Abstract
An initial step in the genetic transformation of Phalaenopsis violacea orchid species was investigated in the plant-Agrobacterium interaction. Agrobacterium tumefaciens strains EHA 101 and 105, harboring the pCAMBIA 1304 plasmid contains gfp gene as the reporter gene marker, were used in this transformation study. The spectrophotometric GFP assay provides information on the amount of inoculated Agrobacterium tumefaciens that effectively bound to various orchid tissues. Different temperatures during co-cultivation period, the concentration of Lcysteine, calcium (CaCl2) and silver nitrate (AgNO3) in co-cultivation medium during cocultivation period were identified to be major and important factors in enhancing the increase percentage of transient gfp gene expression in PLBs. Agrobacterium tumefaciens EHA 105 was proved to be better bacterial strain in transforming the targeted PLBs than EHA 101, based on the notably higher transient expression of gfp gene in all the optimization parameters that were tested. Highest T-DNA delivery efficiencies were obtained when P. violacea PLBs were co-cultivated with Agrobacterium tumefaciens strain EHA 105 in half-strength MS medium supplemented with 5% of banana cultivar, Mas extract containing 200 mgL-1 L-cysteine, 60µM silver nitrate, without calcium in the medium during co-cultivation in the dark condition at 24°C. The results from the transient gfp gene expression of PLBs suggested that Agrobacterium-mediated transfer of T-DNA to the naturally recalcitrant P. violacea is feasible and is highly efficient. Hence, the use of the gfp marker gene during in vitro screening of the transgenic cells has enabled the visual selection of orchid transformed by Agrobacterium tumefaciens at higher frequency rates.
Highlights
Phalaenopsis violacea have greenish white flowers with purple pigment around the sepals and the lip
Agrobacterium is able to attach to different types of P. violacea orchid cells based on the Green fluorescent protein (GFP) spectrometric assay
Once Agrobacterium reaches the neighbourhood of wounded tissues, the step required for the development of plant tumors is its attachment to plant cells
Summary
Phalaenopsis violacea have greenish white flowers with purple pigment around the sepals and the lip. P. violacea are native to the peninsular Malaysia and are closely related to P. bellina of the Borneo. P. violacea are important parental varieties to produce novel Phalaenopsis hybrids with special fragrance. In order to improve the quality, modern molecular biology techniques could be applied to transfer desired gene(s) into orchid genome instead of using conventional breeding method which is time consuming and lack of genetic variability. Agrobacterium tumefaciens mediated gene transfer system, microprojectile bombardment and direct gene transfer into the orchid protoplast, are the three major gene transformation methods applied in orchid biotechnology. Agrobacterium mediated transformation has several advantages over other direct methods such as the transfer of relatively large segments of DNA with little rearrangement and the integration of low copy numbers of T-
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