Method for cultivating catharanthus roseus transgenic plants induced by agrobacterium tumefacien

A technology of Agrobacterium tumefaciens and periwinkle, applied in the field of cultivating transgenic plants of periwinkle mediated by Agrobacterium tumefaciens, can solve the problems of limited application value, unstable metabolites and genetics of cell culture, and reduced TIAs content, etc.

Active Publication Date: 2012-10-17
成都上交致远生物科技有限公司
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Problems solved by technology

In 2004, Choi PS et al reported the genetic transformation method of Vinca mediated by Agrobacterium rhizogenes, but since the hairy root of Vinca does not contain vinblastine and vincristine, its application value is limited
The genetic transformation of periwinkle mediated by Agrobacterium tumefaciens has only been reported on the transformation of periwinkle suspension cells. The content of TIAs in the

Method used

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  • Method for cultivating catharanthus roseus transgenic plants induced by agrobacterium tumefacien
  • Method for cultivating catharanthus roseus transgenic plants induced by agrobacterium tumefacien
  • Method for cultivating catharanthus roseus transgenic plants induced by agrobacterium tumefacien

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Experimental program
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Embodiment

[0030] (1) Cultivation of periwinkle aseptic seedlings

[0031] The periwinkle seeds used in the present invention are purchased from PanAmerican Seed Company, Pacifica Cherry Red (PCR). The periwinkle seeds were sterilized with 75% alcohol for 1 min, 20% NaClO for 5 min, rinsed with sterile water for 3 times, blotted the excess water with filter paper, then inoculated on MS medium, cultured for 3-4 days, and cut to obtain 5 mm long Bacterin hypocotyls.

[0032] Culture room conditions: temperature (25±2)°C, light time 16h / d, light intensity 2,000lx.

[0033] (2) Preparation of Agrobacterium tumefaciens bacterial liquid

[0034] Plant expression vector pCAMBIA2300-GUS (such as figure 1) as the test material, which is preserved by the Plant Biotechnology Research Center of Shanghai Jiaotong University. The expression vector can be obtained through publicly available commercial channels, for example, it can be purchased from CAMBIA Company in Australia, and the vector number...

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Abstract

A method for cultivating catharanthus roseus transgenic plants induced by agrobacterium tumefacien comprises the steps as follows: taking aseptic seedling of the catharanthus roseus, and obtaining hypocotyl of the aseptic seedling; preparing bacteria solution of the agrobacterium tumefacien and utilizing the agrobacterium tumefacien for inducing transformation of the hypocotyl of catharanthus roseus; taking and placing the hypocotyl on a callus induction culture medium for culture to obtain a callus; inoculating the callus onto a differential culture medium for culture to obtain a green callus; inoculating the green callus onto an indefinite bud induction culture medium for culture to obtain an indefinite bud; and taking and placing the indefinite bud into a radication culture medium for culture to obtain the catharanthus roseus transgenic plants. In the method, a stable genetic transformation system of the catharanthus roseus induced by the agrobacterium tumefacien is established, therefore the method lays a foundation for further study on metabolic pathway control net and for the improvement of TIAs content, and can be further applied to gene engineering seed breeding for producing TIAs metabolite.

Description

technical field [0001] The invention relates to a recipe for cultivating periwinkle transgenic plants mediated by Agrobacterium tumefaciens, belonging to the field of biotechnology. Background technique [0002] Vinca (Catharanthus roseus) is a plant of the genus Vinca in the family Apocynaceae, containing Vinblastine, Vincristine, Vindoline, Ajmalicine, and Serpentine (Serpentine) and more than 100 kinds of terpenoid indole alkaloids (Terpenoid indole alkaloids, TIAs). Most of these alkaloids have pharmacological activity, especially vinblastine and vincristine are currently the most widely used natural anti-tumor drugs. However, under normal circumstances, the content of diterpene indole alkaloids (vinblastine and vincristine) in plants is extremely small, and hairy roots and cell culture can only produce monoterpene indole alkaloids. At present, both at home and abroad, most of them extract Vindolin and Vinblastine, which are relatively abundant in Vinca, and then semi-...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84
Inventor 唐克轩王荃潘琪芳袁芳王国丰刑世海赵静雅
Owner 成都上交致远生物科技有限公司
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