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Method for setting early-maturing rice gene transformation system on grassland

A bluegrass, gene transformation technology is applied in the field of establishing a bluegrass gene transformation system, which can solve the problems of high gene silencing, high cost, poor transformation frequency and the like

Inactive Publication Date: 2004-01-28
北京春雨林业技术研究所 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is costly, the transformation frequency is not good, and the frequency of gene silencing in transgenic offspring is high

Method used

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  • Method for setting early-maturing rice gene transformation system on grassland
  • Method for setting early-maturing rice gene transformation system on grassland
  • Method for setting early-maturing rice gene transformation system on grassland

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Using Agrobacterium transfection method to transform SacB gene into bluegrass to obtain drought-resistant bluegrass

[0058] 1. Construction of target gene and vector: pKP plasmid containing SacB gene (see Patent Application No. 98101336.8) was donated by Professor Zhang Yinmei. The pKP plasmid contains the rice CAMV35S promoter, the vacuolar localization leader sequence CPY gene, the kanamycin resistance gene and the SacB gene from Bacillus subtilis.

[0059] 2. Transformation of bluegrass: The bluegrass seeds are wrapped in gauze, soaked in 70% ethanol solution for 30 seconds, taken out and placed in 0.1% mercuric chloride solution for sterilization for 5-15 minutes, and then shaken and rinsed with sterile water 3 -6 times. The sterilized seeds were inserted into the callus induction medium, and cultured in the dark, and white or pale yellow callus swelled from the seed roots in about 20 days (see figure 1 ). Subculture with callus induction medium every ...

Embodiment 2

[0068] Example 2 Transformation of callus by gene gun bombardment

[0069] 1. DNA-wrapped microbombs and gene gun bombardment

[0070] (1) The plasmid pKP with SacB was extracted from the host E. coli DH5α, and standard procedures and methods were used for extraction and purification (see "Molecular Cloning" Second Edition). The plasmid quality is between OD2601.7-1.8. The plasmid DNA concentration was diluted to 1 μg / μl and stored at -20°C for future use.

[0071] (2) 60mg of tungsten powder is placed in a centrifuge tube, and anhydrous ethanol is added, and it is sonicated with an ultrasonic pulverizer until it feels hot to the touch.

[0072] (3) Centrifuge and discard the supernatant. Add 1 ml of absolute ethanol and vortex for 3-5 minutes. Let stand for 1 minute. Discard the supernatant by centrifugation.

[0073] (4) 1 ml of sterile distilled water was added, vortexed, centrifuged for precipitation, and the supernatant was discarded (repeated 3 times).

[0074] (5...

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Abstract

The invention refers to a method to set up an grassland annual bluegrass gene-transforming system, including sterilizing and sprouting, embryonic callus inducing sub-differentiation differential culture and small-seedling rooting, and test-tube seedling transplanting, and so on; the use of the transforming system to set up transgenic drought-resistant glassland annual bluegrass to obtain the grassland annual bluegrass.

Description

Field of Invention [0001] The present invention relates to the field of agricultural biotechnology or the field of plant genetic engineering, more specifically, the present invention relates to a method for establishing a gene transformation system of Bluegrass Steps such as rooting of seedlings and transplanting of test-tube seedlings; application of the transformation system in the establishment of transgenic drought-resistant bluegrass and the transgenic drought-resistant bluegrass obtained by applying the transformation system. Background of the Invention [0002] Poa pratensis L. is a perennial turfgrass of the Poaceae family and is an important economic crop. According to statistics, my country's urban population has an average lawn area of ​​less than 10 square meters, while the United States has 200 million hectares of lawn in the 1990s, including more than 50 million lawns, 15,000 golf courses, and 6,442 lawn parks. With a lawn area of ​​800 square meters, the lawn ...

Claims

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Application Information

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IPC IPC(8): A01H4/00
Inventor 胡赞民陈宇红孙明杰盛立朱诗忠张红梅章银梅陈正华
Owner 北京春雨林业技术研究所
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