Method for establishing agrobacterium-mediated high-efficient transformation system of Lanzhou lily

An Agrobacterium lily, mediated technology, applied in the field of genetic engineering, to achieve the effects of increasing Vc content and enhancing stress resistance

Inactive Publication Date: 2010-06-23
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method effectively overcomes the defect that the existing Agrobacterium-me

Method used

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  • Method for establishing agrobacterium-mediated high-efficient transformation system of Lanzhou lily
  • Method for establishing agrobacterium-mediated high-efficient transformation system of Lanzhou lily
  • Method for establishing agrobacterium-mediated high-efficient transformation system of Lanzhou lily

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Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1 tissue culture seedling scale preparation

[0033] Wash off the soil on the surface of lily bulbs, peel off the outer scales with lesions or mechanical damage, take the middle and outer layers without lesions, and soak them in detergent, then rinse them under tap water for about 12 hours, and then use them before sterilization. Wash with distilled water for 3 times, disinfect with 70% ethanol for 40 seconds on the ultra-clean bench, put into 0.1% mercuric solution for disinfection for 15 minutes, rinse with sterile water for 5 times, cut the sterilized scales into 1.0~2.0cm×1.0~2.0cm Cubes were inoculated upside down on the differentiation medium, and cultivated at 26° C., 2000 lx, 16 hours per day, and light for 30 to 60 days to obtain scales of tissue culture seedlings.

Embodiment 2

[0035] 1) Cut out the scales of the tissue culture seedlings and culture them in the dark for 2 days on the differentiation medium 1 / 2MS+2.0mg / LBA+0.2mg / LNAA+30g / L sucrose+6g / L agar as the acceptor material, and the culture condition is 26°C;

[0036] 2) Inoculate the donor strain Agrobacterium EHA105 into kanamycin 50mg / L, streptomycin 30mg / L and rifampicin 40mg / L, overnight at 28°C on a constant temperature shaker at 200rpm, and collect the bacterial solution in In a 10ml centrifuge tube, centrifuge at 5000rpm for 10 minutes at room temperature, pour off the supernatant, dissolve and dilute the precipitate with liquid MS, and carry out infection transformation when the concentration OD600 is 0.4, and add 20mM acetosyringone (AS) at the same time;

[0037] 3) Put the scales pre-cultured for two days into the liquid MS containing the bacteria solution, shake on the shaker at 200 rpm at 28°C for 20 minutes, put the infected scales on the filter paper to blot dry, and then inocul...

Embodiment 3

[0040] 1) The scales of the tissue culture seedlings were cut and cultured in the dark for 2 days on the differentiation medium as the receptor material, and the culture condition was 26°C;

[0041] 2) Inoculate the donor strain Agrobacterium EHA105 into 150mg / L, 70mg / L streptomycin and 800mg / L rifampicin, overnight at 28°C on a constant temperature shaker at 200rpm, and collect the bacterial solution into a 10ml centrifuge tube , centrifuge at 5000rpm at room temperature for 10 minutes, discard the supernatant, dissolve and dilute the precipitate with liquid MS, and carry out infection transformation when the concentration OD600 is 0.6, and add 20mM acetosyringone at the same time;

[0042] 3) Put the scales pre-cultured for two days into the liquid MS containing the bacteria solution, shake on the shaker at 200 rpm at 28°C for 20 minutes, put the infected scales on the filter paper to blot dry, and then inoculate them with 20mg / L AS and 200mg / L cephalosporin in the different...

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Abstract

The invention discloses a method for establishing an agrobacterium-mediated high-efficient transformation system of Lanzhou lily, which comprises the steps of cutting scales of tissue culturing seedlings, carrying out dark culture on a differentiation culture medium for 2 days, and being used as a receptor material; inoculating a donor strain onto an LB liquid culture medium with kanamycin, streptomycin and rifampicin, collecting bacterial liquid, centrifugalizing, tossing away supernatant liquid, using liquid MS for dissolving the sediment and diluting till the concentration that OD600 is 0.4-0.6, adding into the liquid Ms, adding the receptor material for infection, placing the scales after the infection on filter paper for drying by absorption, inoculating onto the differentiation culture medium containing 20mg/L AS and 200mg/L cefotaxime for carrying out the dark culture for 3d, then inoculating onto the differentiation culture medium containing 2mg/L representative hygromycin, and carrying out the dark culture at 26 DEG C till the outgrowth of resistant shoots; and further screening and culturing the resistant shoots, carrying out rooting culture when 4-5 leaves are grown, and then transplanting. The method opens up a new way for culturing good rice varieties.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a method for establishing a high-efficiency transformation system mediated by Agrobacterium lanzhou lily. Background technique [0002] Lanzhou lily (Lilium davidii var. unicolor) is a variety of Lilium davidii var. unicolor, also known as king lily, sky lily and flat land lily. It is a perennial bulbous herb. The cultivation of Lanzhou lily began in the Tongzhi period of the Qing Dynasty, about 130 years ago. Due to the unique soil conditions and the cultivation techniques accumulated over the years, the bulbs are huge, white in color, plump and tender in scale, delicate in texture, rich in nutrition, and sweet in taste. ; Its flowers are solitary, large and beautiful, rich in fragrance, and have high edible, medicinal, health care and ornamental values. It is not only the top grade of Chinese lilies, but also the only sweet lily in China. Professor Kong ...

Claims

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Application Information

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IPC IPC(8): C12N15/82
Inventor 马锋旺师守国冯凤娟李永红李善菊梁东王爱莲
Owner NORTHWEST A & F UNIV
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