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

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, Orchid

Early detection of the orchid flowering gene PaFT1 in tobacco cells using a GFP reporter

Here we describe a novel method of using green fluorescence protein (GFP) as a reporter gene for early detection of an integrated T­DNA containing the orchid flowering gene, PaFT1 (Phalaenopsis aphrodite Flowering locus T1) in the tobacco genome. Functional assays that report the presence of exogenous DNA early in development are especially useful in plants where the desired phenotype is only apparent after long periods of vegetative growth. The objective of this study is to establish a method for detecting an inserted Phalaenopsis orchid flowering gene and examining its function in tobacco. The p35S::PaFT1­ 35S::GFP construct was introduced into Agrobacterium tumefaciens strain EHA101. Transformed tobacco leaves were cultured on MS medium with addition of 1 mgL-1 NAA+3 mgL-1 BAP+50 mgL-1 Kanamycin+300 mgL-1 timentin for selection. Results showed bright green GFP fluorescent signals in 11 out of 15 (73%) tobacco leaf cells at a 2­month time point after transformation. GFP and PaFT1 fragments were amplified in genomic PCR using GFP and PaFT1 specific primers. The accumulated PaFT1 transcripts were observed in 3 month­old transgenic tobacco plants containing p35S::PaFT1­35S::GFP. Green florescence was observed only in the transgenic plants at the 5 month­old stage but not in the wild type controls