The effect of Agrobacterium densities and inoculation times on gene transformation efficiency in rubber tree

Abstract

Rubber tree belonging to the genus Hevea is an economically important crop of Thailand and South-east Asia. To optimize its agronomical trait for glyphosate-resistant, in vitro gene transformation through Agrobacterium tumefaciens was investigated. The bacteria carrying plasmid pCAMBIA 1304, harboring gus as screenable marker genes and EPSPs gene was used. The shoot tips were immersed in A. tumefaciens suspension at optical densities (OD₆₀₀) at 0.3, 0.6 and 0.9 for various times (15, 30 and 60 min). The results revealed that shoot explants immersed in A. tumefaciens suspension at OD₆₀₀ of 0.6 for 30 min gave the higher survival rate after being cultured on glyphosate containing MS medium for one and half months. Assessment of transformed shoots revealed positive results in GUS histochemical assay. The presence of the gus and EPSPs genes in transformed rubber tree were confirmed by polymerase chain reaction (PCR) technique, dot blot hybridization and Southern PCR hybridization. Specific primers for the gus and EPSPs genes were designed to amplify a 919 and 1,600 bps DNA fragment, respectively.

Keywords: Transgenesis, glyphosate, inoculation time, Agrobacterium density, Hevea

African Journal of Biotechnology, Vol 13(23) 2321-2329