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Culture method of highly yielding vinblastine from catharanthus roseus cells

A culture method, the technology of periwinkle, which is applied in the field of cell culture, can solve the problems such as the low content of vinblastine and the inability of vinblastine to meet the market demand, and achieve the effects of increasing biosynthesis, fast cell growth, and shortening the induction period

Inactive Publication Date: 2019-01-04
覃家日
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the vinblastine content in the vinca plant is extremely small, so that the vinblastine extracted from the vinca plant is far from meeting the market demand

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] A method for culturing vinblastine cell high-yield vinblastine, comprising the following steps:

[0022] 1) Pick plump periwinkle seeds, soak them in weak acid electrolyzed water for 10 seconds, wash them with sterile deionized water, and use them on B5 base containing 10mg / L vitamin C, 7g / L agar and 0.1g / L oligosaccharin The sterile seedlings are obtained by culturing on the medium, the hypocotyls of the sterile seedlings are used as explants, and the embryonic axes of the sterile seedlings are inserted into the callus induction medium for induction culture. Induce callus, then transfer callus into subculture medium, subculture once every 25 days, and subculture 3 times to obtain stable periwinkle callus;

[0023] 2) choose the described periwinkle callus with vigorous growth, loose texture, stable and uniform state, inoculate in the liquid suspension medium, then add glass beads in the liquid suspension medium, and carry out high-speed vibration 8min in a shaking shak...

Embodiment 2

[0027] A method for culturing vinblastine cell high-yield vinblastine, comprising the following steps:

[0028] 1) Pick plump periwinkle seeds, soak them in weak acid electrolyzed water for 10 seconds, wash them with sterile deionized water, and use them on B5 base containing 10mg / L vitamin C, 7g / L agar and 0.1g / L oligosaccharin The sterile seedlings are obtained by culturing on the medium, the hypocotyls of the sterile seedlings are used as explants, and the embryonic axes of the sterile seedlings are inserted into the callus induction medium for induction culture. Induce callus, then transfer the callus into subculture medium, subculture once every 25 days, and subculture 3 times to obtain stable periwinkle callus; wherein, the callus induced The medium is B5 basal medium containing 1mg / L cytokinin, 00.5mg / L 6-benzylaminopurine, 30g / L sucrose, 3mg / L licorice flavonoids and 8g / L agar;

[0029] 2) choose the described periwinkle callus with vigorous growth, loose texture, sta...

Embodiment 3

[0033] A method for culturing vinblastine cell high-yield vinblastine, comprising the following steps:

[0034] 1) Pick plump periwinkle seeds, soak them in weak acid electrolyzed water for 10 seconds, wash them with sterile deionized water, and use them on B5 base containing 10mg / L vitamin C, 7g / L agar and 0.1g / L oligosaccharin The sterile seedlings are obtained by culturing on the medium, the hypocotyls of the sterile seedlings are used as explants, and the embryonic axes of the sterile seedlings are inserted into the callus induction medium for induction culture. Induce callus, then transfer the callus into subculture medium, subculture once every 25 days, and subculture 3 times to obtain stable periwinkle callus; wherein, the callus induced The medium is the B5 basal medium containing 1mg / L cytokinin, 00.5mg / L 6-benzylaminopurine, 30g / L sucrose, 3mg / L licorice flavonoids and 8g / L agar; The wound tissue induction medium was added with phenylalanine, and the concentration o...

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PUM

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Abstract

The invention provides a culture method of highly yielding vinblastine from catharanthus roseus cells. The culture method comprises the following steps: 1) culturing a catharanthus roseus callus; 2) culturing a stable genetic catharanthus roseus cell line; and 3) inoculating the catharanthus roseus cell line obtained in the step 2) to a liquid suspension culture medium for induced culture to simulate accumulation of alkaloid in catharanthus roseus, wherein the inducing conditions are as follows: from the 10-15 days of culturing the catharanthus roseus cell line in the liquid suspension culturemedium, reducing the culture temperature from 32-35 DEG C to 25-28 DEG C, introducing carbon monoxide gas and adding nicotinic acid, ecdysone and silver thiosulfate into the liquid suspension culturemedium, maintaining the temperature to culture the cell line continuously for 2-3 days, recovering the culture temperature to 32-35 DEG C, culturing the cell line for 4-5 days, and carrying out heating and cooling culture alternately till the culture is finished to obtain the catharanthus roseus cells which yield vinblastine highly. The culture method has the characteristics of being high in cellgrowth speed, high in vinblastine accumulative content and the like.

Description

technical field [0001] The invention belongs to the technical field of cell culture. More specifically, the present invention relates to a method for culturing vinblastine cells with high yield of vinblastine. Background technique [0002] Periwinkle, alias Calendula, Four Seasons Spring, Ri Rixin, Yantouhong, Sanwanhua, is a plant of the genus Periwinkle of the oleander family. At present, more than 100 kinds of terpenoid indole alkaloids have been isolated from periwinkle, such as vinblastine, vincristine, serpentine, and vindoline, etc. Many alkaloids isolated from periwinkle have Among them, vinblastine is the alkaloid with the greatest medicinal value and the most widely used in periwinkle. However, the content of vinblastine in the periwinkle plant is extremely small, so that the vinblastine extracted from the periwinkle plant is far from meeting the market demand. SUMMARY OF THE INVENTION [0003] As a result of various extensive and meticulous studies and experi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00
CPCA01H4/001A01H4/005
Inventor 覃家日
Owner 覃家日
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