Sonication-assisted Agrobacterium-mediated transformation of the ACC gene to interfere the production of ethylene in spring Dendrobium cv. ‘Sanya’

Abstract

In the present study, a protocol was developed to obtain stable lines of the spring Dendrobium cv. ‘Sanya’ via Sonication-Assisted Agrobacterium-mediated Transformation (SAAT) of protocorm-like bodies (PLBs). Agrobacterium tumefaciens strain LBA4404 was used with the binary vector YF9078 containing the inverted repeat (IR) of ACC gene (IRACC) for the inhibition of ethylene production, the neomycin phosphotransferase II gene (NPTII) gene as a selectable marker for kanamycin resistance, and an intron-containing β-glucuronidase gene (gus-int) as a reporter gene. PLBs were treated for 10 min with ultrasound (40 kHz) and subsequently inoculated with Agrobacterium suspension for 60 min. The addition of 100 μM acetosyringone (AS) during the preculturing, inoculation and co-cultivation resulted in the highest efficiency of gene transformation (1.04%). Following co-cultivation, PLBs were cultured on selective medium containing 200 mg/l kanamycin and 250 mg/l cefotaxime. Proliferating PLBs were repeatedly selected for kanamycin resistance, and stable transformed lines were generated. Incorporation and expression of transgenes were confirmed by GUS histochemical assay, PCR analysis, Southern blot analysis, and real-time reverse transcription-polymerase chain reaction (RT-PCR). A total of five Southern blot positive transgenic lines were obtained. The ability for ethylene suppression by small potted transgenic plants was measured. The results showed that it was significantly higher in transgenic lines than in untransformed control; different strains exhibited marked variation; and this was related to their cDNA expression.