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Method of regenerative plants from hairy root of woody mandala having hyoscyamine and scopolamine

A technology for regenerating plants and scopolamine, which is applied in botany equipment and methods, biochemical equipment and methods, plant regeneration, etc., and can solve undiscovered problems

Inactive Publication Date: 2008-07-23
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The complete plants regenerated from transgenic Datura japonica, and the identification of transformed Datura woody plants with high content of scopolamine and scopolamine through screening can provide great impetus and broad prospects for theoretical research and production practice, especially for the final solution Hyoscyamine and scopolamine drugs are scarce to provide high-quality new varieties of Datura woody, but no reports related to the hairy root regeneration plants of Datura woody mentioned in the subject of the present invention have been found

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Induction of Callus and Differentiation of Adventitious Buds from Hairy Roots of the Woody Datura datura

[0025] Cut the hairy roots of Datura woody into segments about 2-3cm long, and transfer them to the callus induction medium, the formula of which is MS medium + 4.0mg·L -1 6-Benzyladenine (6-BA)+0.5mg·L -1 Kinetin (KT)+0.5mg·L -1 2,4-Dichlorophenoxyacetic acid (2,4-D). Cultured at a constant temperature of 25°C±1.0°C, with 15h of light / day, most of the hairy root callus will be formed after 15 days, and the volume of callus will increase rapidly with the prolongation of culture time; from the induced hairy root callus Part of the hairy roots with good growth and no browning were selected and transferred to the adventitious bud induction medium, the formula of which was MS medium + 4.0mg·L -1 6-Benzyladenine (6-BA)+0.2mg·L -1 Naphthaleneacetic acid (NAA)+30g·L -1 sucrose. After 14 days of light culture, buds began to appear on the callus, and with the prolon...

Embodiment 2

[0027] Rooting Culture of Adventitious Buds of Woody Datura datura

[0028] Cut the green adventitious buds differentiated from the hairy roots of Datura woody, and transfer them to rooting and rooting medium, the formula of which is 1 / 2MS medium + 0.1mg L -1 Indole-3-butyric acid (IBA)+30g·L -1 sucrose. Placed at 25°C ± 1.0°C for constant temperature cultivation, 15h of light per day, adventitious roots began to grow after 6 days, and complete regenerated plants gradually formed.

Embodiment 3

[0030] Molecular detection of regenerated plants from the hairy roots of the woody Datura datura

[0031] 1. The extraction of genomic DNA from the regenerated plant of Datura woody root, the method is as follows:

[0032] (1) Take 200 mg leaves of the regenerated plants of the hairy roots of Datura japonica, filter them, wash them with 10 mL of distilled water, blot them dry, quick-freeze them in liquid nitrogen, and grind them into powder.

[0033] (2) Add 500 μL of extraction buffer (3% mercaptoethanol) to a 1.5 mL Eppendorf tube, fully shake in a 65°C water bath for 50 minutes, and mix by inverting every 5 minutes.

[0034] (3) Centrifuge at 4°C, 12,000 rpm for 10 minutes.

[0035] (4) Aspirate the supernatant, add 500 μL of phenol: chloroform: isoamyl alcohol (25:24:1), mix gently, and let stand for 5 minutes to separate layers.

[0036] (5) Centrifuge at 12,000 rpm for 10 minutes at room temperature.

[0037] (6) Aspirate about 350 μL of the supernatant, add an equa...

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PUM

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Abstract

The invention provides a method for obtaining a regeneration plant of the woody stramonium with high yield of hyoscyamine and scopolamine by utilizing tissue culture technology to induce woody stramonium hairy root and relates to the method for inducing the regeneration plant of the woody stramonium hairy root to obtain the full woody stramonium plant which can produce hyoscyamine and scopolamine. The process of the invention is that first proper tissue culture technology is used for inducing the woody stramonium hairy root to obtain the woody stramonium regeneration plant. The regeneration plant is determined to be genetic transformation hairy root regeneration plant by molecule test and an HPLC test shows that the woody stramonium hairy root regeneration plant obtained from the tissue culture technology can produce hyoscyamine and scopolamine with a remarkably higher yield than wild plants. The method can provide a novel and sustainable medicinal herbs resource for the production of hyoscyamine and scopolamine.

Description

technical field [0001] The invention belongs to the fields of molecular biology, pharmacognosy, breeding, cell engineering and the like, and relates to a method for obtaining high-yielding scopolamine and scopolamine woody Datura hairy root regenerated plants using tissue culture technology, in particular to inducing woody Specific procedure for regeneration of Datura datura hairy roots and obtaining high hyoscine and scopolamine producing woody Datura datura plants. The invention also provides the regenerated plants of Datura woody hairy roots with high hyoscine and scopolamine yield obtained by tissue culture technology and the cultured offspring. Background technique [0002] Woody Datura belongs to the Solanaceae Datura genus perennial medicinal arbor plants. There are about 16 kinds of plants in this genus, most of which are distributed in tropical and subtropical regions, and less in temperate regions. There are 4 species in my country, distributed in all provinces in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00C12Q1/68
Inventor 孙敏雷桅杨雪清
Owner SOUTHWEST UNIV
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