Study on In vitro Regeneration and GFP Gene Transfer in Lilium longiflorum via Agrobacterium tumefaciens

Abstract

The in vitro regeneration and GFP gene transfer to Lilium longiflorum via Agrobacterium tumefaciens were studied with the aim to determine optimal regeneration medium and influences of technical stages on gene transformation. The combination of 2.4D and 6-benzyladenine (BA) in Murashige and Skoog (1962) basic medium proved effective in inducing callus formation. The MS medium containing BA at 0.5 mg/liter and 2.4D at 0.5 mg/liter was found suitable for callus induction, while the MS basic medium supplemented with 0.25 mg BA/liter, 20 gram sucrose, with pH adjusted to 5.8 before autoclave appeared optimal for shoot induction from callus. To successfully transform GFP gene the callus should be pre-cultured on regeneration medium before co-culture with A. tumefaciens. The callus should then be cut on filter paper, dipped in A. tumefaciens solution for 5 minutes and co-cultivated with A. tumefaciens for 3 days on regeneration medium. GFP gene expression was clear with high expression rate (52.38 to 62.35% in calli, 31.25 to 52.94% in roots and 1.25% in shoots)