Preparation and cultivation method of hairy roots of tongkat ali

A cultivation method and hairy root technology are applied in the field of preparation and cultivation of Dongge Ali-shaped root system to achieve the effects of saving manpower and fast growth.

Inactive Publication Date: 2012-10-31
DALIAN POLYTECHNIC UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no relevant literature report on the preparation of Dongge Ali hairy root system by biological cell engineering technology

Method used

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  • Preparation and cultivation method of hairy roots of tongkat ali
  • Preparation and cultivation method of hairy roots of tongkat ali
  • Preparation and cultivation method of hairy roots of tongkat ali

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1 Mannoline paper chromatography analysis

[0024] (1) Material: Dongge Ali hair root (NTR) newly induced by the present invention; hair root (TR) of the present invention through subculture; organ root (NR), leaf (L) or callus cell (C) , Agrobacterium rhizogenes A4 bacterial liquid;

[0025] (2) Extraction of agromine: take 1g of the above samples, add 1ml (0.1M) HCl to grind at room temperature, centrifuge at 12,000rpm for 5min, take 10μl of the supernatant, and apply the sample with a micro-sampler.

[0026] (3) Chromatography: preparation of chromatographic filter paper: cut chromatographic filter paper into 8×8 (cm) size, soak in 0.4mol / L HCl solution for 24 hours, then rinse with distilled water, and then wash with ethanol and diethyl ether in turn After washing, lay flat on a tray, dry at 30°C, and set aside.

[0027] (4) Sample spotting: draw a straight line 1-1.5 cm away from the bottom edge of the chromatography filter paper, and mark the equidist...

Embodiment 2

[0032] (1) Cut the young stems of Dongge Ali into small pieces of 2cm, and use HgCl in the aseptic operation table 2 Treat for 7 minutes, soak in alcohol for 6 seconds, rinse with sterile water for 4 times, transfer to MS+6-BA0.5mg / L+NAA0.3mg / L medium, culture in dark at 25℃±2 for 12d, and obtain Dongge Aliyu damage tissue.

[0033] (2) After diluting the Agrobacterium rhizogenes DC-AR2 strain with 1 μL of sterile water, spread the bacterial solution on 20 mL of YEB solid medium, culture in the dark at 25°C±3 for 4 days to grow a single colony, and obtain hairy roots Agrobacterium A4 clone.

[0034] (3) Pick the Agrobacterium rhizogenes A4 clone and place it in YEB medium, culture it with shaking at 25°C±2, the OD value is 0.5, take 1ml of the bacterial liquid and place it in 100ml YEB liquid medium for shaking culture, the OD value is 0.5 , collect the bacterial liquid, and centrifuge at 3000 rpm for 7 minutes to collect the bacterial cells.

[0035] (4) Place the thalline...

Embodiment 3

[0040] (1) Cut the young stems of Dongge Ali into small pieces of 4cm, and use HgCl in the aseptic operation table 2 Treat for 10 minutes, soak in alcohol for 8 seconds, rinse with sterile water for 7 times, transfer to MS+6-BA0.5mg / L+NAA0.3mg / L medium, culture in dark at 25℃±2 for 30d, and obtain Dongge Aliyu damage tissue.

[0041] (2) After diluting the Agrobacterium rhizogenes DC-AR2 strain with 1 μL of sterile water, spread the bacterial solution on 20 mL of YEB solid medium, culture in the dark at 25°C±3 for 5 days to grow a single colony, and obtain hairy roots Agrobacterium A4 clone. .

[0042] (3) Pick Agrobacterium rhizogenes A4 clone and place it in YEB medium, culture it with shaking at 25°C±2, the OD value is 1, take 1ml of the bacterial liquid and place it in 100ml YEB liquid medium for shaking culture, the OD value is 0.7 , collect the bacterial liquid, and centrifuge at 3000rpm for 10min to collect the bacterial cells.

[0043] (4) Place the thalline collec...

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PUM

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Abstract

The invention discloses a preparation and cultivation method of hairy roots of tongkat ali and belongs to the biological cell engineering technology. The preparation and cultivation method includes: tender stems of tongkat ali are used as explants which subjected to dedifferentiation to obtain fresh callus of tongkat ali, the callus and Agrobacterium rhizogenes containing Ri (root-inducing) plasmid are co-cultured, co-cultured bacteria liquid is transferred to an inductive medium for induced culture after excess bacteria liquid is absorbed with aseptic paper, fairy roots of tongkat ali grow at the callus of tongkat ali; and the explants with hairy roots are laced in an amplification medium for extended culture of the hairy roots after biostatistics culture. The tongkat ali hairy root culture system is established by the biological cell engineering technology and used to prepare the hairy roots of tongkat ali, standard production of tongkat ali is realized, wild resources are replaced, increasing market demands on tongkat ali are eased, and the problem that tongkat ali relies on import is solved. The preparation and cultivation method is important to industrial and commercial development of hairy roots of medicinal plants.

Description

technical field [0001] The invention relates to a method for preparing and cultivating a hairy root system of a plant, in particular to a method for preparing and cultivating a plant hairy root system, which belongs to biological cell engineering technology. Background technique [0002] The scientific name of Tongkat Ali is Eurycoma longifolia Jack, which belongs to Simaroubaceae. Dongge Ali is a slow-growing tropical rainforest shrub plant, mainly distributed in Southeast Asian countries and regions such as Malaysia, the Philippines, Indonesia and Thailand. Its roots are used as medicine, and are often used to treat diseases such as malaria, dysentery, oral ulcers, scarlet fever, headaches, etc. It also has the effect of enhancing male sexual function, and is called "Malaysian ginseng". Recent studies have shown that quassinoids extracted from this plant species also have good anti-tumor and anti-HIV effects. Dongge Ali is a dioecious plant. Because Dongge Ali is a worl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00
Inventor 张宗申刘同祥于振艳叶乾堂
Owner DALIAN POLYTECHNIC UNIVERSITY
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