Method for inducing transgenic hairy roots of dioscorea composita

A technology of diosminum chrysanthemum and hairy root, applied in the field of induction of transgenic hairy root of diosminum chrysanthemum, can solve the problem of low induction efficiency

Active Publication Date: 2020-10-16
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

According to the relevant research reports on the induction of hairy roots, there are few relevant research reports on the successful induction of hairy roots in monocotyledonous plants, and the relevant research reports on the induction of hairy roots in Dioscorea genus are only found in the hairy roots of Dioscorea scutellaria. Preliminary research on root induction (Chen Yongqin, Shen Junhao, Pan Jun. Induction of hairy roots of Dioscorea scutellaria and production of diosgenin[J]. Journal of Hubei University (Natural Science Edition), 2009(01):79-83.) , which applied R1601, A4 and other Agrobacterium rhizogenes to induce hairy roots with the stems, leaves and callus of Dioscorea scutellaria as explants, and found that only the callus could be induced to produce hairy roots, and The induction efficiency is low, being 34.5%, and the relevant technology about the hairy root induction of Dioscorea chrysanthemi has not been reported yet

Method used

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  • Method for inducing transgenic hairy roots of dioscorea composita
  • Method for inducing transgenic hairy roots of dioscorea composita
  • Method for inducing transgenic hairy roots of dioscorea composita

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] (1) Cultivation of tissue culture seedlings

[0050] Put the rooted tissue culture seedlings that have grown for about one month on the ultra-clean workbench, remove the roots with a scalpel, and transfer them to MS+0.1mg / L NAA+1.5mg / L 6-BA subculture medium after removing the roots Pre-cultivate for about 2 weeks in a tissue culture room at 25°C, with a light intensity of 2000 lx and a light time of 16 hours per day. Rooted tissue culture seedling materials see figure 1 -a.

[0051] (2) Preparation of genetically modified engineering bacteria

[0052] After two activations, Agrobacterium rhizogenes C58C1 was expanded and cultivated according to the inoculum size of 1%, and the bacterial liquid was cultivated to an absorbance of about 0.6, and then treated with 20mmol / L CaCl 2 Make competent cells, and then use the freeze-thaw method to transform the pCanG-GFP expression vector into the competent cells, culture at 28°C and 180r / min for 3 hours to recover the strains,...

Embodiment 2

[0073] (1) Cultivation of tissue culture seedlings

[0074] Put the rooted tissue culture seedlings that have grown for about one month on the ultra-clean workbench, remove the roots with a scalpel, and transfer them to MS+0.1mg / L NAA+1.5mg / L 6-BA subculture medium after removing the roots Pre-cultivate for about 2 weeks in a tissue culture room at 25°C, with a light intensity of 2000 lx and a light time of 16 hours per day.

[0075] (2) Preparation of transgenic infection bacterial liquid

[0076] Activate the genetically modified engineered bacteria C58C1+pCanG-GFP, and then expand the culture according to the inoculum size of 1%, and detect the absorbance OD of the bacterial solution with a UV spectrophotometer 600 About 0.6, centrifuge to collect the bacteria, then resuspend the bacteria with 1 / 2MS liquid medium containing 200 μmol / L acetosyringone (AS), and use an ultraviolet spectrophotometer to detect the absorbance OD of the resuspension 600 It is suitable to be 0.5-...

Embodiment 3

[0081] The AS concentration optimization of embodiment 3 hairy root induction

[0082]The concentration of AS used to induce hairy roots of Dioscorea chrysanthemum was optimized, and seven AS concentration gradients of 0 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, and 600 μM were designed. Root induction. After infection and co-cultivation in the dark, the explants were transferred to 250 mg / L Cef-MS solid medium for culture. Within the first week of culture, the hairy roots of each experimental group were observed, except for the AS concentration of 0 μM In the experimental group, the remaining 6 experimental groups all began to grow roots. At the third week of cultivation, the number of hair roots and the number of pollution were counted for each experimental group, and the hair root rate and pollution rate of each experimental group were calculated. The results are shown in Table 1. Only the experimental group with an AS concentration of 0 had low pollution, and the rest ...

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Abstract

The invention provides a method for inducing transgenic hairy roots of dioscorea composita. The method comprises the steps of tissue culture seedling cultivation, preparation of transgenic infecting bacterium liquid, infection and co-culture, induction of hairy roots, sterilization culture of the hairy roots and liquid amplification culture of the hairy roots. According to the method for inducingthe transgenic hairy roots of the dioscorea composita, by taking root-removed tissue culture seedlings of the dioscorea composita as explants and agrobacterium rhizogenes C58C1 as an induction strain,the hairy roots can be efficiently induced under the condition of acetosyringone with specific concentration, and the induction rate can reach 50-85%; and meanwhile, the transgenic hairy roots containing different target genes can be induced according to requirements. The method is of great significance for identification of functional genes of the dioscorea composita, research on biological metabolic pathways of dioscin synthesis and the like, molecular breeding, industrial production of secondary metabolites and the like.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a method for inducing transgenic hairy roots of Dioscorea chrysanthemi. Background technique [0002] Dioscorea composita Hemsl. is a perennial twisting herb of the Dioscorea family, native to Mexico, and is one of the main cultivars for the production of diosgenin, a raw material for hormone drugs in Mexico. my country successfully introduced it in Xishuangbanna Tropical Botanical Garden for the first time in 1978. It is characterized by high diosgenin tuber yield and high diosgenin and starch content. It is the preferred raw material for the production of diosgenin and fuel ethanol by fermentation. However, due to the over-exploitation of wild diosgenin resources, its regeneration speed cannot keep up with the market demand, and the output of diosgenin is far from meeting the production demand of the steroid drug industry, and the contradiction between supply ...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/06A01H6/00
CPCC12N15/8205C12N15/8261
Inventor 谢君周建婵钟春梅
Owner SOUTH CHINA AGRI UNIV
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