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An Agrobacterium-mediated method for cultivating transgenic willow plants

An Agrobacterium-mediated, transgenic technology, applied in botany equipment and methods, horticultural methods, genetic engineering, etc., can solve the problems of inability to obtain willow transgenic plants, inability to induce resistant buds, etc., and achieve high transformation rate and short induction time Short, fast-growing buds

Inactive Publication Date: 2014-10-08
NORTHEAST FORESTRY UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] The present invention aims to solve the problem that the existing method cannot induce resistant buds from the transgenic callus of willow, so the transgenic plant of willow cannot be obtained, and provides a method for cultivating transgenic willow plant mediated by Agrobacterium

Method used

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  • An Agrobacterium-mediated method for cultivating transgenic willow plants
  • An Agrobacterium-mediated method for cultivating transgenic willow plants
  • An Agrobacterium-mediated method for cultivating transgenic willow plants

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specific Embodiment approach 1

[0020] Specific embodiment one: the method for cultivating transgenic Salix sativa plant mediated by Agrobacterium in the present embodiment is carried out according to the following steps:

[0021] 1. Sterilize the mature willow seeds, then inoculate the sterilized seeds in the pre-cultivation medium and cultivate them for 4-8 days to obtain germinated seeds;

[0022] 2. Pick a single Agrobacterium tumefaciens colony carrying the pBI121 vector, insert it into LB liquid medium containing 50mg / L kanamycin, and culture it overnight at 28°C to obtain OD 600 0.2-1.2 Agrobacterium liquid;

[0023] 3. The germinated seeds obtained by pre-cultivation in step 1 are excised from the base to germinate the terminal bud position, and 2 cotyledons are reserved, and the germinated seeds with excised terminal buds are used as explants, and the explants are placed in step 2 for preparation The OD600 is 0.2-1.2 Agrobacterium bacteria solution soaked for 30-40 minutes;

[0024] 4. Then the ex...

specific Embodiment approach 2

[0030] Specific embodiment two: This embodiment is a further description of the pre-culture medium in step one of the method for cultivating transgenic Salix salix plants mediated by Agrobacterium described in specific embodiment one, and the pre-culture medium described in step one is MS medium containing 1.0 mg / L zeatin, 0.1 mg / L naphthaleneacetic acid, 30 g / L sucrose and 8 g / L agar.

specific Embodiment approach 3

[0031] Specific embodiment three: This embodiment is a further description of the method for disinfecting mature willow seeds in step one of the method for cultivating transgenic willow plants mediated by Agrobacterium described in specific embodiment one. The method for disinfecting mature willow seeds is as follows: soak mature willow seeds in an ultra-clean workbench with an ethanol solution with a volume concentration of 70% for 30 seconds, rinse with distilled water twice, and then transfer them to a 1% volume concentration. Soak in sodium hypochlorite solution for 10 minutes, rinse with distilled water 5 times.

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Abstract

The invention discloses a method for culturing agrobacterium-mediated transgenic salix matsudana plants and relates to the method for culturing the transgenic salix matsudana plants. The method aims to solve the problems that the existing method cannot induce resistant buds from transgenic transgenices of salix matsudana so that the transgenic salix matsudana plants cannot be obtained. The method comprises the following steps of: firstly, sterilizing and preculturing salix matsudana seeds; secondly, selecting agrobacterium tumefaciens with pBI121 carriers, and culturing to obtain a bacteria solution; thirdly, taking sprouted top bud parts cut from basic parts as explants, and then soaking in the bacteria solution; fourthly, taking out the explants and soaking up through filter papers, and inoculating the explants into a co-culture culture medium; fifthly, switching to a selective medium till the resistant buds grow, switching to a light condition, and screening the resistant buds; sixthly, transferring the resistant buds into an elongation culture medium; seventhly, cutting off stem sections, and switching to a rooting culture medium for culture till roots grow; and finally, carrying out polymerase chain reaction (PCR) and glucuronidase (GUS) staining detection, transplanting to earth, and obtaining the transgenic salix matsudana plants. The method for culturing the agrobacterium-mediated transgenic salix matsudana plants is applied to culturing the transgenic salix matsudana plants.

Description

technical field [0001] The invention relates to a method for cultivating transgenic dry willow plants. Background technique [0002] There are hundreds of species of willows in the world, mainly distributed in the northern hemisphere, especially in the northern temperate zone and arctic regions. Many willow tree species have strong adaptability, large biomass, fast growth, water and humidity resistance, and high absorption capacity for heavy metals, organic matter and eutrophication. Facing the increasingly serious problems of energy depletion and environmental pollution, the economic importance of willow for pollution control and energy production is increasing day by day. However, willow still has certain limitations for soil remediation. For example, willow is not a hyperaccumulator plant, has a long cycle, and heavy metals are mainly concentrated in the roots, which is not good for absorption. In view of these limitations, the application of transgenic breeding technol...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84A01H4/00A01H5/00
Inventor 李成浩杨静莉许雪梅
Owner NORTHEAST FORESTRY UNIVERSITY
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