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High-efficiency nonglutinous rice genetic transformation method

A genetic transformation method, the technology of indica rice, is applied in the field of plant tissue culture and transgenic engineering, which can solve the problems of difficult transformation, poor tissue culture characteristics, and the establishment of an efficient genetic transformation system. good condition

Inactive Publication Date: 2017-09-08
湖南隆平高科种业科学研究院有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for indica rice with a large planting area, its tissue culture characteristics are generally poor due to differences in genotypes, and the transformation difficulty is significantly greater than that of japonica rice.
In recent years, the use of Agrobacterium-mediated transformation of indica rice has also achieved some success, but a relatively stable and efficient genetic transformation system like japonica rice has not been established so far.

Method used

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  • High-efficiency nonglutinous rice genetic transformation method
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  • High-efficiency nonglutinous rice genetic transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] (1) Induction of callus

[0042]Select the rice grains of the well-matured and complete indica rice variety indica type two-line restorer line 2537, first sterilize with 75% alcohol for 3 minutes, then soak in 25% (V / V) sodium hypochlorite solution, and place in a shaker at 150rpm and 25°C. Sterilize the bed for 20 minutes; wash the sterilized seeds 5 times with sterile water on the ultra-clean workbench, then dry the water with sterile filter paper, and then inoculate the seeds in NB medium; cultivate them under light at 28°C-30°C for 8 days, callus induction ( figure 1 a), wherein, NB culture medium component is: N 6 Inorganic salts and organic matter + inositol 0.1g / L+glutamine 0.5g / L+acid hydrolyzed casein 0.5g / L+proline 2.878g / L+sucrose 30.0g / L+agar powder 8.0g / L+2,4-D 2.0mg / L, pH 5.8.

[0043] (2) Pre-cultivation of callus

[0044] Select callus with compact structure, bright color and granular shape and transfer it to the pre-medium, and culture it under ligh...

Embodiment 2

[0056] (1) Select the rice grains of well-matured and complete indica rice variety 628S, disinfect them with 75% alcohol for 3 minutes, then soak them in 25% (V / V) sodium hypochlorite solution, and place them on a shaking table at 150rpm and 25°C for disinfection 20 minutes. The sterilized seeds were washed 5 times with sterile water on the ultra-clean workbench, then blotted dry with sterile filter paper, and then inoculated the seeds in NB medium. Cultured under light at 28°C-30°C for 10 days to induce callus ( figure 1 b); NB medium components are: N 6 Inorganic salts and organic matter + inositol 0.1g / L+glutamine 0.5g / L+acid hydrolyzed casein 0.5g / L+proline 2.878g / L+sucrose 30.0g / L+agar powder 8.0g / L+2,4-D 2.0mg / L, pH 5.8. .

[0057] (2) Pre-cultivation of callus

[0058] Select callus with compact structure, bright color and granular shape and transfer it to the pre-medium, and culture it under light at 28°C-30°C for 3 days for transformation ( figure 2 b); the pre...

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Abstract

The invention discloses a high-efficiency nonglutinous rice genetic transformation method. The method comprises the following steps: inducing nonglutinous rice mature embryo callus, pre-culturing the callus, culturing agrobacterium tumefaciens, performing agrobacterium tumefaciens-mediated transformation, co-culturing agrobacterium tumefaciens and callus, screening resistant callus, differentiating, and rooting regenerated seedlings. The method is simple and efficient; the obtained callus is good in quality and suitable for transformation; the transformation operation process is simple; by transforming nonglutinous rice two-line sterility-line 628S and nonglutinous rice two-line restoration-line 2537 of the nonglutinous rice variety, a positive transformed plant of the nonglutinous rice can be obtained in about 60 days, the transformation efficiency reaches up to 35 percent or higher, the transformation rate is relatively high, and the genetic transformation efficiency of the nonglutinous rice is increased.

Description

technical field [0001] The invention belongs to the technical field of plant tissue culture and transgenic engineering, and specifically relates to a method for genetic transformation of high-efficiency indica rice. Methods of genetic transformation of injured tissue. Background technique [0002] Rice (Oryza sativa L.) is one of the most important food crops in my country. It is divided into two subspecies: indica rice and japonica rice. The use of transgenic technology to improve rice varieties and cultivate new varieties with high yield, high quality, multi-resistance, and wide adaptability will not only ensure global food security, but also the development direction of rice breeding in the future. Therefore, genetic transformation of rice, especially indica rice, is playing an increasingly important role in variety improvement, gene function detection, and creation of mutants, and the establishment of an efficient and stable indica rice genetic transformation system is ...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H4/00
CPCA01H4/008C12N15/8205
Inventor 杨远柱周延彪唐晓丹
Owner 湖南隆平高科种业科学研究院有限公司
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