INDUCTION OF IN VITRO FLOWERING OF INDONESIAN WILD ORCHID, Phalaenopsis amabilis (L.) Blume

Abstract

Orchids are generally cultivated for their flower. To induce flower initiation in Phalaenopsis amabilis orchid, genetic and physiological approaches were developed. Genetic modification by insertion of P. amabilis Flowering Locus T (PaFT) gene driven by Ubiquitin promoter into orchid genome using Agrobacterium tumefaciens, whereas physiological approach was conducted by the use of growth regulators: N6­benzyladenine (BA) (1, 3, 9) mg.L-1 or gibberellic acid (GA3) (5, 10, 15) mg.L-1 alone or in combination in culture medium. Orchid seeds were sown on New Phalaenopsis (NP) medium for 8 weeks, then subcultured on NP liquid medium + BA + GA3 with shaking for 9 weeks. Developping protocorms were spread on NP solid medium, then supplemented with NP liquid medium + BA + GA3 (5:2). Cultures were maintained at 25oC with a photoperiod of 8 hrs light/16 hrs dark. For genetic transformation, 3 weeks old protocorms were immersed overnight in cultures of A. tumefaciens with T-DNA harboring Ubipro::PaFT and Hygromycin phosphotransferase (HPT) gene as selectable marker. Phenotypic analysis was carried out from 5-20 plants, each of them was observed for leaf and root number and lengths, comparing with untreated plants. Shoots with normal phenotype were generated from all treatments. RT-PCR analysis from 3 plants each of 4 weeks-24 months old-WT plants, 6 months old phytohormone treated plants and also 12 and 24 months old transgenic plants showed that POH1 juvenile gene transcript can be detected at juvenile stage of WT and PaFT mRNA was expressed in late stage after 6 months old WT plants. In all phytohormone treated plants and transgenic orchid both POH1 anf PaFT transcripts can be detected in 5, 12 and 24-months old plants, but no flower initiation was occurred. It indicates that post transcriptional inhibition might be occurred, and it needs to be explored. Keywords : in vitro, flower, PaFT, growth regulators, Orchids