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Genetic transformation method of gene gun mediated elite maize inbred line

A technology of genetic transformation and biolistic method, applied in the fields of biochemical equipment and methods, horticultural methods, genetic engineering, etc.

Active Publication Date: 2020-05-15
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But for the corn backbone inbred line Jing 724, there is no report on its efficient tissue culture and genetic transformation methods

Method used

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  • Genetic transformation method of gene gun mediated elite maize inbred line
  • Genetic transformation method of gene gun mediated elite maize inbred line
  • Genetic transformation method of gene gun mediated elite maize inbred line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0082] Example 1, the medium involved in the tissue culture and genetic transformation of the corn backbone inbred line Jing 724

[0083] 1. Callus induction medium

[0084] The medium designed for the callus induction of the corn backbone inbred line Jing 724 includes callus induction medium MC-1, callus induction medium MC-2 and callus induction medium MC-3.

[0085] The callus induction medium MC-1 is composed of solute and solvent, the solvent is double distilled water, the solute and its concentration are: 0.5ml / L 1000X N6 basic salt, 2g / L MS basic salt, 1ml / L 1000X N6 organic, 0.2g / L Calcium Chloride Dihydrate, 20g / L Sucrose, 1.4g / L Proline, 0.5g / L Hydrolyzed Casein, 0.1g / L Inositol, 0.5mg / L 2,4-D, 2.5mg / L dicamba, 0.2mg / L 6-benzylaminopurine, 3g / L plant gel. The pH value of callus induction medium MC-1 was 5.8.

[0086] Callus induction medium MC-2 is composed of solute and solvent, the solvent is double distilled water, the solute and its concentration are: 0.35ml / ...

Embodiment 2

[0098] Embodiment 2, the tissue culture method of corn backbone inbred line Beijing 724

[0099] 1. Acquisition and picking of young ears

[0100] According to the method reported in the literature "Jones T, Lowe K, Hoerster G, et al. Maize Transformation Using the Morphogenic Genes Baby Boom and Wuschel2[M] / / Transgenic Plants. HumanaPress, New York, NY, 2019:81-93." The corn backbone inbred line Jing 724 was sown in the field, and artificial pollination was carried out 50-55 days after sowing. 9-12 days after artificial pollination, young ears are picked when the maximum diameter of corn kernels is 4-4.5 mm.

[0101] 2. Peel off the immature embryos

[0102] According to the method reported in the literature "Jones T, Lowe K, Hoerster G, et al. Maize Transformation Using the Morphogenic Genes Baby Boom and Wuschel2[M] / / Transgenic Plants. HumanaPress, New York, NY, 2019:81-93." The immature embryos are peeled off from the young ears obtained in step one.

[0103] 3. Callus...

Embodiment 3

[0115] Example 3. Genetic Transformation Method of Young Embryos of Maize Backbone Inbred Line Jing 724

[0116] 1. Carrier Construction

[0117] Insert the pr2X35s::GFP::t35s / / prZmUbi-01::HPT::tNos fragment shown at position 131-5144 of Sequence 1 into the T-DNA region of the transformed pCAMBIA3301 vector to obtain the pY105 recombinant vector. The nucleotide sequence of the pY105 recombinant vector is shown as sequence 1 in the sequence listing.

[0118] 2. Gene gun transformation

[0119] After the immature embryos were obtained according to the method in steps 1 to 3 in Example 2, the immature embryos were cultured in a hyperosmotic medium for about 4-6 hours, and transformed by a gene gun-mediated transformation method to obtain immature embryos after shooting. For the gene gun-mediated transformation method, please refer to the specific steps in Agrobacterium-and Biolistic-Mdediatedtransformation of Maize B104 Inbred reported by Jennifer A.Raji et al. In the experimen...

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Abstract

The invention discloses a genetic transformation method of a gene gun mediated elite maize inbred line. The method comprises the steps as follows: 1) a vector containing a target gene is shot into a young embryo of a maize inbred line Jing 724 by a gene gun method to obtain a shot young embryo; 2) a callus induction medium is inoculated with the shot young embryo for culture to obtain embryonic callus; 3) a screening medium is inoculated with the embryonic callus for culture to obtain resistant callus; 4) a regeneration medium is inoculated with the resistant callus for culture to obtain seedlings; and 5) the seedlings are cultured in a rooting medium to obtain a transgenic plant of the maize inbred line Jing 724.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a genetic transformation method of a corn backbone inbred line, in particular to a gene gun-mediated genetic transformation method of the corn backbone inbred line Jing 724. Background technique [0002] Corn is an important crop for the supply of food, feed, and industrial raw materials. It plays an important role in the modern era of population growth, animal husbandry, and high-speed industrial development. According to the statistics of the Ministry of Agriculture, the gap between my country's corn supply and demand in 2017 / 2018 was 4.3 million tons. According to the analysis of the corn industry development trend report, the growth rate of China's corn production will decline during 2016-2030, while the growth rate of total corn consumption It will obviously exceed the growth rate of production, and the gap between domestic supply and demand will further expand. If t...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H6/46A01H4/00
CPCC12N15/8207A01H4/008
Inventor 武莹李宏潮杨进孝冯峰
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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