Protocorm wounding enhanced Agrobacteriummediated transformation of Hygrochilus parishii (Veitch & Rchb.f.) Pfitz

This study was aimed to encourage the use of protocorm wounding as a substitute ofacetosyringone in an attempt to reduce cocultivation time and to increase possibility of successful genetictransformation of Hygrochilus parishii(Veitch & Rchb.f.) Pfitz. Agrobacterium tumefaciens strain LBA4404harboring the plasmid pBI121 was employed as a transgene carrier. The plasmid pBI121 contained β-glucuronidase (gus) and kanamycin resistance (nptII) genes, both of which were driven by the CaMV35Spromoter. It was found that wounded protocorms cocultivated with A. tumefaciens for 30 min andacetosyringone-treated protocorms cocultivated for 30 min exhibited high gene expressions of 75 and80%, respectively, while normal protocorms (control) showed gene expression of 60% when cocultivatedwith A. tumefaciens for 45 min. The presence of CaMV35S promoter and NOS terminator detected byPCR technique confirmed a successful T-DNA integration. This suggested that protocorm wounding couldbe used as a substitute of acetosyringone in enhancement of genetic modification.