In vitro regeneration and Agrobacterium mediated transformation in gladiolus

In vitro regeneration and transformation studies were conducted on two cult ivars of gladiolus. Cormels of 1.0 to 1.5 cm diameter cut into 2-3 mm thick slices of top, middle and bottom, and in vitro derived bisected shoot tips were used as explants on MS medium supplemented with 18.6 mu M kinetin for multiple shoot induction. Amongst the cormel slices, the top slice gave be tter shoot induction response of 89% with an average of 2.4 shoots per expl ant over both cultivars. In vitro derived bisected shoot tips were inoculat ed on the medium oriented cut-side up, cut-side down and vertically both wi th and without the cormel base attached. Bisected shoot tips without attach ed cormel base and inoculated in the cut-side down orientation showed an av erage of 90% shooting response. In vitro derived shoot tips were used as ex plants for transformation. Explants were wounded by scalpel and particle bo mbardment with 1.6 mu m naked gold particles by the biolistic delivery syst em. The wounded explants, after 3 d of recovery period, were cocultivated w ith Agrobacterium strain LBA4404 harbouring the binary vectors pBI141 and p TOK233 which contained gus reporter gene with rice actin and 35S promoters respectively. GUS expression frequencies of 5.3% and 23% was obtained from scalpel and particle bombardment wounded explants, respectively. Particle w ounded explants showed an average of 63 and 103 GUS spots when co-cultivate d with pBI141 and pTOK233 binary vectors respectively. Explants co-cultivat ed with pBI141, after three weeks of selection on antibiotic containing med ium showed blue streaks of GUS expression. It was concluded that Agrobacter ium could infect the monocot gladiolus and transform the tissue efficiently when tissues were prewounded with naked gold particles delivered by partic le gun.