Transformation of three genera of orchid using the bar gene as a selectable marker

Protocorms or protocorm-like bodies (PLBs) of three orchid genera, Brassia, Cattleya, and Doritaenopsis, were genetically transformed via microprojectile bombardment using the bar gene from Streptomyces hygroscopicus. Selection for transformed cells was accomplished using bialaphos. PLBs that proliferated on selection medium containing 1 mg/l bialaphos were minced and transferred to selection medium containing 3 mg/l of the herbicide. This selection was repeated twice at 2-month intervals. Putatively transformed plantlets were regenerated in the absence of bialaphos for Doritaenopsis, or on regeneration medium containing 3 mg/l bialaphos for Brassia and Cattleya. Presence of the bar gene in the transformed plants was confirmed by polymerase chain reaction and Southern blot analysis. Transcription of bar was assessed by northern blot analysis. Plantlets of all three genera exhibited functional expression of bar as determined by assaying for resistance to bialaphos applied directly to leaves.