Method for culturing siraitia grosvenorii callus cell suspension system

A technology of callus and cell suspension, applied in tissue culture, plant cells, biochemical equipment and methods, etc., to achieve the effect of increasing yield

Active Publication Date: 2011-09-07
浙江康德药业集团股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mogroside is the main active ingredient in Luo Han Guo, which has health care and medicinal value. However, since mogroside is extracted from the fruit of Luo Han Guo, the extraction yield is only about 1%. Therefore, it is necessary to find a new meth

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] The cultivation method of Luo Han Guo callus cell suspension system comprises the steps:

[0013] In this experiment, the stem section of the healthy plant of Luo Han Guo was used as the experimental material.

[0014] 1) Inoculate and induce the callus of the stem section of Luo Han Guo, and subculture on MS liquid medium (pH 5.8) supplemented with 0.2mg / l 6-BA+0.04mg / l NAA hormone, 2.5% sucrose, and 4.0mg / l agar Culture, select the fresh, soft and friable yellow-white callus that has been continuously subcultured for 3 times, 4 times or 5 times;

[0015] 2) The callus obtained in step 1) is transferred to the aseptic Erlenmeyer flask containing MS+0.2mg / l 6-BA+0.02mg / l NAA, 2.0% sucrose as the liquid medium of carbon source (inoculation 300g / l), the pH of the initial medium was 5.0-5.4, placed in a 120r / min shaking incubator at 25°C in the dark for 24 hours and then left to stand, then transferred the upper layer of medium together with single cells and small cell cl...

Embodiment 2

[0018] The cultivation method of Luo Han Guo callus cell suspension system comprises the steps:

[0019] In this experiment, the stem section of the healthy plant of Luo Han Guo was used as the experimental material.

[0020] 1) Inoculate and induce the callus of the stem segment of Luo Han Guo, and subculture on MS medium (pH 5.8) supplemented with 0.2mg / l 6-BA+0.04mg / l NAA hormone, 2.5% sucrose, and 4.0mg / l agar , select fresh, soft and friable yellow-white callus that has been continuously subcultured for 3 or 4 or 5 times;

[0021] 2) The callus obtained in step 1) is transferred to the aseptic Erlenmeyer flask containing MS+0.2mg / l 6-BA+0.06mg / l NAA, 3.0% sucrose as the liquid medium of carbon source (inoculation 300g / l), the initial pH of the medium was 5.5-6.0, placed in a 120r / min shaking incubator at 25°C in the dark for 24 hours and then left to stand, then transferred the upper layer of medium together with single cells and small cell clusters to a sterile Continu...

Embodiment 3

[0024] The cultivation method of Luo Han Guo callus cell suspension system comprises the steps:

[0025] In this experiment, the stem section of the healthy plant of Luo Han Guo was used as the experimental material.

[0026] 1) Inoculate and induce the callus of the stem segment of Luo Han Guo, and subculture on MS medium (pH 5.8) supplemented with 0.2mg / l 6-BA+0.04mg / l NAA hormone, 2.5% sucrose, and 4.0mg / l agar , select fresh, soft and friable yellow-white callus that has been continuously subcultured for 3 or 4 or 5 times;

[0027] 2) The callus obtained in step 1) is transferred to the aseptic Erlenmeyer flask containing MS+0.2mg / l 6-BA+0.10mg / l NAA, and 4.0% sucrose is the liquid medium of carbon source (inoculation 300g / l), the initial pH of the medium was 6.1-6.4, placed in a 120r / min shaking incubator at 25°C in the dark for 24 hours and then left to stand, then transferred the upper layer of medium together with single cells and small cell clusters to a sterile Con...

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Abstract

The invention relates to a method for culturing a siraitia grosvenorii callus cell suspension system. The method comprises the following steps of: 1) inoculating and inducing siraitia grosvenorii stem calluses, performing subculture on MS liquid medium added with 1-naphthaleneacetic acid (NAA) hormone, sucrose and agar, and selecting the calluses; 2) transferring the calluses obtained in the step 1) to a sterile triangular flask, putting the triangular flask into a shaking incubator, performing suspension culture for 24 hours under the dark condition of 25 DEG C, transferring the culture medium of the upper layer, single cells and small cell masses to a triangular flask, and continuously performing shaking culture, performing transfer again after 3 days by adopting the same method, filtering the suspended cells through a screen with meshes of 40 microns after 7 days, inoculating the suspended cells to fresh culture medium for culturing, transferring the cultured cells to a big triangular flask after a certain volume is reached, and continuously adding the culture medium and performing shaking culture by using the same method; and 3) culturing for 21 days, harvesting, flushing for 3 times, performing vacuum filtration, weighing and drying. The method has the characteristics of high propagation speed and large culture scale, and provides a large amount of uniform plant cell cultures.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for cultivating a Luo Han Guo callus cell suspension system. Background technique [0002] Luo Han Guo is the fruit of Siraitia grosvenorii (Swingle) C. Jeffery ex Lu et Z. Y. Zhang, a plant of the genus Siraitia of the family Cucurbitaceae. Luo Han Guo is a precious medicinal and edible plant unique to Guilin, Guangxi. Mogroside is the main active ingredient in Luo Han Guo, which has health care and medicinal value. However, since mogroside is extracted from the fruit of Luo Han Guo, the extraction yield is only about 1%. Therefore, it is necessary to find a new method to obtain mogroside It is imperative that plant cell culture technology to produce secondary metabolites is one of the effective ways to obtain mogroside. At present, there is no report about the suspension cell culture of Luo Han Guo at home and abroad. Contents of the invention [0003] The object of t...

Claims

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Application Information

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IPC IPC(8): C12N5/04C12N5/02
Inventor 段小群曾建红申响宝宋波卢曦李云秋
Owner 浙江康德药业集团股份有限公司
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