PJB-2018-239
Efficient Genetic Transformation and Post-Infection Regeneration System of Alfalfa Mediated by Agrobacterium Tumefaciens
Yi Shi
Abstract
The hypocotyl segments of three alfalfa varieties Gannong No.1 (G1), Gannong No.3 (G3) and Xinjiang Big Leaf (XB), were chosen as recipients of Agrobacterium-mediated transformation of the lysozyme (LYZ) gene. Kanamycin at 75 mg L-1 was suitable for the selection of transformed tissue of G1 and XB, and 50 mg L-1 was suitable for G3. Cefotaxime at 250 mg L-1 efficiently inhibited bacterial growth for the G3 and XB, while 350 mg L-1 was required for G1. The alfalfa explants were incubated in Agrobacterium solution (OD600=0.4) for 10 minutes on a shaker set at 100 rpm at room temperature in preparation for transformation. Expression of the green fluorescent protein (GFP) and LYZ genes was tested to confirm the transformation efficiency of this system by PCR and Southern blot. Disease resistance of transgenic plants was increased against the pathogen Rhizoctonia solani.
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