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

Genetic transformation method for acquiring Taxus chinensis transgenic callus

A technique for callus and embryogenic callus, applied in the field of obtaining yew transgenic callus, can solve the problems of unsuccessful reports and undiscovered yew transgenic callus, etc.

Inactive Publication Date: 2012-09-26
FUDAN UNIV
View PDF0 Cites 8 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 aseptic seedlings of Taxus chinensis

[0022] Peel off the skin of the yew seeds and sterilize the intact endosperm. The procedure is as follows: 75% ethanol sterilization for 2 min, 0.1% mercury liter sterilization for 10 min, rinse with sterile water 3 times, then strip the seed embryos and inoculate them in B5 culture Base+6-BA1.0 mg / l+2,4-D 0.1 mg / l+hydrolyzed casein 1.0 g / l+active charcoal 1 g / l+sucrose 20 g / l+plant gel 2.6 g / l in a medium ( Among them, the number after the component indicates the added amount (g) of the component in the unit volume (l) of the B5 medium. First, let the seeds germinate in a dark environment, then gradually increase the light, and use weaker slow light to cultivate , The sterile seedlings of Taxus chinensis can be obtained. After the seedlings grow to 2-3 cm, the hypocotyls are cut and used for callus induction.

[0023] 2. Induction of Embryo...

Embodiment 2

[0026] Obtaining of the plant expression vector pCAMBIA1304 Agrobacterium tumefaciens engineering strain containing GUS gene

[0027] The plant expression vector pCAMBIA1304 was transferred to Agrobacterium tumefaciens (such as EHA105, which is a publicly available biological material on the market, which can be purchased from CAMBIA, Australia, with the strain number Gambar 1), and the PCR verification was performed. Specifically, first prepare competent Agrobacterium tumefaciens (such as EHA105): cultivate Agrobacterium tumefaciens to O.D. 600 =0.5, after ice bathing the bacteria for 30 min, take the bacteria in a 1.5 ml Eppendorf tube, centrifuge at 5000 rpm at 4℃ for 5 min, and remove the supernatant; use 0.1M CaCl sterilized by suction filtration 2 (Pre-cooling) 400 μl of suspended bacteria (slightly beaten with a pipette), place on ice for 30 min, centrifuge at 5000 rpm for 5 min, remove the supernatant, and use 100 μl of pre-cooled 0.1M CaCl 2 After suspending the cells, sto...

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 on a culture medium (its composition is: YEB liquid medium + streptomycin (Str) 25 μg / ml + kanamycin (Kan) 100 μg / ml+rifampicin (Rif) 40 μg / ml), shake at 180 rpm on a shaker at 28°C overnight, the next day when the bacterial concentration reaches OD 600 =0.5, centrifuge at 5000 rpm for 10 minutes, discard the upper culture medium, resuspend the Agrobacterium that settles on the bottom, add 100 μM acetosyringone, activate it on a shaker (180 rpm) at 28°C for 2 hours and then use it to transform red beans Embryonic callus of fir.

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

[0033] Infect the embryogenic callus mass of Taxus chinensis with the Agrobacterium tumefaciens strain, and place the callus with a small amount of Agrobac...

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 specifically relates to a method for obtaining yew transgenic callus. Background technique [0002] Yew ( Taxus sp.), also known as yew, is Taxaceae (Taxaceae) Taxus ( Taxus ) Of 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 T.cuspidata, T.yuennanensis, T.chinensis, T.chinensis var.mairei, Tibet yew (T.wallichiana) (Chinese Academy of Sciences Chinese Flora Editorial Committee, 1978) is mainly distributed in various provinces in Northeast, Northwest, North, Southwest, Central South and South China. Later, T.x media was introduced, which is a hybrid of Taxus cuspidata and T.baccata. Its female parent is Taxus cuspidata and its male parent is Taxus cuspidata. [0003] Taxol (taxol) is a natural active ingredient extracted from the bark of Taxus chine...

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): C12N15/84C12N5/10
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