Ixora S. Mercuriani

Overexpression of PaFT gene in the wild orchid Phalaenopsis amabilis (L.) Blume

To shorten vegetative stage and induce transition from vegetative to reproductive stage in orchids, we overexpressed Phalaenopsis amabilis Flowering LocusT (PaFT) gene under the control of Ubiquitin promoter into protocorm of Indonesian Wild Orchid Phalaenopsis amabilis (L.) Blume. The dynamic expression of vegetative gene Phalaenopsis Homeobox1 (POH1) and flowering time gene PaFT has been analyzed. Accumulation of mRNA was detected in shoot and leaves of both transgenic and non transgenic plants by using Reverse transcriptase-PCR (RT-PCR) with specific gene primers for POH1 and PaFT in 24 months old plants. To analyze the POH1 and PaFT genes, three pairs of degenerate primers PaFT degF1R1, F2R2 and F3R3 that amplified 531 bp PaFT cDNA were used. We detected 700 bp PaFTcDNA from leaves and shoots of transgenic plants, but not in NT plants. POH1 mRNA was detected in plants. PaFT protein consists of Phospatidyl Ethanolamine-Binding Protein (PEBP) in interval base 73-483 and CETS family protein at base 7-519, which are important motif for transmembrane protein. We inserted Ubipro::PaFT/pGAS101 into P. amabilis protocorm using Agrobacterium. Analysis of transgenic plants showed that PaFTmRNA was accumulated in leaves of 12 months after sowing, although it is not detected in non transgeic plants. Compare to the wild type (NT plants), ectopic expression of PaFT shows alter phenotype as follows: 31% normal, 19% with short-wavy leaves, 5% form rosette leaves and 45% produced multishoots. Analysis of protein profiles of trasgenic plants showed that a putative PaFT protein (MW 19,7 kDa) was produced in 1eaves and shoots.This means that at 12 months, POH1 gene expression gradually decreased/negatively regulated, the expression of PaFT gene was activated, although there is no flower initiation yet. Some environmental factors might play a role to induce inflorescens. This experiment is in progress.

Dynamic expression of POH1 gene in shoot development during in vitro culture of Phalaenopsis orchid

Phalaenopsis Orchid Homeobox1 (POH1) gene is a KNOTTED1 homologous gene from Indonesian wild orchid Phalaenopsis amabilis. As a member of homeobox gene family in orchid, POH1 may function in the initiation and development of shoot in orchid. To understand the roles of POH1 gene, we analyzed the expression and function of POH1 gene during in vitro culture of P. amabilis orchids. The method was conducted by growing seeds of P. amabilis orchids on New Phalaenopsis (NP) medium to grow protocorms, and then in turned it grew into plantlets. Phenotypic characters such as a number of shoots, number and size of leaves were analysed in (4, 8, 16 and 24) wk after sowing (WAS) plantlets. Molecular analysis including the expression of POH1 gene was analysed by Reverse Transcriptase (RT)-PCR using POH1F1R1 primers on various ages of (4, 8, 16 and 24) WAS plantlets following by protein profile analysis using 10 % SDS-PAGE. The results showed that during in vitro condition, embryos were developed into a single and multip...

In Planta Transformation Method for T-DNA transfer in Orchids

Transgenic plant technology is an efficient tool to study the function of gene(s) in plant. The most popular and widely used technique is Agrobacterium-mediated transformation in which cocultivation was done by immersing the plant tissues/organ in overnight bacterial cultured for about 30 minutes to one hour under in vitro condition. In this experiment, we developed more easier technique that omitted the in vitro step during cocultivation with Agrobacterium, namely in planta transformation method. Pollinaria (compact pollen mass of orchid) of Phalaenopsis amabilis and Spathoglottis plicata orchids were used as target explants that were immersed into bacterial culture for 30 minutes, then dried up the pollinaria, the transformed pollinaria was used to pollinate orchid flowers. The T-DNA used for this experiments were Ubipro∷PaFT/A. tumefaciens GV3101 for P. amabilis and MeEF1α2 pro∷GUS/ A. tumefaciens LBA 4404 for S.plicata. Seeds that were produced from pollinated flowers were grown onto 10 mg/l hygromici...