Agrobacterium tumefaciens-mediated barley leaf base transformation method

An Agrobacterium-mediated, Agrobacterium-based technology, applied in biochemical equipment and methods, botany equipment and methods, genetic engineering, etc., can solve problems such as differences and high technical requirements

Inactive Publication Date: 2013-12-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, although shoot tip transformation does not require callus induction, it requires...

Method used

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  • Agrobacterium tumefaciens-mediated barley leaf base transformation method
  • Agrobacterium tumefaciens-mediated barley leaf base transformation method
  • Agrobacterium tumefaciens-mediated barley leaf base transformation method

Examples

Experimental program
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Embodiment 1

[0076] Embodiment 1: Construction of plasmid vector and transformation of Agrobacterium tumefaciens

[0077] Carrier used in the present invention is PMBL-9 (at the specification page 13 of patent application number is 201110257189.9 patent document and is attached figure 2 In the final expression plasmid, it is named PMBL-9, and the construction of the expression plasmid refers to the applicant's patent application "A method for transformation of barley shoot tip mediated by Agrobacterium tumefaciens", the patent application number is 201110257189.9, the publication number It is CN102304544A, the publication date is 2012.01.04).

[0078] Transformation of Agrobacterium tumefaciens: the PMBL-9 vector was used to transform the Agrobacterium GV3101 strain by electroporation (Agrobacterium GV3101 strain was purchased from Invitrogen Company, and the electroporation transformation method was referred to the method reported by Sambrook et al., see: Sambrook et al., Molecular Clonin...

Embodiment 2

[0079] Example 2: Callus Induction Using Barley Leaf Cut Sections as Explants

[0080] 1. Sterilization of barley kernels and germination of mature embryos

[0081]Select healthy and plump barley grains (the variety is E 32380, E Beer No. 2 (gifted by Hubei Academy of Agricultural Sciences)), remove the chaff, soak in 70% alcohol for 2 minutes, then soak in 0.1% mercury liter for 15 minutes, pour off the alcohol and mercuric chloride solution, soak the seeds with sterilized water for 3-4 times, 3-5 minutes each time; soak the sterilized seeds in sterile water, and place them at room temperature for 5-10 hours. Pour off the sterilized water in the ultra-clean bench, peel off the complete mature embryos, put the scutellum down, and place them in MS basic medium (Murashige T, Skoog F.1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15, 473-497) at 25°C in the dark for 3 days.

[0082] 2. Cut barley leaf base to induce ca...

Embodiment 3

[0084] Example 3: Agrobacterium-mediated genetic transformation of barley

[0085] 1. Hypertonic treatment of callus

[0086] The calli cultured for 27 days were transferred to the hyperosmotic medium, and hyperosmotic treatment was performed at 25°C for 4 hours.

[0087] 2. Infection with Agrobacterium

[0088] 1 mL of preserved Agrobacterium liquid containing PMBL-9 plant expression vector was inoculated in 50 mL of YEB medium, which was supplemented with 100 mg / L rifampicin (Rif), 100 mg / L carbenicillin (Carb), 25 mg / L carbenicillin Namycin (Kan), 2mmol / LMgSO 4 medium, 28°C, 200r / min dark culture to OD 600 = about 0.8, then centrifuge at 5000r / min at 4°C, collect the bacteria, and resuspend the liquid (that is, the resuspension) to OD 600 = about 0.6.

[0089] The hyperosmotically treated callus was transferred to the resuspension solution, and then placed on a decolorizing shaker at 100 r / min for 30 minutes for transformation. 500-600 calluses are transformed each ti...

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Abstract

The invention belongs to the technical field of plant transgenosis, and particularly relates to an agrobacterium tumefaciens-mediated method for obtaining a transgenic plant by adopting a barley seedling leaf base to induct a callus. The agrobacterium tumefaciens-mediated method has the key points as follow: a barley leaf base serves as an explant to induct the callus; an agrobacterium tumefaciens-mediated genetic transformation method is adopted, a Bar gene is used as a screening gene, and a herbicide of glufosinate ammonium is used as a selective agent; the transformed callus and differentiated plants are screened; a foreign gene is confirmed to be integrated into a barley genome through PCR and Southern hybridization analysis; a transgenic barley plant is obtained. Compared with the prior art, the invention has the advantage that the explant (callus) of the transgenic plant can be supplied all the year round; compared with a classic transformation method in which a young embryo is taken as an acceptor, the agrobacterium tumefaciens-mediated method has the advantage that the acceptor obtaining time is shortened to be 3 days form 150 days, and the time limit of young embryo sampling is eliminated; compared with a stem tip transformation method, the agrobacterium tumefaciens-mediated method is lower in technical requirement and simpler to operate.

Description

technical field [0001] The invention belongs to the technical field of plant transgenesis, and in particular relates to a method for inducing callus using barley leaf base as an explant material and cultivating transgenic plants mediated by Agrobacterium tumefaciens. The invention relates to a method for preparing barley leaf-based materials and inducing callus, which is mediated by Agrobacterium tumefaciens and used for genetic transformation of barley cultivars. Background technique [0002] Barley is one of the most important crops in the world. Its planting area and yield are second only to rice, wheat and corn, and it ranks fourth among cereal crops. It is mainly used as food, feed, raw materials for beer industry, raw materials for pharmaceutical industry and health care food etc. The genetic improvement of barley has been paid much attention. [0003] Tingay et al. successfully transformed barley immature embryos with Agrobacterium-mediated method for the first time...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H5/00
Inventor 廖玉才李和平黄涛杨鹏程伟
Owner HUAZHONG AGRI UNIV
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