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

Abstract. This study was aimed to encourage the use of protocorm wounding as a substitute of acetosyringone in an attempt to reduce cocultivation time and to increase possibility of successful genetic transformation of Hygrochilus parishii (Veitch & Rchb.f.) Pfitz. Agrobacterium tumefaciens strain LBA4404 harboring the plasmid pBI121 was employed as a transgene carrier. The plasmid pBI121 contained fi-glucuronidase (gus) and kanamycin resistance (nptII) genes, both of which were driven by the CaMV35S promoter. It was found that wounded protocorms cocultivated with A. tumefaciens for 30 min and acetosyringone-treated protocorms cocultivated for 30 min exhibited high gene expressions of 75 and 80%, respectively, while normal protocorms (control) showed gene expression of 60% when cocultivated with A. tumefaciens for 45 min. The presence of CaMV35S promoter and NOS terminator detected by PCR technique confirmed a successful T-DNA integration. This suggested that protocorm wounding could be used as a substitute of acetosyringone in enhancement of genetic modification.