Method for gene gun-mediated genetic transformation of moso bamboos

A technology of genetic transformation and gene gun method, applied in the field of moso bamboo mediated genetic transformation by gene gun method, can solve the problems of difficult Agrobacterium transformation, poor repeatability, limited scope of application, etc., and achieve shortened cycle, high controllability, The effect of increasing the probability

Inactive Publication Date: 2017-07-21
SOUTHWEAT UNIV OF SCI & TECH
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Problems solved by technology

The genotypes of Phyllostachys pubescens seeds vary greatly, and the callus induction rate and quality of callus of different genotypes of Phyllostachys pubescens are also quite different, and the repeatability is not good. At present, there are only a small number of reports about the induction and regeneration of Phyllostachys pubescens callus; and Agrobacterium bacteriostatic agents such as carbenicillin and cephalosporins are commonly used in the process of screening and cultivating, which can cause great damage to the callus of Phyllostachys pubescens, and no successful genetic transformation of Phyllostachys pubescens has been reported so far.
[0005] Th

Method used

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  • Method for gene gun-mediated genetic transformation of moso bamboos
  • Method for gene gun-mediated genetic transformation of moso bamboos
  • Method for gene gun-mediated genetic transformation of moso bamboos

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Embodiment 1

[0068] Embodiment 1, a kind of method of biolistic-mediated genetic transformation Moso bamboo

[0069] 1. Preparation of experimental materials

[0070] 1. Preparation of pCAMBIA1303-N vector plasmid

[0071] The plant binary vector pCAMBIA1303-N (Beijing Dingguo Changsheng Biotechnology Co., Ltd., MCV035-N) was designated as the target vector, which carried the GUS reporter gene, kanamycin and hygromycin resistance genes.

[0072] The target vector was transformed into E. coli DH5a by heat shock method. Pick a single colony of Escherichia coli and culture overnight in LB medium containing 100mg / L kanamycin at 37°C and 150rpm. After verification by bacterial liquid PCR, use the plasmid mini-extraction kit to extract the plasmid containing the target vector, concentrate or dilute to the required concentration, and set aside.

[0073] 2. Disinfection of Phyllostachys pubescens seeds

[0074] Soak the bamboo seeds in water at 25°C ( Figure 4 A) 24h, then soak 30S with 70% ...

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Abstract

The invention discloses a method for gene gun-mediated genetic transformation of moso bamboos. The method includes the steps: moso bamboo mature embryo acquisition and pre-culture; extraction of plasmid containing target genes; gene gun parameter setting; resistance screening; transformed plant identification; positive plant culture; transplantation. The method further includes a formula of a special culture medium in processes of transformation and induction. The method solves the problem of difficulty in genetic transformation of the moso bamboos, the period of acquiring transgenic bamboos can be effectively shortened (resistant seedlings are acquired after screening is performed for one month, and positive plants can be acquired for two to four months), and the probability of acquiring the transgenic bamboos is increased. The method is good in repeatability, plays an important role in industrial bamboo genetic improvement, bamboo function genomics research and the like, and has a wide application prospect.

Description

technical field [0001] The invention relates to a method for genetically transforming moso bamboo by means of a gene gun method, and belongs to the technical field of plant transgenics. Background technique [0002] Bamboo is a perennial evergreen plant in the subfamily Bambusoideae of Gramimineae. There are more than 70 genera and 1,250 species of bamboo resources in the world. There are 39 genera and more than 500 species of bamboo resources in my country. One of the forest resources, but also an important renewable resource. There are two main methods for improving the quality of bamboo fiber: one is the conventional breeding method, but due to the uncertainty of bamboo flowering, and the characteristics of death after flowering, there are huge restrictions; the other is through genetic engineering at the molecular level. Improve it. However, so far, the superiority of bamboo fiber has been verified mainly through some physical and chemical analysis, but no in-depth res...

Claims

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Application Information

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IPC IPC(8): C12N15/89
CPCC12N15/895
Inventor 胡尚连黄艳廖倩姜勇曹颖徐刚卢学琴
Owner SOUTHWEAT UNIV OF SCI & TECH
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