Indirect somatic embryogenesis and <i>Agrobacterium-</i>mediated transient transformation of ginger (<i>Zingiber officinale</i> Rosc.) using leaf sheath explants

Abstract

This study has established an efficient and simple Agrobacterium-mediated transient transformation method for a valuable spice crop ‘ginger’ via indirect somatic embryogenesis. Leaf sheath explants were transformed using Agrobacterium tumefaciens strains EHA105 and LBA4404 harbouring vector pGFPGUSPlus containing β-glucuronidase (gus) reporter and hygromycin phosphotransferase (hptII) selection marker. Different parameters enhancing agro-infection were investigated to optimise transformation efficiency. High transformation frequency was achieved when explants were co-cultivated with bacterial cell density of 0.6 OD600 on medium containing 150 µM acetosyringone for 2 days, followed by selection regime of 40 mg/l hygromycin. Histochemical GUS analysis confirmed the transient expression of gus gene, which was further confirmed by PCR analysis of hptII and nptII genes specific primers. The origin and developmental stages of somatic embryos from leaf sheath explants were investigated with the help of scanning electron micrographs. This newly developed transient transformation system provides a basis for quick expression of marker genes in regenerated plantlets.Abbreviation: GUS - β-glucuronidase; PCR - Polymerase chain reaction; LB - Luria broth; BA - 6- Benzylaminopurine; 2,4-D - 2,4-Dichlorophenoxyacetic acid; TDZ - Thidiazuron; NAA - 1-Naphthaleneacetic acid