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Genetic transformation method of creeping type ground-cover chrysanthemum

A technology of genetic transformation and groundcover chrysanthemum, applied in biochemical equipment and methods, horticultural methods, botanical equipment and methods, etc., can solve the problems of low transformation frequency, difficult transformation of transformed cells into seedlings, strong genotype dependence, etc. Achieve high transformant regeneration rate, inhibit the growth of Agrobacterium, and improve the regeneration rate

Inactive Publication Date: 2009-06-10
NANJING AGRICULTURAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to solve the problems of strong genotype dependence, low transformation frequency, difficult regeneration of explants after transformation, poor repeatability, and difficult seedling formation of transformed cells in the transgene of chrysanthemum mediated by Agrobacterium tumefaciens. The genetic transformation conditions of creeping ground cover chrysanthemum have been systematically and deeply studied, and a set of genetic transformation system suitable for creeping ground cover chrysanthemum has been established, laying the foundation for the molecular breeding of creeping ground cover chrysanthemum

Method used

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  • Genetic transformation method of creeping type ground-cover chrysanthemum
  • Genetic transformation method of creeping type ground-cover chrysanthemum
  • Genetic transformation method of creeping type ground-cover chrysanthemum

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Embodiment Construction

[0052] 1) Obtaining sterile vaccines

[0053] At the end of autumn, take the vigorous underground shoots of the creeping ground cover chrysanthemum variety 'Yuhua Xun' (China Chrysanthemum Research Network, http: / / www.chrysanthemum.cn), wash with detergent for 15 minutes, rinse with running water for 2 hours, and absorb the water with filter paper , under aseptic conditions, soak in ethanol with a volume ratio of 70% for 30s, sterilize with mercuric chloride solution with a mass percentage of 0.1% for 6min, rinse with sterile water for 4 times, cut out 0.5cm stems with axillary buds, and inoculate them in MS+ cell division Prime 6-BA 1.0mg·L -1 + Auxin NAA 0.1mg·L -1 Induce the germination of axillary buds on the medium, cut off the adventitious buds when they grow to about 2cm, and transfer them to the rooting medium 1 / 2MS+auxin NAA 0.1mg·L -1 obtain sterile vaccines;

[0054] 2) Preparation of Agrobacterium suspension

[0055] Cloning of DREBa gene of chrysanthemum 'Zhon...

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Abstract

The invention relates to a method for genetic transformation of creeping ground-cover chrysanthemum, which belongs to the field of molecular breeding of chrysanthemum. RNA of a chrysanthemum variety zhongshanhongfeng is taken as a template to amplify to synthesize a DREBa gene, the gene is cloned to a T-vector, a target fragment of the DREBa gene obtained by double digestion is connected with pCAMBIA 1301, and then agrobacterium is transformed to obtain a bacterial liquid of the agrobacterium containing a recombinant plasmid pCAMBIA1301-DREBa; and a leaf disc of an aseptic seedling is infected by the bacterial liquid of the agrobacterium for 10min, and a rooted seedling with hygromycin resistance is obtained through the total culture for 3d, the debacteria culture for 3d and the screening culture. The PCR detection proves that a foreign gene has been transferred into DNA of a genome of the ground-cover chrysanthemum. The method aims at the creeping ground-cover chrysanthemum to establish a genetic transformation system of the creeping ground-cover chrysanthemum for the first time, provides a theoretical basis and an experimental evidence for molecular breeding work of the ground-cover chrysanthemum, and can effectively promote the progress of the molecular breeding of the ground-cover chrysanthemum.

Description

1. Technical field [0001] The invention relates to a genetic transformation method of creeping ground cover chrysanthemum, which is specially used for the molecular breeding of creeping ground cover chrysanthemum and belongs to the field of chrysanthemum molecular breeding. 2. Background technology [0002] Ground cover chrysanthemum is a kind of unique ground cover plant with comprehensive functions of greening, beautifying, coloring and aromatizing at the same time. department concerned. Therefore, screening and cultivating new varieties of creeping ground cover chrysanthemum with excellent characters has great practical value and economic significance. [0003] In recent years, with the development of plant genetic engineering, molecular breeding has overcome the limitations of conventional breeding, providing new means and ways for improving chrysanthemum varieties with new genetic resources. The Agrobacterium-mediated method has become the preferred method in the tran...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82A01H4/00A01H5/00
Inventor 陈发棣崔新利陈素梅房伟民缪恒彬姜贝贝管志勇刘兆磊滕年军
Owner NANJING AGRICULTURAL UNIVERSITY
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