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A method for Agrobacterium infection during callus transformation of Taxus chinensis

A technology of callus transformation and yew, applied in the field of bioengineering, can solve the problems of low transformation efficiency and survival rate, and achieve the effects of avoiding poisoning, high transgenic efficiency, and wide application value

Inactive Publication Date: 2017-11-10
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the efficiency of transformation and the survival rate of callus are not high when using the conventional Agrobacterium infection method.

Method used

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  • A method for Agrobacterium infection during callus transformation of Taxus chinensis

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Induction and Proliferation of Embryogenic Callus of Taxus chinensis

[0023] 1. Propagation of yew aseptic seedlings

[0024] 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.

[0025] 2. Induction of Embryogenic Callus of Taxus chinensis

[0026]The hypocotyls were inoculated o...

Embodiment 2

[0028] Obtaining Plant Expression Vector pCAMBIA1304 Containing GUS Gene in Agrobacterium tumefaciens

[0029] 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 bacterial solution on ice for 30 minutes, take the bacteria into a 1.5 ml Eppendorf tube, centrifuge at 5000 rpm at 4°C for 5 minutes, 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 ...

Embodiment 3

[0031] 1. Agrobacterium tumefaciens-mediated transformation

[0032] 1.1. Cultivation of Agrobacterium tumefaciens

[0033] 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, and activate it on a shaker (180rpm) at 28°C for 2 hours before transforming Taxus Embryogenic callus.

[0034] 1.2. Co-culture of Agrobacterium tumefaciens and explants

[0035] Infiltrate the sterile blade with the Agrobacterium tumefaciens bacteria solution, cut the embryogenic callus block of about 1 cubic centim...

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Abstract

The invention belongs to the technical field of bioengineering, in particular to a method for Agrobacterium infection in the process of yew callus transformation. In the present invention, the yew hypocotyl is used as an explant to induce callus, and the embryogenic callus is obtained through culture; mediated by Agrobacterium tumefaciens, the yew callus is cut with a blade infiltrated by Agrobacterium, and the GUS The pCAMBIA1304 plasmid of the gene was introduced into the callus of Taxus chinensis, the integration of exogenous hygromycin phosphotransferase gene (HPT) was detected by PCR, the expression of GUS gene in the tissue was detected by histochemical method, and the transgenic callus of Taxus chinensis was obtained ; Among them, the contact of the knife surface forms a certain bacterial liquid gradient on the cut surface of the callus, thereby covering the optimal bacterial liquid concentration required for transformation, ensuring that the cells are successfully transformed, improving the success rate of Agrobacterium transformation, and providing yew. It lays the foundation for efficient genetic transformation engineering of injured tissue.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for infecting yew callus with Agrobacterium. 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 Northeas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84C12N5/10
Inventor 皮妍蒋科技赵东利廖志华孙小芬唐克轩
Owner FUDAN UNIV
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