Induction and in-vitro culture method of plant stem cells derived from apical meristem of catharanthus roseus roots

A technology of apical meristem and plant stem cells, applied in plant cells, cell culture medium, etc., to achieve important application value and market prospects, stable genetics, and high yield

Inactive Publication Date: 2020-11-03
LINGNAN INST OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether the vinblastine stem cells derived from the stem cambium can produce vinblastine, and what is the ability to produce vinblastine, these issues are not covered by the above-mentioned patents

Method used

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  • Induction and in-vitro culture method of plant stem cells derived from apical meristem of catharanthus roseus roots
  • Induction and in-vitro culture method of plant stem cells derived from apical meristem of catharanthus roseus roots
  • Induction and in-vitro culture method of plant stem cells derived from apical meristem of catharanthus roseus roots

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] A method for inducing stem cell lines from periwinkle root top meristem, comprising the steps of:

[0052] S1: Collection and sterilization of explants

[0053] In the early morning, the root tips of the 1-2 year-old Apocynaceae Vinca genus Vinca were collected, rinsed with tap water for 4 hours, and gently wiped off the water with filter paper to be used as explants. Rinse explants with sterile double distilled water for 2 h in a biological safety cabinet, sterilize with 75% (V / V) ethanol solution for 30 s, and wash with 0.06% HgCl 2 The solution was treated for 5 minutes, rinsed with sterile double distilled water for 10 minutes, and then the sterilized explants were placed in the anti-browning preservation solution for 1-3 hours and stored for use;

[0054] The above-mentioned anti-browning preservation solution is an MS medium containing 250 mg / L citric acid, 300 U / mL penicillin, and 300 mg / L streptomycin, with a pH value of 5.75.

[0055] The MS medium formula is...

Embodiment 2

[0067] The Erlenmeyer flask suspension culture method of periwinkle stem cell line described in embodiment 1

[0068] Add 200mL MS liquid medium to a 500mL Erlenmeyer flask, inoculate the periwinkle stem cell line described in Example 1 at an inoculation amount of 60g / L, and then carry out suspension shaking culture.

[0069] Suspension culture conditions are: temperature 26°C, rotation speed 120rpm, completely dark culture, dissolved oxygen 0.30mg / L, culture time 15 days; MS liquid medium is supplemented with 2.0mg / L naphthaleneacetic acid, 2.0mg / L 2,4 -D MS medium.

Embodiment 3

[0071] The bioreactor culture method of periwinkle stem cell line described in embodiment 1

[0072] 8L MS liquid medium is placed in 10L bioreactor, then the periwinkle stem cell line of suspension culture in embodiment 2 is subcultured in 10L bioreactor and carries out suspension culture, inoculum size is 60g / L; Suspension culture condition is : The temperature is 26°C, the rotation speed is 120rpm, the aeration ratio is 0.50vvm, the pH value of the suspension medium is adjusted to 5.70, the culture is completely dark, the dissolved oxygen amount is 0.50mg / L, and the culture is continuous for 15 days.

[0073] The liquid medium used in the bioreactor culture is: MS medium supplemented with 35g / L sucrose, 2.5mg / L naphthaleneacetic acid, and 2.0mg / L 2,4-D.

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Abstract

The invention discloses an induction and in-vitro culture method of plant stem cells derived from apical meristem of catharanthus roseus roots. The induction method comprises the following steps: cutting off a root cap from the root tip of the catharanthus roseus, performing inoculating to an induction culture medium, performing culturing in dark, and proliferating and growing a stem cell layer inan explant to form a cell cluster; transferring the layered stem cell layer to a subculture medium for culturing for 10-15 days, and performing complete culturing in dark; and performing transferringto a new subculture medium, and performing culturing for 10-15 days under the same condition to obtain a catharanthus roseus stem cell line with stable cell morphology. A catharanthus roseus stem cell culture system is established by inducing apical meristem of the catharanthus roseus roots, stable subculture can be performed under certain conditions, and epigenetic variation is avoided. The catharanthus roseus stem cell culture system established by the invention has the characteristics of being high in growth speed, high in genetic stability and the like, and culture with a 10L bioreactor is preliminarily realized.

Description

technical field [0001] The invention relates to a method for inducing and in vitro culturing of plant stem cells derived from the top meristem of periwinkle root. Background technique [0002] Periwinkle is a plant of the genus Periwinkle in the family Apocynaceae, from which more than 130 alkaloids have been discovered, which have various pharmacological activities. The drugs vinblastine and vincristine. [0003] At present, the sources of these drugs are mainly extracted from the original plants, but periwinkle has a long growth cycle and is affected by the environment and climate, and the content of alkaloids such as vinblastine and vincristine in plants is extremely low, only A few parts per million to a few ten thousandths. In addition, there are more than 130 kinds of alkaloids in periwinkle, and the composition of alkaloids is extremely complex. These factors make the cost of obtaining a single medicinal active ingredient extremely high. At the same time, the chemi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/04
CPCC12N5/0025C12N5/04
Inventor 李海华李岩兰小群曾琳玲陈静敏
Owner LINGNAN INST OF TECH
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