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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     M 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.

Agrobacterium-Mediated Transformation of the RecalcitrantVandaKasem's Delight Orchid with Higher Efficiency