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Method for producing alkannin by utilizing Arnebia euchroma(Royle)Johnst hairy root

A Xinjiang comfrey and hairy root technology, applied in the field of plant biology, can solve the problems of ecological environment damage in Xinjiang, low seed maturity and germination rate, slow natural restoration, etc., and achieve the effects of saving electricity, reducing costs, and increasing production

Inactive Publication Date: 2012-08-29
XINJIANG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Xinjiang comfrey relies on seeds to reproduce, and it takes 3-5 years to bloom and bear fruit, and the seed maturity and germination rate are low, and natural restoration is slow. The amount of excavation is less than 20 tons, which puts the wild resources of Xinjiang comfrey on the verge of extinction and has caused great damage to the ecological environment of Xinjiang. For this reason, my country has listed Xinjiang comfrey as a national second-class protected plant as early as 1996.
There is no report on the hairy root of Sinica xinjiang in China

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] a. Sterilize the Xinjiang comfrey seeds with 75% alcohol for 30 seconds, then sterilize the surface with 0.1% mercury liter for 5 minutes, fully rinse with sterile water overnight, then soak in 500mg / L cefotaxime sodium for 60 minutes, and inoculate in 1 / 2MS Cultivate on solid medium at 25°C±1°C in a dark environment for 15 days to obtain the required sterile seedlings for use, cut out 0.2cm 2 The young cotyledons of the small aseptic seedlings were inoculated on 1 / 2MS solid medium, the temperature was 25°C±1°C, and the dark environment was pre-cultivated for 2 days before use;

[0044] b. Inoculate Agrobacterium rhizogenes on the YEB solid medium according to the conventional method. After 2 days of dark culture, pick a single colony and inoculate it in the YEB liquid medium. The solution was placed in a centrifuge at a temperature of 4°C and a speed of 5000r / min for 5 minutes, and the obtained bacteria were resuspended in MS liquid medium and used for infection;

[0...

Embodiment 2

[0053] a. Sterilize the seeds of Xinjiang comfrey with 75% alcohol for 45 seconds, then sterilize the surface with 0.1% mercury liter for 8 minutes, fully rinse with sterile water overnight, then soak with 500 mg / L cefotaxime sodium for 60 minutes, inoculate in 1 / 2MS solid medium until germination, then cut off the young cotyledon leaves of the sterile seedlings, and inoculate them on 1 / 2MS solid medium at a temperature of 25°C±1°C, cultivate in a dark environment for 18 days, and cut out 0.5cm 2 The young cotyledons of the small aseptic seedlings were inoculated on 1 / 2MS solid medium, the temperature was 25°C±1°C, and the dark environment was pre-cultivated for 2 days before use;

[0054] b. Inoculate Agrobacterium rhizogenes on the YEB solid medium according to the conventional method, and after cultivating in the dark for 2 days, pick a single colony and inoculate it in the YEB liquid medium at a temperature of 28°C and a rotation speed of 135r / min. The solution was placed...

Embodiment 3

[0063] a. Disinfect Xinjiang comfrey seeds with 75% alcohol for 60 seconds, then sterilize them with 0.1% mercury liter surface for 10 minutes, fully rinse with sterile water overnight, then soak them in 500 mg / L cefotaxime sodium for 60 minutes, and inoculate them in 1 / 2MS solid medium until germination, then cut the young cotyledons of sterile seedlings into 0.5cm 2 and inoculated on 1 / 2 MS solid medium at a temperature of 25°C±1°C, and cultivated in a dark environment for 20 days to obtain the required sterile vaccine for use;

[0064] Cut out the young cotyledons of the aseptic seedlings from around the cotyledons, inoculate them on 1 / 2MS solid medium at a temperature of 25°C±1°C, pre-cultivate them in a dark environment for 2 days, and set aside;

[0065] b. Inoculate Agrobacterium rhizogenes on the YEB solid medium according to the conventional method. After 2 days of dark culture, pick a single colony and inoculate it in the YEB liquid medium. The solution was placed ...

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Abstract

The invention relates to a method for producing alkannin by utilizing Arnebia euchroma(Royle)Johnst hairy root. The method includes selection and processing of Arnebia euchroma(Royle)Johnst explant, activation culture of agrobacterium rhizogene, infection of cotyledon explant and induction of hairy root, obtaining of sterile Arnebia euchroma(Royle)Johnst hairy root system and liquid culture of Arnebia euchroma(Royle)Johnst hairy root as well as alkannin production. The used hairy root has the characteristic of rapid and autonomous growth on culture medium without exogenous hormones, thus the method not only can overcome the defects that cell culture grows slowly and hormone is required to be additionally added to maintain the growth thereof when utilizing cell culture to produce alkannin,and the method has the advantages of stable alkannin throughput, simple operation in cultivation, lower cost and no restriction of climate and land natural conditions. Especially the invention provides a stable and continuable novel medicine source for producing alkannin by utilizing hairy root culture method and provides reliable source and material basis for alkannin mass production by applyingbioreactor in the future.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, and relates to a method for producing shikonin by utilizing the hairy roots of Xinjiang comfrey. Background technique [0002] Arnebia euchroma (Royle) Johnst is a perennial medicinal plant. The main component of Arnebia euchroma (Royle) Johnst is shikonin, which is a naphthoquinone compound. Pigments and their derivatives have significant pharmacological effects such as anti-fertility, anti-inflammation, anti-tumor, bactericidal and anti-virus, liver protection and immune regulation. At the same time, they can be used as pigments in cosmetics, food and printing and dyeing. In medicine, food, cosmetics, printing and dyeing The field has huge market potential. Xinjiang comfrey accounts for more than 80% of the country's resources, and is the main source of commercial comfrey at home and abroad. However, Xinjiang comfrey relies on seeds to reproduce, and it takes 3-5 years to bloom and bear fru...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82A61K131/00
Inventor 王芳麻浩陈永芳芦苇华
Owner XINJIANG AGRI UNIV
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