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Method for producing podophyllotoxin by using callus adventitious roots of sinopodophyllum hexandrum royle

A technology of podophyllotoxin and peach seven, applied in horticultural methods, botanical equipment and methods, plant regeneration and other directions, can solve farmers' lack of enthusiasm for planting, large consumption of peach seven seeds, slow callus growth, etc. problems, to achieve the effect of low degree of non-subgeneration browning, short culture period, and promotion of accumulation and content improvement.

Active Publication Date: 2019-07-26
GANSU ACAD OF SCI INST OF BIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There have been many reports on the artificial domestication and cultivation of Taoerqi, but the cultivation period is long, the yield is low, and the purchase price of medicinal materials is low. Farmers have no enthusiasm for planting
There are sporadic reports on the production of podophyllotoxin through the radicle and callus of T. chinensis tissue culture seedlings, but it is difficult to take root, the number of radicles is small, the yield is low, and this method consumes a lot of seeds of T. chinensis; Although callus culture can increase yield through subculture of callus, callus grows slowly, tends to brown after subculture, and subcultures up to 2 times, which limits the effective application of this method
It has been reported that podophyllotoxin can be produced by the endophyte of P. chinensis, but the content of podophyllotoxin produced by endophyte fermentation is low, the stability of the strain is poor, and the ability to produce podophyllotoxin is lost with the passage of the strain.

Method used

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  • Method for producing podophyllotoxin by using callus adventitious roots of sinopodophyllum hexandrum royle
  • Method for producing podophyllotoxin by using callus adventitious roots of sinopodophyllum hexandrum royle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] The acquisition of embodiment 1 sterile seed embryo

[0044]Remove moth-eaten and mildewed seeds from fresh peach seven seeds, wash them with water, corrode the seed coats with a little concentrated sulfuric acid until the color turns black, soak them in 0.5% sodium carbonate at room temperature for 20 hours to remove oily, waxy and other hydrophobic components, 80mg / L gibberellin (GA4+7) was soaked at 4°C for 28 hours, and 0.2% mercury chloride surface was disinfected for 6 minutes.

[0045] In the experimental group, the above-mentioned pretreated seeds were blotted dry with sterilized filter paper, and the seed embryos were extruded from the germination hole of the hilum with sterilized needle-nose pliers, and inoculated in MS medium; The pretreatment method of group seeds is the same as above, only the seed embryos are taken out using the following method: the seeds are cut open with a scalpel, and half seeds with embryos are inoculated on MS medium. The two group...

Embodiment 2

[0048] Effect of Example 2 Different Hormones on Embryogenic Callus Induction

[0049] Using seed embryos as explants, culture them at a temperature of 23°C in the dark for about 40 days until adventitious root primordia are clearly visible and embryogenic callus is obtained. Embryogenic callus induction medium formula: MS basic culture medium, sucrose 50g / L, acid hydrolyzed casein 1g / L, agar 5g / L, biotin 0.2mg / L, pH6.5, and add different hormones for culture , see Table 1 for the ratio of different hormones.

[0050] Table 1 Embryogenic callus induction medium

[0051]

[0052] After the culture, the growth state of the explants was observed, the callus induction rate was calculated, and the number of adventitious root primordia was counted. The results are shown in Table 2.

[0053] test group explant growth status callus induction rate average adventitious root cardinality 1 After about 20 days, the explants expanded obviously 80% callus inductio...

Embodiment 3

[0056] The growth of embodiment 3 adventitious roots

[0057] The callus with root primordia obtained in Example 2 was transferred into the adventitious root growth medium, and cultured for 40 days under dark conditions at a temperature of 20° C., and the length of the adventitious root was 5+1 cm. At this time, the adventitious roots were divided into two groups, and one group continued to grow in the liquid 1 / 2MS medium (sucrose 20g / L) without agar under sterile conditions; Cultivate in a flask, immerse the roots in 0.2g / L of water-soluble fertilizer with a nitrogen, phosphorus and potassium content ratio of 20:20:20 (with 0.1g / L of sucrose added), and cover the callus with sterilized peat soil.

[0058] Adventitious root growth medium formula is as follows: 1 / 2MS basic culture medium, sucrose 20g / L, agar 5g / L, naphthaleneacetic acid (NAA) concentration of 0.2mg / L, indoleacetic acid (IAA) concentration of 0.2mg / L.

[0059] The experimental results show that adventitious roo...

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Abstract

The invention discloses a method for producing podophyllotoxin by using callus adventitious roots of sinopodophyllum hexandrum royle. The method comprises the steps of obtaining sterile embryos, carrying out embryonic callus induction, adventitious roots growing, induction production of the podophyllotoxin, and the like. The set of method for producing the podophyllotoxin by coupling cell engineering with fermentation engineering is established. The method provided by the invention is simple to operate and high in yield, subculture does not occure to the calluses, browning degree is low; the adventitious roots are treated by using endophyte of the sinopodophyllum hexandrum royle, clonostachys rosea (fhnzm) fermentation liquor and some plant growth regulators, so that the content of the sinopodophyllum hexandrum royle is effectively increased.

Description

technical field [0001] The invention relates to the field of agricultural and industrial biotechnology, in particular to a method for producing podophyllotoxin from the adventitious roots of the seven calli. Background technique [0002] Sinopodophyllum hexandrum Royle is a herbaceous plant of the genus Sinopodophyllum hexandrum Royle in the family Berberidaceae, a national third-level protected plant, and is mainly distributed in forest margins and shrubs above 2800 meters in western my country. Due to the scarcity and endangerment of natural resources, it was included in the List of Rare and Endangered Plants in China and the Red Book of Plants in China. [0003] Taoerqi is mainly used in the folk for pain, boils, swollen poison, rheumatic bone pain, tracheitis and other diseases. Modern pharmaceutical research shows that its roots and rhizomes mainly contain lignans, flavonoids, saponins, polysaccharides and other components, of which The lignans had the highest podophyl...

Claims

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Application Information

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IPC IPC(8): A01H4/00
CPCA01H4/001A01H4/008
Inventor 杨涛王治业杨晖李鑫李珊方彦昊
Owner GANSU ACAD OF SCI INST OF BIOLOGY
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