The presented study established Agrobacterium-mediated genetic transformation using protocorm-like bodies (PLBs) for the production of transgenic Vanda Kasem's Delight Tom Boykin (VKD) orchid. Several parameters such as PLB size, immersion period, level of wounding, Agrobacterium density, cocultivation period, and concentration of acetosyringone were tested and quantified using gusA gene expression to optimize the efficiency of Agrobacterium-mediated genetic transformation of VKD's PLBs. Based on the results, 3-4 mm PLBs wounded by scalpel and immersed for 30 minutes in Agrobacterium suspension of 0.8 unit at A 600nm produced the highest GUS expression. Furthermore, cocultivating infected PLBs for 4 days in the dark on Vacin and Went cocultivation medium containing 200 𝜇M acetosyringone enhanced the GUS expression. PCR analysis of the putative transformants selected in the presence of 250 mg/L cefotaxime and 30 mg/L geneticin proved the presence of wheatwin1, wheatwin2, and nptII genes.

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Physical contact between A. tumefaciens and the target plant cell walls is essential to transfer and integrate the transgene to introduce a novel trait. Chemotaxis response and attachment of Agrobacterium towards Vanda Kasem's Delight (VKD) protocorm-like bodies (PLBs) were studied to analyse the interaction between Agrobacterium and PLB during the transformation event. The study shows that initially A. tumefaciens reversibly attached to PLB surface via polar and lateral mode of adherence followed by the irreversible attachment which involved the production of cellulosic fibril by A. tumefaciens. Cellulosic fibril allows formation of biofilm at the tip of trichome. Contrarily, attachment mutant Escherichia coli strain DH5a was significantly deficient in the attachment process. Spectrophotometric GUS assay showed the mean value of attachment by A. tumefaciens was 8.72 % compared to the negative control E. coli strain DH5a that produced 0.16 %. A. tumefaciens swarmed with sharper and brighter edge when severe wounding was applied to the PLBs producing the highest swarming ratio of 1.46 demonstrating the positive effect of the plant exudates on bacterial movement. The study shows that VKD's PLBs are the suitable explants for Agrobacterium-mediated transformation since the bacteria expressed higher competency rate.

Growing orchids has been classified as an international business since it covers 8% of the world floriculture trade. Thus, large-scale micropropagation of orchid using tissue culture techniques and improvement of some essential traits, such as resistances to various diseases and pests, and tolerances to environmental stresses, such as low temperatures and low light intensities, via genetic engineering acknowledged the orchids as one of the top ten cut flowers. Protocorm-like bodies (PLBs) are excellent explants for clonal propagation, artificial seeds development and genetic engineering since they are organized and easily regenerable somatic embryos that propagate rapidly. Vanda Kasem's Delight hybrid orchid's PLBs derived from shoot tip culture were examined in order to understand the fundamental ultrastructure of PLB. Examination of the cross sections of PLB revealed that PLB is made of a discrete bipolar structure that contains anterior and posterior meristems. Actively dividing meristem cells and outer layer lined by several rows of small and isodiametric cells with a dense cytoplasm and a prominent nucleus were also observed via cytological studies. Scanning Electron Microscope investigations revealed that the surface of PLBs is occupied by vertically positioned branched trichome appendages and regularly spaced stomatal openings with two guard cells. Organelles such as mitochondria of various sizes, shapes and biconvex chloroplast in the cytoplasm were revealed by Transmission Electron Microscope analysis. Hence, inheriting actively dividing cells, and bestowed with elements related to transpiration, photosynthesis and energy synthesizing power house makes PLB a suitable explant for micropropagation and genetic engineering studies.

The aim of this work was to analyse the morphological aspects of ABB's PLBs and interaction between ABB's PLBs and Agrobacterium. Methodology and results: Aranda Broga Blue's (ABB) protocorm-like bodies (PLB) proliferated on Vacin and Went medium by incubating at 25 ± 2°C under 16 h semi-photoperiod was used to study the micromorphology of PLB, bacterial attachment and chemotaxis studies. The anatomy and micromorphology study indicates that ABB's PLBs provide the platform for attachment of Agrobacterium tumefaciens strain LBA 4404. Successful propagation of ABB's PLBs was achieved on modified Vacin and Went solid media supplemented with organic homogenates. Histological analysis of the development of ABB's PLBs shows it is a meristematic tissue and contains actively dividing cells. Transmission electron microscope (TEM) studies signify the presence of specialized organelles to indicate PLB is similar to a mature plant. Scanning electron microscope (SEM) observation on ABB's PLBs and bacterial attachment studies proves that trichomes and stomata on the surface facilitate the interaction with A. tumefaciens. Quantification of the attachment of Agrobacterium to ABB's PLBs using spectrophotometric system displayed higher affinity compared to the attachment mutant Escherichia coli DH5α. The interaction of Agrobacterium to ABB's PLBs was also evaluated based on chemotaxis studies. Highest motility respond of Agrobacterium was recorded (1.6 unit) towards severely wounded PLBs indicating wounding is necessary for the interaction between bacterium and PLB. Conclusion, significance and impact study: Micromorphology of protocorm-like bodies (PLBs) enhances the physical interaction with bacteria proved that PLB is a suitable explant for the Agrobacterium-mediated genetic transformation.