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Agrobacterium tumefaciens-mediated genetic transformation method of zoysia japonica

A genetic transformation method, Agrobacterium-mediated technology, applied in biochemical equipment and methods, botanical equipment and methods, applications, etc., can solve the problems of difficult application of genetic transformation of gramineous plants, to avoid waste, save costs, The effect of reducing workload

Inactive Publication Date: 2012-11-14
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention overcomes the defect that the existing Agrobacterium-mediated technology is difficult to apply to the genetic transformation of grasses, and provides a method for the genetic transformation of Zoysia japonica mediated by Agrobacterium

Method used

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  • Agrobacterium tumefaciens-mediated genetic transformation method of zoysia japonica
  • Agrobacterium tumefaciens-mediated genetic transformation method of zoysia japonica
  • Agrobacterium tumefaciens-mediated genetic transformation method of zoysia japonica

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Experimental program
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Embodiment 1

[0026] Example 1 Agrobacterium-mediated screening of optimal conditions for Zoysia japonica genetic transformation system

[0027] 1 recipient plant material

[0028] Japanese Zoysia cultivar 'Zenith' mature seeds ('Zenith' cultivar seeds purchased from U.S. TMI company (TurfMerchants, Inc)) were soaked in sodium hypochlorite solution (purchased from Beijing Chemical Plant, >5% active chlorine, adding 1-2 drops In Tween 20), sterilize on a magnetic stirrer for 1 hour, rinse with sterile water 3-5 times, soak at 4°C for 3 days, then sterilize with sodium hypochlorite solution for 20 minutes, rinse with sterile water for 3 times, and use sterile filter paper Drain excess water, inoculate on callus induction medium, and induce callus at 28°C in the dark. The callus was subcultured once a month, and the white or yellow, granular callus was selected and transferred to the subculture medium for culture as the recipient material for gene transformation.

[0029] 2 strains and vecto...

Embodiment 2

[0072] Example 2 Agrobacterium-mediated transfer of RNA interference (RNAi) plant expression vector pc1301-ubi-ZjGA20-dsRNAi into Zoysia japonica to obtain transgenic plants

[0073] 1 recipient plant material

[0074] Mature seeds of Japanese Zoysia cultivar 'Zenith' were soaked in sodium hypochlorite solution (purchased from Beijing Chemical Plant, >5% active chlorine, added 1-2 drops of Tween 20), sterilized on a magnetic stirrer for 1 hour, and sterile Rinse with water 3-5 times, soak at 4°C for 3 days, sterilize the soaked seeds with sodium hypochlorite solution for 20 minutes, rinse with sterile water for 3 times, blot excess water with sterile filter paper, and inoculate on callus induction medium , callus was induced at 28°C in the dark. The callus was subcultured once a month, and the white or yellow, granular callus was selected and transferred to the subculture medium for culture as the recipient material for gene transformation.

[0075] 2 strains and vectors

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Abstract

The invention provides an agrobacterium tumefaciens-mediated genetic transformation method of zoysia japonica. The method comprises the steps of infecting calluses of the zoysia japonica with agrobacterium tumefaciens suspended in a AAM-AS culture medium; putting the infected calluses in a N6D2-AS culture medium and co-culturing for 2-3 days; washing off the agrobacterium tumefaciens; screening and culturing the calluses. Aiming at three important influence factors which influence conversion efficiency of the agrobacterium tumefaciens-mediated genetic transformation system of the zoysia japonica, the best bacterium concentration (OD600 value being 0.3-0.4), infecting time (1.5-2 h) and co-culturing time (2-3 days) are found out. Combining with an improvement of the culture medium, final conversion efficiency reaches more than 50%. Besides, by improving washing method of the agrobacterium tumefaciens, pollutions of the agrobacterium tumefaciens can be eliminated in the screening and culturing process. Compared with previous agrobacterium tumefaciens-mediated genetic transformation methods, the method greatly reduces work-load, saves cost and prevents waste of callus materials.

Description

technical field [0001] The field of genetic engineering of the present invention, in particular, relates to a method for genetic transformation of Zoysia japonica mediated by Agrobacterium. Background technique [0002] Zoysia Japonica Steud. is the only turfgrass species rich in resources in my country that does not need to be imported. It has many excellent characters such as strong adaptability, drought resistance, heat resistance, cold resistance, trampling resistance and barren resistance. Its disadvantages are mainly slow germination and slow emergence into lawn, which makes it easy to be invaded by weeds. Therefore, the general breeding goals are focused on prolonging the green period, improving its texture and cold resistance, accelerating seedling growth, germination rate, etc., but there are few studies in this area, and only some basic research has been done on breaking seed dormancy at home and abroad. Research. Due to the complexity of plant stress resistance ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84
Inventor 韩烈保代小梅孙鑫博王怡杰
Owner BEIJING FORESTRY UNIVERSITY
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