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Aquilaria sinensis hairy root induction and genetic transformation method

A method of genetic transformation and a technology of genetic transformation, applied in the field of induction of hairy roots of A. chinensis and its genetic transformation, can solve the problems of no induction of hairy roots of A. argentina and no genetic transformation of A. argentina

Active Publication Date: 2018-11-20
INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

) have not yet seen the research report on the induction of the hairy roots of A. argentina and its genetic transformation, nor the research report on the genetic transformation (transgenic) of A. arborinae

Method used

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  • Aquilaria sinensis hairy root induction and genetic transformation method
  • Aquilaria sinensis hairy root induction and genetic transformation method
  • Aquilaria sinensis hairy root induction and genetic transformation method

Examples

Experimental program
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Effect test

Embodiment 1

[0090] The embodiment of the present invention provides a method for inducing the efficiency of the hairy root of A. sinensis, comprising the following steps:

[0091] 1. Acquisition of exogenous

[0092] 1. Rinse the seeds with distilled water and peel off the seed coat;

[0093] 2. Soak the seeds in 75% ethanol for 30-50 sec, 20% sodium hypochlorite (NaClO) solution for 15 min, rinse with sterile water for 3-5 times, and dry the water with sterile filter paper on a clean bench;

[0094] 3. The treated seeds were placed on solid MS medium and cultured at 25°C under 8h light / 16h dark conditions;

[0095] 4. After being cultivated for 3 days, the seeds will start to germinate and be used as explant materials for subsequent infection.

[0096] 2. Agrobacterium rhizogenes infection (hairy root induction)

[0097] 1. Agrobacterium rhizogenes was cultured at 28°C in LB liquid medium containing 50mg / L Rif.

[0098] 2. Collect the bacteria by centrifugation, and resuspend the pel...

Embodiment 2

[0125] The embodiment of the present invention provides a method for inducing the efficiency of the hairy root of A. sinensis, comprising the following steps:

[0126] 1. Acquisition of exogenous

[0127] 1. Rinse the seeds with distilled water and peel off the seed coat;

[0128] 2. Soak the seeds in 75% ethanol for 30-50 sec, 20% sodium hypochlorite (NaClO) solution for 15 min, rinse with sterile water for 3-5 times, and dry the water with sterile filter paper on a clean bench;

[0129] 3. The treated seeds were placed on solid MS medium and cultured at 25°C under 8h light / 16h dark conditions;

[0130] 4. After being cultivated for 3 days, the seeds will start to germinate and be used as explant materials for subsequent infection.

[0131] 2. Agrobacterium rhizogenes infection (hairy root induction)

[0132] 1. Agrobacterium rhizogenes was cultured at 28°C in LB liquid medium containing 50mg / L Rif.

[0133] 2. Collect the bacteria by centrifugation, and resuspend the pel...

Embodiment 3

[0143] The embodiment of the present invention provides a method for inducing the efficiency of the hairy root of A. sinensis, comprising the following steps:

[0144] 1. Acquisition of exogenous

[0145] 1. Rinse the seeds with distilled water and peel off the seed coat;

[0146] 2. Soak the seeds in 75% ethanol for 30-50 sec, 20% sodium hypochlorite (NaClO) solution for 15 min, rinse with sterile water for 3-5 times, and dry the water with sterile filter paper on a clean bench;

[0147] 3. The treated seeds were placed on solid MS medium and cultured at 25°C under 8h light / 16h dark conditions;

[0148] 4. After being cultivated for 3 days, the seeds will start to germinate and be used as explant materials for subsequent infection.

[0149] 2. Agrobacterium rhizogenes infection (hairy root induction)

[0150] 1. Agrobacterium rhizogenes was cultured at 28°C in LB liquid medium containing 50mg / L Rif.

[0151] 2. Collect the bacteria by centrifugation, and resuspend the pel...

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Abstract

The invention provides an aquilaria sinensis hairy root induction and genetic transformation method. According to the method, on the basis of research on conditions such as explant types, infection modes, infection times and hairy root culture temperatures, conditions applicable to aquilaria sinensis hairy root induction and culture are tested and screened, and most appropriate infection conditions for different explants of aquilaria sinensis are obtained. In addition, optimal induction conditions for inducing hairy roots from different parts (cotyledon, stems, roots and leaves) of SW101 aquilaria sinensis are provided. The invention provides a method for improving aquilaria sinensis hairy root genetic transformation efficiency. A green fluorescent protein (fgp) is introduced into aquilaria sinensis hairy roots, whether aquilaria sinensis hairy root genetic transformation succeeds or not can be visibly identified, and meanwhile, the effectiveness of a transformation system can be doubly verified in a PCR (Polymerase Chain Reaction) detection mode. Good, efficient and simple options are provided for aquilaria sinensis function gene study and / or industrialization of aquilaria sinensis hairy root metabolite.

Description

technical field [0001] In the field of biological technology, the invention specifically relates to a method for inducing and genetically transforming the hairy roots of A. sinensis. Background technique [0002] Hairy roots, also known as hairy roots, hairy roots, are inserted into the nuclear genome of plants or organs, tissues (including callus), single cells, and protoplasts by T-DNA on the Ri plasmid contained in Agrobacterium rhizogenes , and a large number of by-products are produced at the site of infection. The induced culture of hairy roots can be used as a bioreactor to produce valuable plant metabolites. In addition, exogenous genes can also be introduced by Agrobacterium rhizogenes to regulate the synthesis of target metabolites. [0003] Agarwood is formed when the trunks or roots of Aquilaria spp. and Gyrinops spp. plants in the family Aquilaria spp. secrete special oils during the self-defense response after various injuries. Both Agarwood and Pseudo-Agarw...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H4/00
CPCA01H4/005C12N15/8205
Inventor 魏建和隋春徐娇
Owner INST OF MEDICINAL PLANT DEV CHINESE ACADEMY OF MEDICAL SCI
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