关键词: 药用大黄(Rheum officinale Baill.), 遗传转化, 叶柄, GUS染色, GFP荧光检测

Abstract: An efficient and stable genetic transformation system for Rheum officinale Baill. was established. Using aseptic seedlings as explant materials, key parameters including explant type for infection, hormone ratios in the co-culture medium, and kanamycin concentration for transformant selection were optimized. The results showed that calli induced from petioles were suitable for Agrobacterium-mediated transformation. After infection, the explants were cultured on co-culture medium (MS + 2.0 mg/L 6-BA + 0.8 mg/L NAA + 0.5 g/L CH + 33 g/L Sugar + 6.5 g/L Agar,pH=5.9) in darkness at (23 ± 2) ℃ for 2 days. The co-cultured calli were then transferred to selection medium (MS + 0.8 mg/L NAA + 33 g/L Sugar + 6.5 g/L Agar + 25 mg/L Kan + 250 mg/L Carb + 250 mg/L Tim,pH=5.6) for resistance screening, from which adventitious buds of Rheumofficinale were successfully regenerated. These buds were excised at the base and inoculated onto proliferation medium (MS + 0.8 mg/L NAA + 1.6 mg/L KT + 33 g/L Sugar + 6.5 g/L Agar,pH=5.9) for propagation, yielding resistant plantlets. Putative transgenic plants were verified at multiple levels via GUS histochemical staining, PCR amplification, and qRT-PCR analysis. After rooting, acclimatization, and transplantation, the positive plants could be applied in industrial production. Based on GFP fluorescence detection, the positive transformation efficiency of this optimized system reached 12.5%.

Key words: Rheum officinale Baill., genetic transformation, petiole, GUS histochemical staining, GFP fluorescence detection