Chinese yew cell culture and method for large-scale subculture of same

A yew cell and subculture technology, applied in the field of yew fines, can solve the problems of failing to find and screen high-yield cell lines, reducing the content of active ingredients, restricting application, etc., and achieving easy control of product quality, reduced probability of contamination, and equipment. simple effect

Inactive Publication Date: 2010-12-08
DALIAN PRACTICAL BIOTECH +2
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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 amp...

Method used

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Examples

Experimental program
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Embodiment 1

[0023] Example 1 Obtaining of Taxus chinensis high-yield and high-quality cell lines

[0024] Take the seeds of natural yew, soak in 75% alcohol solution for 30 seconds, soak in 0.1% mercuric solution for 10 minutes and sterilize with vibration, wash with sterile water 4-5 times; use gibberellin 1.0mg / L- Soak in 0.2mg / L solution for 24 hours; after soaking for 24 hours, soak the seeds again in 0.1% mercuric chloride solution for 10 minutes, wash with sterile water for 4-5 times; peel off the seeds under aseptic conditions Shell, tear off the inner membrane of the seed, inoculate the peeled seed on the prepared plant induction B5 medium (gibberellin 1.0mg / L-0.2mg / L), and sow 4 seeds in every 40 milliliters of medium; Cultivate in the dark for 4-6 days at ℃, and grow into yew plants after the seeds germinate.

[0025] Take aseptic seedling roots, stems, leaves, cotyledons, and embryos as explants, and under aseptic conditions, use sterile surgical scissors and sterile tweezers ...

Embodiment 2

[0027] Embodiment 2 production method

[0028] 1 Production of yew callus

[0029] 1.1 Selection

[0030] Select the yew callus that has been cultured for 25-35 days, grows well, is white or yellow-white, has obvious structure, and has no browning characteristics as seeds.

[0031] 1.2 Medium

[0032] B5 medium (B5 basic medium+2, 4-D 0.5mg / L-2.0mg / L+6-BA 0.1mg / L-1.0mg / L) was inoculated.

[0033] 1.3 transfer

[0034] Under sterile conditions, pick the yew callus with white or yellow-white color, obvious structure, and no browning characteristics and divide it into small pieces of tissue with tweezers, and transfer the small pieces of tissue to B5 medium (B5 basic medium + 2. In 4-D 0.5mg / L-2.0mg / L+6-BA 0.1mg / L-1.0mg / L), the inoculation block size is 6-10mm.

[0035] 1.5 Taxus callus culture

[0036] Seed culture conditions:

[0037] Culture temperature: 22-27°C, dark culture; culture period: 30-40 days.

[0038] Harvesting Culture Conditions:

[0039] Cultivation tem...

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Abstract

The invention provides a technology for generating paclitaxel from Chinese yew cells. In the technology, Chinese yew callus cells are produced in large-scale in a mode of combing light culture and dark culture. The invention is characterized in that callus induction is carried out on Chinese yew on a B5 improved culture medium to induce the Chinese yew cells which do not have brown stain and can be continuously subcultured; the light culture and the dark culture are combined to be used for culturing the Chinese yew cells to obtain high yield cells with paclitaxel the content of which is above 0.12 %; and the Chinese yew cells are subjected to subculturing multiplication so that a great amount of Chinese yew cells are produced. The cell culture can be directly used for extracting the paclitaxel, thereby overcoming the defects of deficient resources, low content, damage to ecological environment and the like of the process for producing the paclitaxel from plant bark. Compared with the existing technology for producing the paclitaxel through the cell culture, the technology of the invention has the characteristics that the content of the paclitaxel can be stably improved without degradation and the operation is simple and convenient.

Description

technical field [0001] The present invention relates to the cell culture of medicinal plant and its culture method, especially relates to a kind of yew cell culture [0002] The invention relates to a cell culture and a method for cultivating it, belonging to the field of plant biotechnology engineering. Background technique [0003] 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. [0004] At present, paclitaxel is mainly obtained from bark, and the sour...

Claims

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Application Information

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IPC IPC(8): A01H4/00
Inventor 刘汉石
Owner DALIAN PRACTICAL BIOTECH
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