Encapsulation-based a novel antibiotic selection technique for Agrobacterium-mediated genetic transformation of Dendrobium Broga Giant orchid

Abstract

Dendrobium Broga Giant (Dendrobium Bobby Messina × Dendrobium superbiens) is a new hybrid orchid in Malaysia. This study has established encapsulation based a novel antibiotic selection technique to determine the minimal inhibitory concentration of potential antibiotic for Agrobacterium-mediated transformation of Dendrobium Broga Giant (DBG) orchid. For determination of minimal inhibitory concentration, two steps of antibiotic selection techniques were used in this experiment such as potential antibiotic was selected before encapsulation and minimal inhibitory concentration was determined after encapsulation. The effectiveness of three antibiotics (kanamycin, geneticin and neomycin) with their different concentrations were evaluated as a selection agent for transformation studies. The effectiveness was determined by identifying the minimal concentration of the selection agent required to fully inhibit the growth of Dendrobium Broga Giant orchid PLBs. Non transformed PLBs were cultured on hormone-free ½MS selection medium containing varying concentrations (0–300 mg/l) of the antibiotics. The growth of PLBs were recorded every week up to four weeks. Among the selection agents evaluated, geneticin proved to be the most effective antibiotic which could inhibit the growth of PLBs at 50 mg/l. Neomycin requires 250 mg/l for inhibition of all PLBs after four weeks of cultured but Kanamycin is the least effective as it only inhibits 80% of the PLBs grown at 300 mg/l. For stringent minimal inhibitory concentration of antibiotic selection, encapsulated PLBs were cultured on similar concentrations of geneticin containing ½MS selection media and growth of PLBs evaluated every week up to four weeks of cultured. It was observed that 50 mg/l geneticin fully inhibited PLBs after two weeks. Hence, encapsulation based novel antibiotic selection techniques revealed that geneticin is the potential antibiotics and 50 mg/l concentration is the minimal inhibitory concentration for Dendrobium Broga Giant orchid PLBs.