Tissue culture system based on chloroplast transformation in rape cotyledons and method for obtaining transformed plant

A technology of chloroplast and rapeseed, which is applied in the field of obtaining rapeseed chloroplast transgenic plants by the gene gun transformation method, which can solve the problems of limited explant types, short regeneration time, and low transformation frequency

Inactive Publication Date: 2011-05-11
HUAZHONG AGRI UNIV
View PDF0 Cites 5 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the explants used by Hou Bingkai are the cotyledon petioles of rapeseed that have germinated for 4-5 days. Although they have the characteristics of high regeneration rate and short regeneration time, rapid entry into the differentiation state is unfavorable for the homogenization of subsequent transformed plants (Hou BK, Zhou YH, Wan LH, Zhang ZL, Shen GF, Chen ZH, Hu ZM (2003) Chloroplast transformation in oilseed rape. Transge Res 12: 111-114)
At the same time, in the transformation of rapeseed chloroplasts, the current outstanding problems are that the transformation is limited by the type of explants, and the transformation frequency is not high.

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Tissue culture system based on chloroplast transformation in rape cotyledons and method for obtaining transformed plant
  • Tissue culture system based on chloroplast transformation in rape cotyledons and method for obtaining transformed plant
  • Tissue culture system based on chloroplast transformation in rape cotyledons and method for obtaining transformed plant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Embodiment 1: The cultivation method of the chloroplast transgenic plant with rape cotyledon as explant

[0067] 1. Construction of transformation vector

[0068] (1) Cloning of homologous recombination fragment trnI / trnA

[0069] Referring to the Arabidopsis thaliana chloroplast genome sequence (sequence accession number: NC000932) published by Genebank, two pairs of specific primers were designed, and the primer pairs were used to amplify rapeseed chloroplast genes trnI and trnA as homologous recombination fragments.

[0070] The numbering and DNA sequence of the primer pair are as follows:

[0071] F-trnI: 5'-ATAGAGCTCTGGAACCCTGAACAGACTG-3'; (SEQ ID NO: 3 in the sequence listing)

[0072] R-trnI: 5'-ATAGCGGCCGCATTTGAACCAGAGACCTC-3'. (Sequence Listing SEQ ID NO: 4)

[0073] F-trnA: 5'-ATAGTCGACGGGGATATAGCTCAGTTGG-3'; (SEQ ID NO: 5 in the sequence listing)

[0074] R-trnA: 5'-TAAGGGCCCCGGTACTACTTCGCTATCG-3'. (Sequence Listing SEQ ID NO: 6)

[0075] The reaction ...

Embodiment 2

[0136] Embodiment 2: comparative experiment embodiment

[0137] 1) Differences in callus induced by different callus induction media on cotyledons of Brassica napus

[0138] In this embodiment, the applicant designed 6 culture media for inducing callus (as described later), using three rapeseed varieties Huayouza No. 6, Huayouza No. 7, and Huayouza No. 9 (the All three varieties are selected and bred by Huazhong Agricultural University, and the cotyledons of rapeseed new varieties widely used in China) are used as explants. Callus induction medium for callus.

[0139] The formula and numbering of 6 kinds of mediums designed by the present invention are as follows:

[0140] B5D-1: B5 basic medium + NAA 5mg / l + 6-BA 5mg / l;

[0141] B5D-2: B5 basic medium + 0.4% MES;

[0142] B5D-3: B5 basic medium + 2,4-D 0.2mg / l;

[0143] B5D-4: B5 minimal medium + 2,4-D 0.4mg / l;

[0144] B5D-5: B5 basic medium+2,4-D 0.6mg / l+KT0.2mg / l;

[0145] B5D-6: B5 basic medium+2,4-D 0.8mg / l+KT0.3m...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention belongs to the technical field of plant transgenosis, and particularly relates to a Brassica napus transgenic method. A chloroplast transformation tissue culture system suitable to take the rape cotyledons as an explant is constructed, and a chloroplast transformation transgenic plant is obtained through a gene gun transformation method. The invention also discloses a method for constructing a rape chloroplast specificity expression vector, transforming the rape chloroplast and obtaining the transgenic plant by taking rape chloroplast fragments as homologous recombination fragments. The invention provides a novel pathway for transgenosis of Brassica napus.

Description

technical field [0001] The invention belongs to the field of plant transgenic technology. It specifically relates to a method for establishing a chloroplast transformation tissue culture system in which rape cotyledons are explants, and obtaining rape chloroplast transgenic plants through a gene gun transformation method. The invention also relates to a method for using rapeseed chloroplast fragments as homologous recombination fragments to construct rapeseed chloroplast-specific expression vectors, transforming rapeseed chloroplasts and obtaining transgenic plants. Background technique [0002] Chloroplast genetic engineering has many unique advantages, including high-efficiency expression of genes, no need for modification of prokaryotic genes, site-specific integration can be achieved, foreign genes will not spread with pollen, and so on. Moreover, chloroplast genetic engineering also overcomes the phenomena of gene inactivation, gene silencing and position effect that a...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00
Inventor 廖玉才李和平程琳傅庭栋瞿波黄涛涂金星
Owner HUAZHONG AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap