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Method for inducing Polygonum ciliinerve to generate hairy roots

A technique for hairy roots and Polygonum trichomes, which is applied in the field of inducing Polygonum triclosanus to produce hairy roots, and achieves the effects of high proliferation coefficient, convenient material collection, and short culture time.

Inactive Publication Date: 2012-06-27
NORTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the medicinal value of Polygonum trichotilloides has been clinically confirmed. Most of the research on Polygonum trichotilloides has focused on the detection and separation of its medicinal active ingredients. No hairy roots have been obtained from Polygonum trichotillophorus transformed with Agrobacterium rhizogenes. Reports on the utilization of its medicinal active ingredients

Method used

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  • Method for inducing Polygonum ciliinerve to generate hairy roots
  • Method for inducing Polygonum ciliinerve to generate hairy roots
  • Method for inducing Polygonum ciliinerve to generate hairy roots

Examples

Experimental program
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Embodiment 1

[0032] Example 1: Polygonum trichotilloides hairy root induction

[0033] 1. Bacterial strains and culture After dark culture of Agrobacterium rhizogenes W.T15834 on YEB solid medium, pick a single colony, activate at 25°C, 28°C, 200 r / min dark shaking culture for 12h, transfer to fresh YEB liquid culture culture medium to OD 600 When it is 0.6, it is used for infection.

[0034] 2. Preparation of explants The young leaves of 20-day-old aseptic seedlings of Polygonum trichotilloide were cut into about 1.0 cm 2 Petiole-free leaf explants were placed on MS solid medium and pre-cultured for 24 hours for transformation.

[0035] 3. Induction, transformation and induction of hairy roots: immerse the explants of Polygonum trichophyllum leaf explants pre-cultured for 24 hours in the suspension of Agrobacterium rhizogenes W.T15834 diluted 5 times with MS medium and shake gently for 10 minutes, take out For the explants, use sterile filter paper to suck off the excess bacterial solu...

Embodiment 2

[0037] Example 2: Detection of mannoline in the hairy roots of Polygonum trichotilloides

[0038] The roots of non-transformed plants were used as the negative control, and the mannopine standard (1.02 mg / m / L) was used as the positive control. Referring to the method of Wang Guanlin et al., the mannopine in the transformed hairy roots was detected. The gene encoding opine synthase in the Ri plasmid TR-DNA (T-DNA right arm) region of Agrobacterium rhizogenes W.T15834 can be integrated into the Genomic DNA of Polygonum trichotilloides and expressed in Polygonum trichophyllum cells Specific product mannoline ( figure 2 ).

Embodiment 3

[0039] Embodiment 3: PCR detection of the hairy roots of Polygonum trichotilloides

[0040] 1. Plant DNA extraction: extract hairy root DNA with micro-CTAB method, use non-transformed plant hairy root total DNA as a control, and use it as a template for PCR amplification after purification.

[0041] 2. Extraction of Ri plasmid DNA from Agrobacterium rhizogenes: Take 200 μl of activated Agrobacterium rhizogenes strain W.T15834, centrifuge at 6000 r / min for 5 minutes, collect the precipitated bacteria, add 40 μl of lysate (Shanghai Sangong Technology Co., Ltd.), Suspend and mix well, lyse at 94°C for 15 minutes, centrifuge at 13,000 r / min for 5 minutes, and take the supernatant for later use. And take a small amount of supernatant to measure its OD 600 The value is 0.6.

[0042] 3. PCR reaction: 20 μL of reaction system, including 1 μg of DNA template, 1 μL of each upstream and downstream primers (final concentration 20 pmol / L), the primers used were based on the genes related...

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Abstract

The invention discloses a method for inducing Polygonum ciliinerve to generate hairy roots, which comprises the following steps of: inducing laminae of the Polygonum ciliinerve through an Agrobacterium rhizogenes strain W.T15834 to form the hairy roots, and culturing to obtain a great number of hairy roots of the Polygonum ciliinerve, wherein the laminae of the Polygonum ciliinerve serve as explants. According to the method for inducing the Polygonum ciliinerve to generate the hairy roots, the Polygonum ciliinerve is converted by utilizing Agrobacterium rhizogenes on the basis of establishing a sterile rapid propagation system by utilizing isolated culture, thereby, a great number of hairy roots of the Polygonum ciliinerve can be obtained. The method for inducing and culturing the hairy roots of the Polygonum ciliinerve has the characteristics that hormones autonomously and rapidly grow and differentiate, the proliferation coefficient is high, the inheritance is stable, materials can be conveniently fetched without season limitations, and the culture time is short, and has a positive effect on perfecting the Polygonum ciliinerve transgenic technology, performing mass culture on the hairy roots of the Polygonum ciliinerve and utilizing the biotransformation of secondary metabolic products of the hairy roots of the Polygonum ciliinerve in the later period.

Description

technical field [0001] The invention relates to a method for inducing Polygonum trichophyllum to produce hairy roots, which uses plant tissue culture technology and transgenic technology to obtain the hairy roots of Polygonum trichotilloides through Agrobacterium-mediated test-tube seedling leaves, and belongs to the field of biotechnology. Background technique [0002] In 1982, Chilton et al first reported Agrobacterium rhizogenes ( Agrobacterium rhizogenes ) When the Ri plasmid infects plant cells, through the attachment between bacteria and plant cells, the activation and expression of Vir gene (virulencegene), its T-DNA (transferred DNA) fragment is transferred, inserted and stably in the plant cell genome Expression induces plant cells to produce hairy roots. Compared with cultured cells, hairy root culture has the advantages of hormone-autonomous rapid growth and differentiation, many types of secondary metabolites, high content, stable heredity, and no need for marke...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/06
Inventor 王英娟步怀宇
Owner NORTHWEST UNIV
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