Sonication assisted Agrobacterium-mediated transformation of chalcone synthase (CHS) gene to Spring Dendrobium cultivar ‘Sanya’

Abstract

In this study, a protocol was developed to obtain stable lines of the Spring Dendrobium cultivar ‘Sanya’ via sonication assisted Agrobacterium-mediated transformation (SAAT) of protocorm-like bodies (PLBs). Agrobacterium tumefaciens strain LBA4404 was used with the binary vector AG205 containing the chalcone synthase (CHS) gene for flower color change and the neomycin phosphotransferase II gene (NPTIIgene) as a selectable marker for kanamycin resistance. PLBs were treated for 10 min using ultrasound (40 KHz) and subsequently immersed in the Agrobacterium suspension for 60 min. The addition of 100 M acetosyringone (AS) during the pre-culture and co-culture achieved the highest efficiency of gene transformation (0.5%). Following co-cultivation, PLBs were cultured on selective medium containing 200 mgL^-1 kanamycin and 250 mgL^-1 cefotaxime. Proliferating PLBs were repeatedly selected for kanamycin resistance and stable transformed lines were generated. Incorporation and expression of transgenes were confirmed by PCR analysis, southern blot analysis and reverse transcription-quantitative real-time PCR (RT-PCR). A total of five southern blot positive lines were obtained from co-cultivated PLBs.

Key words: Spring Dendrobium cultivar ‘Sanya’, chalcone synthase (CHS) gene, transformation, sonication assisted Agrobacterium-mediated transformation (SAAT), protocorm-like bodies (PLBs).