A method for transformation of barley leaf base mediated by Agrobacterium tumefaciens
A technology based on Agrobacterium tumefaciens and Agrobacterium tumefaciens, which can be used in biochemical equipment and methods, botanical equipment and methods, genetic engineering, etc., and can solve problems such as high technical requirements and differences
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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 electric shock (Agrobacterium GV3101 strain was purchased from Invitrogen Company, and the electric shock transformation method was referred to the method reported by Sambrook et al., see: Sambrook et al., Molecular Cloning:...
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 on MS basic medium (MurashigeT, SkoogF.1962.Arevisedmediumforrapidgrowthandbioassayswithtobaccotissuecultures.PhysiologiaPlantarum, 15, 473-497), 25°C, dark Under culture 3d.
[0082] 2. Cut barley leaf base to induce callus
[0083] Ge...
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 kana Mycin (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 time. Ultrason...
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