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Method for rapid genetic transformation by utilizing mature millet seeds

A genetic transformation and seed technology, applied in the field of plant genetic engineering, can solve the problem of cumbersome induction of callus, achieve the effect of shortening the time and promoting the progress of breeding

Inactive Publication Date: 2014-08-27
刘颖慧 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Based on the above reasons, coupled with the cumbersomeness of the original callus induction and the limitation of seasons, there is an urgent need for a rapid genetic transformation technology that can be done at any time.

Method used

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  • Method for rapid genetic transformation by utilizing mature millet seeds
  • Method for rapid genetic transformation by utilizing mature millet seeds
  • Method for rapid genetic transformation by utilizing mature millet seeds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The seeds of different germination stages were selected for infection, and the results showed that the micro-injury treatment before infection would damage the elongated shoots and roots, and the shaking treatment of co-cultivation would also cause the shoots and roots to fall off. And this damage will be more serious with the increase of germination days, so we choose 1-day-germinated seeds as infected explants.

[0022] Embodiment 2: Agrobacterium strain and cultivation

Embodiment 2

[0023] Agrobacterium strain EHA105 and plasmid pCAMBIA1301 were used for transformation experiments. Transform the plasmid into Agrobacterium EHA105 by heat shock method, inoculate in YEP solid medium and culture in dark for 16-18 hours, pick a single clone and inoculate in 5mL liquid medium with kanamycin, shake at 28°C Cultivate for 12-16 hours to make the bacterial solution reach saturation, then transfer the cultured bacterial solution to fresh YEP liquid medium at a ratio of 1:100 to activate the OD 600 Values ​​between 0.4-0.8 are achieved for infestation.

[0024] Embodiment 3: Transformation, screening and detection of plants

Embodiment 3

[0025] Different concentration gradients of hygromycin were used for screening, and the results showed that most millet could not survive under 25 mg / l hygromycin, especially after transplanting into pots, many seedlings died. The surviving seedlings were selected and transplanted to the field for planting, and finally surviving plants were obtained. In order to study the integration of genes, genomic DNA was extracted, and PCR detection was performed on the transgenic plants.

[0026] Example 4: GUS activity detection

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Abstract

The invention relates to an agrobacterium-mediated transformation system for rapid genetic transformation by utilizing mature millet seeds, and the agrobacterium-mediated transformation system is a rapid genetic millet transformation system without relying on tissue culturing. The transformation efficiency of the agrobacterium-mediated transformation system is 1.77%; the plant regeneration time is 3 months. According to the agrobacterium-mediated transformation system, only 45 days are required from the infection of the mature seeds to the final obtainment of a plant; only 3 months are required up until the seeds are harvested, so that the plant transformation time is greatly shortened. As the agrobacterium-mediated transformation system is used, the clonal variation of cytoplasts during the tissue culturing is reduced; meanwhile, a plurality of important agronomic trait genes can be introduced into millets, thereby promoting the breeding progress rate of millet molecules.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and relates to a method for genetic transformation of millet, in particular to a method for establishing a fast Agrobacterium transformation system that does not rely on tissue culture and is carried out using mature millet seeds. Background technique [0002] Millet is an important food and feed crop, especially in less developed countries and arid and semi-arid areas, where millet occupies a very important position. Agrobacterium-mediated plant transformation technology is becoming more and more mature and perfect, and has been widely used in major crops such as rice, corn, and wheat. However, the genetic transformation of millet is still relatively small, and a mature transformation system has not yet been established. In particular, the callus culture of millet is difficult and tedious, and the materials are also limited by seasons and other factors. [0003] Studies have s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/10
Inventor 刘颖慧董志平袁进成马海莲宋晋辉李志勇宋小青王凌云
Owner 刘颖慧
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