Ephedra crown-gall nodule cell ZHA-2 CGMCC NO.0521 and its inducing method

A ZHA-2CGMCCNO0521, tumor cell technology, applied in plant cells and other directions, can solve the problem of difficult separation of culture medium and cell culture, achieve fast growth ability, improve production capacity and yield effect

Inactive Publication Date: 2005-04-20
INST OF PROCESS ENG CHINESE ACAD OF SCI +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The object of the present invention is to induce ephedra crown gall tumor cell (Ephedra) ZHA-2CGMCC NO.0521 by ephedra plant; Utilize the culture that ephedra crown gall tumor cell (Ephedra) ZHA-2 CGMCC NO.0521 obtains can greatly increase biomass and The content of ephedrine in the culture, purpose two is to overcome existing in the bioreactor in the process of cultivating plant cells to produce metabolites, culture fluid and cell culture are not easy to separate, the growth of callus culture and metabolites Synthesize often contradictory shortcomings, thereby providing a kind of ephedra crown gall tumor cell (Ephedra) ZHA-2 CGMCC NO.0521 and its induction method

Method used

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  • Ephedra crown-gall nodule cell ZHA-2 CGMCC NO.0521 and its inducing method

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Experimental program
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Effect test

Embodiment 1

[0035] Ephedra crown gall tumor cells (Ephedra) ZHA-2 CGMCC NO 0521 were induced according to the following steps:

[0036] 1) Equisetum or Ephedra plants are washed with detergent solution, rinsed with distilled water three times; sterilized with 2% (weight / volume) sodium hypochlorite for 30 minutes, washed with sterilized distilled water, and cut into 0.5 cm lengths , to obtain the sterilized Ephedra plant;

[0037] 2) with the sterilized Ephedra plant, and the OD that has been diluted 20 times 600 Agrobacterium culture solution of 0.6 was co-cultivated for 30 minutes, and the cultivation temperature was 25° C. to obtain Ephedra plants infected with Agrobacterium;

[0038] 3) placing the plants infected with Agrobacterium in the above step 2 in a growth medium containing 2wt% kanamycin and culturing them for 15 days at a culture temperature of 25° C. Ephedra crown gall tumor cells gradually grow on the plants;

[0039] 4) Excise the ephedra crown gall tumor cells obtained ...

Embodiment 2，3，4，5，6，7 and example 8

[0045] The ephedra crown gall tumor cell (Ephedra) ZHA-2 CGMCC NO.0521 cultivated in embodiment 2-8 is identical with embodiment 1 with induction process and the condition of induction, but used ephedra plant can be classified as: horsetail ephedra , Ephedra chinensis, Ephedra mesophylla, Ephedra membranaceus, Casuarina quinquefolius, Ephedra monoephedra, Ephedra biscarina, Ephedra biscarina, Ephedra chinensis, and Ephedra ephedra crown gall tumor cells are induced in different compositions, and the composition of the medium is listed as follows:

[0046]

Embodiment 9

[0047] The ephedra crown gall tumor cell (Ephedra) ZHA-2 CGMCC NO.0521 cultivated in embodiment 9,10,11,12,13,14,15 and 16 is identical with embodiment 1, and, the ephedra crown gall tumor cell is made of The induction process of the plants of the genus Ephedra and the conditions of induction are also the same as those described in Example 1, but the composition of the medium induced by the ephedra crown gall tumor cells is different, as follows:

[0048] Example 9

[0049] On the basis of the method in Example 1, 40 mmol / L phenylalanine was added to the composition of the culture medium. Due to the addition of phenylalanine, the induction rate of ephedra crown gall tumor cells and the productivity of ephedrine are improved.

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Abstract

The invention relates to introducing the Agrobacterium into the Ephedra plant, inducing the Ephedra plant to produce Ephedra crown gall tumor cell (Ephedra) ZHA-2 CGMCC NO.0521 and the induction method. The ephedra crown gall tumor cell (Ephedra) ZHA-2 induced by the present invention has been preserved in China Microorganism Collection Center on December 11, 2000. The ephedra crown gall tumor cells have the characteristics of ephedra crown gall tumor cells on sucrose agar medium containing a small amount of hormones, and the strain is cultured at about 23-32°C for 10-15 days. Light yellow to dark green, 0.5-15 mm in diameter. The induction method of the present invention uses the Ephedra plant, uses the Agrobacterium Ti plasmid as a carrier, and introduces the exogenous gene into the Ephedra plant to induce the Ephedra crown gall tumor cell (Ephedra) ZHA-2 CGMCC NO.0521 in the culture medium; the induced The ephedra crown gall tumor cells can be subcultured in the culture medium of 1.0~5.0% sugar, and cultivated for 5-60 days at 23~32° C., and the proliferation amount of the ephedra crown gall tumor cells obtained is 2~2% of that at the time of inoculation. 8 times, the content of ephedrine is 0.1~25wt%.

Description

technical field [0001] The invention relates to the induction of Ephedra crown gall tumor cells (Ephedra) ZHA-2 CGMCC NO.0521 from plants of the genus Ephedra by using biotechnology. More specifically, the present invention relates to introducing Agrobacterium into Ephedra plants to induce Ephedra plants to produce Ephedra crown gall tumor cells (Ephedra) ZHA-2 CGMCC NO.0521 and its induction method. Background technique [0002] The ephedra plant mainly contains ephedrine, pseudoephedrine, L-N methylephedrine, d-N methylpseudoephedrine, L-desmethylephedrine, d-desmethylpseudoephedrine, etc. Ephedra is a commonly used Chinese herbal medicine, which has the effects of sweating, relieving asthma, and expelling wind. Ephedrine, like adrenaline, can excite sympathetic nerves, but ephedrine can be taken orally, and has a long acting time, which adrenaline cannot. Ephedrine can dilate the bronchi, shrink the nasal mucosa, and increase blood pressure...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/04
Inventor 查丽杭张国政秦川朱维型苏志国欧阳藩
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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