High-frequency induction and genetic transformation method for pear hairy roots of Agrobacterium rhizogene-mediated balsam

An Agrobacterium rhizogenes, high-frequency induction technology, applied in the field of plant genetic engineering and cell engineering

Inactive Publication Date: 2011-03-16
毛清黎
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the active ingredients, pharmacological functions and extraction and separation technology of bitter gourd have been comprehensively studied at home and abroad, but the research on the use of Ri plasmid transformation techn

Method used

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Examples

Experimental program
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Embodiment Construction

[0010] Test tube seedling culture: Take balsam pear seeds (green balsam pear and Changbai balsam pear seeds), peel off the seed coat, soak in distilled water for 1 hour, soak in 75% ethanol for 1 minute, rinse the ethanol with sterile water, and then wash with 0.1% HgCl 2 Disinfect for 10 minutes, rinse with sterile water for 5 times, place on MS0 medium for 12h / d and cultivate under light until the seedling height is about 6-8cm (about 1-3w), which can be used as explant material for transformation.

[0011] Activation of bacteria: R1000 Agrobacterium rhizogenes ( Agrobacterium rhizogenes ) single colony, transferred to contain 100mgL -1 Kanamycin YEB culture solution (25-30ml / 100ml Erlenmeyer flask), incubate in the dark at 25°C, 120 rpm for 24-36 h (bacterial solution OD 600 =0.8-1.0, equivalent to 8X10 6 -10 7 bacteria / ml bacterial solution) for infection.

[0012] Transformation of explants:

[0013] Dipping: Bitter gourd aseptic seedling explants (cut into s...

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Abstract

The invention relates to a high-frequency induction and genetic transformation method for pear hairy roots of Agrobacterium rhizogene-mediated balsam, comprising the following steps: selecting R1000 agrobacterium rhizogene strains to infect the stems or scutellum of seedlings in test tubes with the seedling age of 10-20 days, co-culturing in an MS culture medium containing IBA5.0 for 1-2 days to obtain co-cultured explants, and performing bacteria-free culture on the explants on a 1/2MS0 solid medium containing cephalosporin with the concentration of 500mg L<-1> for 8-10 days to obtain the balsam pear hairy roots. The balsam pear hairy roots acquired by the invention has the rooting frequency and rooting density, which respectively reaches 84.2% and 12.3%, and the total content of balsam pear protodioscin in the balsam is 33.43mg/g, which is respectively 69.1%, 48.6% and 49.8% higher than those in the root, stem and leaf of the seedling in a test tube. The method of the invention lays a foundation for breeding new fine-quality and high-yield balsam pear varieties and utilizing Ri plasmid as a vector to perform genetic transformation, and provides an effective path for adopting the balsam pear hairy roots to culture and biosynthesize medical components such as balsam pear saponin and the like.

Description

technical field [0001] The invention belongs to the technical fields of plant genetic engineering and cell engineering. Specifically, it relates to a method for Agrobacterium rhizogenes mediating high-frequency induction and genetic transformation of bitter melon hairy roots. Background technique [0002] Modern research has confirmed that balsam pear saponin is the main active ingredient in balsam pear, which has pharmacological effects such as hypoglycemic, anti-tumor, anti-AIDS, anti-oxidation, anti-fertility, antibacterial, anti-virus, and improving human immunity. Compared with cell culture and callus culture, Agrobacterium rhizogenes ( Agrobacterium rhizogenes ) Ri plasmid transformation induced hairy roots have the characteristics of rapid growth, hormone autotrophy, simple growth conditions, high content of secondary metabolites and stability, high degree of differentiation, and not easy to mutate. compound of. Therefore hairy root culture technology is considered...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H5/06
Inventor 刘仲华毛清黎王坤波杨新河龚雨顺毛雅慧李顺文
Owner 毛清黎
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