Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Genetic transformation method for acquiring Taxus chinensis transgenic callus

A genetic transformation method and callus technology, applied in biochemical equipment and methods, genetic engineering, plant genetic improvement, etc., can solve the problems of undiscovered yew transgenic callus, no successful reports, etc.

Inactive Publication Date: 2014-02-19
FUDAN UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many laboratories at home and abroad are conducting research in this area, but there is no successful report
At present, there is no relevant report on obtaining the transgenic callus of Taxus chinensis

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Induction and Proliferation of Embryogenic Callus of Taxus chinensis

[0021] 1. Propagation of yew aseptic seedlings

[0022] Peel off the outer skin of the yew seeds, and disinfect the complete endosperm. The procedure is as follows: 75% ethanol for 2 minutes, 0.1% mercuric chloride for 10 minutes, and sterile water for 3 times. Base+6-BA1.0 mg / l+2,4-D 0.1 mg / l+hydrolyzed casein 1.0 g / l+activated carbon 1 g / l+sucrose 20 g / l+phytogel 2.6 g / l ( Among them, the number after the component indicates the addition amount (g) of the component in the unit volume (l) of the B5 medium). First, the seeds are germinated in a dark environment, and then the light is gradually increased, and a weaker slow light is used for cultivation. , the aseptic seedlings of Taxus chinensis can be obtained, and after the seedlings grow to 2-3 cm, the hypocotyls are cut for callus induction.

[0023] 2. Induction of Embryogenic Callus of Taxus chinensis

[0024] The hypocotyls were inoculated ...

Embodiment 2

[0026] Obtaining Plant Expression Vector pCAMBIA1304 Containing GUS Gene in Agrobacterium tumefaciens

[0027] The plant expression vector pCAMBIA1304 was transformed into Agrobacterium tumefaciens (such as EHA105, which is a publicly available biological material in the market and can be purchased from CAMBIA Company in Australia, the strain number is Gambar 1), and PCR verification was performed. Specifically, first prepare competent Agrobacterium tumefaciens (such as EHA105): cultivate Agrobacterium tumefaciens until the bacterial liquid O.D 600 =0.5, put the bacteria solution on ice for 30 min, take the bacteria into a 1.5 ml Eppendorf tube, centrifuge at 5000 rpm at 4°C for 5 min, remove the supernatant; use 0.1M CaCl sterilized by suction filtration 2 (Pre-cooling) 400 μl of suspended bacteria (mixed gently with a pipette gun), placed on ice for 30 minutes, centrifuged at 5000 rpm for 5 minutes, removed the supernatant, and washed with 100 μl of pre-cooled 0.1M CaCl 2Th...

Embodiment 3

[0029] 1. Agrobacterium tumefaciens-mediated transformation

[0030] 1.1. Cultivation of Agrobacterium tumefaciens

[0031] Pick a single colony of Agrobacterium tumefaciens engineering bacteria containing the plant expression vector in the culture medium (it consists of: YEB liquid medium+streptomycin (Str) 25 μg / ml+kanamycin (Kan) 100 μg / ml ml+rifampicin (Rif) 40 μg / ml), shake overnight at 180 rpm on a shaker at 28°C, and when the concentration of the bacterial solution reaches OD the next day 600 = 0.5, centrifuge at 5000 rpm for 10 minutes, pour off the upper culture medium, resuspend the Agrobacterium precipitated at the bottom, add 100 μM acetosyringone, activate it on a shaker at 28°C (180 rpm) for 2 hours, and then use it to transform red beans Embryonal callus.

[0032] 1.2. Co-culture of Agrobacterium tumefaciens and explants

[0033] Infect the embryogenic callus mass of Taxus chinensis with Agrobacterium tumefaciens bacteria solution, and put the callus with a ...

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 biology, and particularly relates to a genetic transformation method for acquiring Taxus chinensis transgenic callus, which comprises the following steps: carrying out induced culture on Taxus chinensis embryo callus, transferring efficient expression vector carrying GUS gene into root-cancer agrobacterium, genetically transforming the embryo callus with the agrobacterium, carrying out enrichment subculture on the transformation receptor material, screening the resistant embryo callus, carrying out enrichment subculture on the resistant embryo callus, carrying out PCR (polymerase chain reaction) detection, detecting the GUS gene, and the like. According to the genetic transformation method, the GUS gene is expressed in the Taxus chinensis embryo callus to successfully acquire the Taxus chinensis embryo callus. The Taxus chinensis transgenic callus acquired by the method provided by the invention can be widely used in Taxus chinensis gene engineering, cell engineering, metabolism engineering and establishment of a Taxus chinensis genetic transformation system.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for obtaining transgenic callus of yew. Background technique [0002] Yew ( Taxus sp.), also known as yew, belongs to the Taxaceae (Taxaceae) genus ( Taxus ) gymnosperms. There are 11 species of Taxus in the world, mainly distributed in the temperate to subtropical regions of the northern hemisphere. There are 4 species and 1 variety in my country, namely Northeast Taxus (T.cuspidata), Yunnan Taxus (T.yuennanensis), Chinese Taxus (T.chinensis), Southern Taxus (T.chinensis var.mairei), Tibetan yew (T.wallichiana) (Editorial Committee of Flora of China, Chinese Academy of Sciences, 1978) is mainly distributed in the provinces of Northeast, Northwest, North China, Southwest, Central South and South China. Later, the Mandia yew (T.x media) was introduced, which is a hybrid of Northeast yew and European yew (T.baccata). The female parent is Northeast yew and the ma...

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 Patents(China)
IPC IPC(8): C12N15/84C12N5/10
Inventor 孙小芬皮妍赵东利蒋科技唐克轩
Owner FUDAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

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

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com