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A kind of yew cell tissue culture

A Taxus chinensis cell and tissue culture technology, which is applied in the field of taxus chinensis cell tissue culture culture, can solve the problems such as failure to find high-yield cell lines for screening, poor cell adaptability, and reduced content of effective components, and is easy to mass-scale production. , to promote rapid growth, improve the effect of effective content

Active Publication Date: 2022-05-17
DALIAN PRACTICAL BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the research reports on the tissue culture of Taxus chinensis, there are many problems that greatly limit its application in actual production. The existing problems include: instability during the amplification process, and the loss of active ingredients when the scale is enlarged and cultivated to a certain scale. The content will be reduced; there is no effective means of screening high-producing cell lines, etc.
But there are still the following deficiencies: (1) low paclitaxel content in cells; (2) poor adaptability of cells to the external environment; (3) poor genetic stability of cells, degeneration, browning and other signs occur after the number of passages increases

Method used

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  • A kind of yew cell tissue culture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The culture method of yew cell tissue of the present invention comprises the following steps:

[0029] 1. Take the seeds of natural yew, soak them in 75% alcohol solution for 30 seconds, soak them in a sodium hypochlorite solution with a mass concentration of 1% and sterilize them by shaking for 10 minutes, wash them with sterile water for 4 to 5 times; Soak in an aqueous solution with a concentration of 0.2mg / L-1.0mg / L for 24 hours; after soaking for 24 hours, soak the seeds in 1% sodium hypochlorite solution for 10 minutes and wash them with sterile water for 4-5 times; Peel off the seed shell and inner film of the seed under aseptic conditions, and inoculate the peeled seed on the prepared induction medium (medium containing salicylic acid concentration of 0.2mg / L-1.0mg / L), every Sow 4 seeds in 40 ml medium; culture at 25°C in the dark for 4-6 days, and after the seeds germinate, cultivate them into yew plants, and continue to cultivate until the height of the yew pl...

Embodiment 2

[0038] The seeds obtained in Step 3 of Example 1 were continued to be subcultured. According to the culture conditions, the culture temperature was 25° C., and the culture period was 35 days; then the callus with vigorous growth, no browning, and yellow-white characteristics was selected and inoculated into The subculture medium is periodically subcultured into one generation. After 60 generations of culture, the callus does not degenerate or brown, and then continue to light culture: the light condition is 4 hours / day, white light, and the temperature is 25°C; After 30 days of cultivation, they were collected and dried in vacuum at low temperature.

[0039] It is determined that the yield is not reduced, and the content of paclitaxel in the dry product culture can reach 15‰ of the dry weight, indicating that the yew callus prepared by the present invention has strong cytogenetic stability and strong adaptability to the external environment.

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Abstract

The invention discloses a method for cultivating yew cell tissue culture. Specifically, yew seeds are sterilized, cleaned, soaked, and seed-coated, inoculated on an induction medium, and cultured in the dark. After the seeds germinate , cultivated into yew plants; select well-grown yew plants as explants; inoculate the explants on the medium, and cultivate them in the dark; then transfer them to a culture room with a temperature of 25°C and 28°C for cultivation; select vigorous growth , no browning, and yellow-white callus are inoculated into the subculture medium for periodic subculture; after that, select seeds, inoculate the seeds into the subculture medium, light culture, and collect after 30 to 35 days of culture , Taxus cell tissue culture. The paclitaxel content of the yew cell tissue culture provided by the present invention can reach 15‰-17‰, and the yew cell tissue does not brown during the passaging process, and the content of paclitaxel produced does not decrease, and the temperature and light conditions can still be changed. Steady growth.

Description

technical field [0001] The invention belongs to the technical field of plant biotechnology engineering, and in particular relates to a method for culturing yew cell tissue culture. Background technique [0002] Taxus is a class of trees and shrubs belonging to the family Taxaceae (Taxaceae) of the genus Taxus. There are 11 species in the world, distributed from the subarctic regions of Eurasia and North America to the subtropical and even tropical regions of Central America and Southeast Asia. . Paclitaxel is a diterpene alkaloid, which has been clinically proven to be effective in the treatment of ovarian cancer, lung cancer, colon cancer, metastatic breast cancer, melanoma, leukemia and other cancers, making paclitaxel one of the most popular anticancer drugs currently one. [0003] At present, paclitaxel is mainly obtained from bark, and the source is extremely limited, which not only destroys the resources and ecological balance of yew, but also cannot meet the growing...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/04
CPCC12N5/0025C12N5/04
Inventor 刘汉石陈梓
Owner DALIAN PRACTICAL BIOTECH
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