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Taxus chinensis cell tissue culture

A yew cell and tissue culture technology, which is applied in the field of yew cell tissue culture culture, can solve the problems of reduced active ingredient content, limited application, browning, etc., to promote rapid growth, increase effective content, and short growth time Effect

Active Publication Date: 2018-11-20
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.
However, the following deficiencies still exist: (1) the paclitaxel content of the cells is low; (2) the cells have poor adaptability to the external environment; (3) the cells have poor genetic stability, and signs of degeneration and browning occur after the number of passages increases

Method used

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  • Taxus chinensis cell tissue culture

Examples

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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 4-5 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 and sterilize the seeds in 1% sodium hypochlorite solution for 10 minutes and wash them with sterile water 4-5 times; Peel off the seed shell and the inner film of the seed under aseptic conditions, and inoculate the peeled seed on the prepared induction medium (the 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 heigh...

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 culture method for a taxus chinensis cell tissue culture. The method specifically comprises the following steps: performing sterilization, cleaning, soaking, and seed coats removing on taxus chinensis seeds, inoculating the seeds on an induction medium, performing dark culture, and culturing the seeds into taxus chinensis plants after the seeds are germinated; selecting taxus chinensis plants with well growth as explants; inoculating the explants on the culture medium, and performing the dark culture; transferring the explants to culture rooms at 25 DEG C and 28 DEG Cfor culturing; selecting callus with strong growth, no browning, yellow-white characteristics, and inoculating the callus on a subculture medium for performing periodic subculture; and then, selecting the seeds, inoculating the seeds into the subculture medium, performing light culture, collecting after the seeds are cultured for 30 to 35 days, and obtaining the taxus chinensis cell tissue culture. A paclitaxel content of the taxus chinensis cell tissue culture can reach 1.5%-1.7%; and during a passage process, taxus chinensis cell tissue does not brown, the content of paclitaxel does not reduce, and the taxus chinensis cell tissue culture can still stably grow when conditions such as temperature and illumination are changed.

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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IPC IPC(8): C12N5/04
CPCC12N5/0025C12N5/04
Inventor 刘汉石陈梓
Owner DALIAN PRACTICAL BIOTECH
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