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Method for high efficiency regeneration and genetic transformation of indica rice

A high-efficiency regeneration and genetic transformation technology, applied in biochemical equipment and methods, plant regeneration, horticultural methods, etc., can solve the problems of lack of high-efficiency regeneration system and genetic transformation method of indica rice, failure of successful transformation, low transformation efficiency, etc. Good bacterial effect, good practicability and high efficiency, and the effect of short transformation period

Inactive Publication Date: 2013-02-27
湖北省农业科学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most indica rice varieties are difficult to differentiate, the transformation cycle is long, the transformation efficiency is low, and even some indica rice varieties cannot be successfully transformed
The main reason is the lack of efficient regeneration systems and genetic transformation methods for indica rice.

Method used

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  • Method for high efficiency regeneration and genetic transformation of indica rice
  • Method for high efficiency regeneration and genetic transformation of indica rice
  • Method for high efficiency regeneration and genetic transformation of indica rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] 1. Callus induction:

[0058] Mature embryos of indica rice variety M5274 were used as explants, soaked in 75% alcohol by volume for 1 min, then soaked in 0.1% mercuric chloride for 12 min, washed 5 times with sterile water, and finally the sterilized explanted The body was inserted into the induction medium, and cultured in the dark at 33°C for 25 days to induce callus.

[0059] 2. Subgeneration of callus:

[0060] From the above-mentioned obtained calli, select vigorously growing pale yellow granular calli and transfer them to the subculture medium, place them under dark culture conditions at 33° C., and subculture for 12 days.

[0061] 3. Preculture of callus:

[0062] Vigorously growing pale yellow callus pieces with a particle diameter of 2-3 mm were inoculated on the pre-culture medium, and cultured in the dark at 33°C for 4 days.

[0063] 4. Agrobacterium-mediated transformation of embryogenic callus:

[0064] The strain used was Agrobacterium tumefaciens...

Embodiment 2

[0075] 1. Callus induction:

[0076] The young embryos of indica rice variety E09-2076 were used as explants, soaked in 75% alcohol by volume for 1 min, then soaked in 0.1% mercuric chloride by mass for 10 min, washed 4 times with sterile water, and finally the sterilized The explants were inserted into the induction medium and cultured in the dark at 33°C for 22 days to induce callus.

[0077] 2. Subgeneration of callus:

[0078] From the callus obtained above, the vigorously growing light yellow granular callus was selected and transferred to the subculture medium, placed in the dark culture condition of 33°C, and subcultured for 14 days.

[0079] 3. Preculture of callus:

[0080] Vigorously growing light yellow callus pieces with a particle diameter of 2-3 mm were inoculated on the pre-culture medium, and cultured in the dark at 33° C. for 5 days.

[0081] 4. Agrobacterium-mediated transformation of embryogenic callus:

[0082] The strain used was Agrobacterium tume...

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Abstract

The invention discloses a method for high efficiency regeneration and genetic transformation of indica rice. The method comprises the following steps of (1) induction of calluses: an indica rice mature embryo or young embryo is used as an explant; (2) subculture multiplication of the calluses: light yellow granular calluses are selected and transferred to a subculture multiplication medium for cultivation; (3) preculture of the calluses; (4) agrobacterium mediated transformation of the embryonic calluses: the precultured calluses are transferred into an agrobacterium work bacterium liquid and immersed at a room temperature; (5) co-culture of the calluses and the agrobacterium; (6) screen of resistant calluses: the co-cultured calluses are screened to grow novel resistant calluses; (7) differentiation of the resistant calluses: the screened resistant calluses are inoculated in a differentiation medium; and (8) rootage of regeneration sprout: rooted transgenic plants are obtained. The method is simple and convenient in screening, has high efficiency, and improves the efficiency of genetic transformation of the indica rice. Three representative indica rice varieties are transferred; and the transformation rate of each indica rice variety is higher than 25%.

Description

technical field [0001] The invention belongs to the technical field of plant tissue culture and genetic engineering, and specifically relates to a method for high-efficiency regeneration and genetic transformation of indica rice. The invention is applicable to callus induction and regeneration of indica rice mature embryo or immature embryo, and transformation of exogenous gene mediated by agrobacterium. Background technique [0002] Rice is one of the most important food crops in the world, and indica rice is the main cultivation type in my country. The genetic transformation mediated by Agrobacterium has good stability, and the exogenous DNA fragments contained in the vector are relatively large, making it the best choice for rice transgenesis. In the method, the target gene fragment is inserted into the Agrobacterium plasmid, and then the callus is co-cultured with the Agrobacterium, so that the target gene is transferred and integrated into the rice genome by the vector...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H4/00A01H5/00
Inventor 向发云李三和游艾青顾玉成韩光明陈志军周雷刘凯杨国才
Owner 湖北省农业科学院
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