120 rpm for 4 hours to obtain the Agrobacterium infectio...

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Abstract

The invention discloses a corn selfing line genetic transformation method. The corn selfing line genetic transformation method comprises the following steps that a recombinant expression vector containing target genes is introduced into agrobacteria, and recombined agrobacteria are obtained; young embryos of the corn selfing line are infected with the recombined agrobacteria; the young embryos of the corn selfing line are taken and placed in a co-culture medium for co-culture; the young embryos of the corn selfing line are taken and placed in a recovery medium I, a screening medium I, a screening medium II, a recovery medium II and a differential medium in sequence for culture, and seedlings are obtained; the seedlings are taken and placed in a rooting medium for culture, and corn selfing line transformation seedlings are obtained. When the corn selfing line genetic transformation method is applied to corn selfing line genetic transformation, a high transformation rate can be obtained, and great significance is achieved on research work of corn breeding and the corn gene function.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for genetic transformation of high-efficiency corn backbone inbred lines mediated by Agrobacterium. Background technique [0002] Corn (Zeamays L.) is an important food and feed crop, and also an important raw material for modern industry. With the development of animal husbandry, industry and the growth of world population, corn plays an increasingly important role in agricultural production. Since 1998, corn has surpassed rice and wheat to become the world's largest crop. Since the founding of the People's Republic of my country, the sown area, per unit yield and total output of corn in China have shown a continuous growth trend. In 2002, the sown area of ​​corn in my country surpassed that of wheat and was second only to rice. The per unit yield of corn increased from 1123.1kg / hm 2 It has increased to 5394.0 kg / ha, exceeding the average yield level of corn in the world. ...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H4/00
CPCC12N15/8205A01H4/001A01H4/008
Inventor 赖锦盛宋伟彬赵海铭
Owner CHINA AGRI UNIV
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