Stable transformation of protocorm-like bodies in Phalaenopsis orchid mediated by Agrobacterium tumefaciens

Genetically transformed plantlets of Phalaenopsis were regenerated after co-cultivation of protocorm-like bodies (PLBs) with Agrobacterium tumefaciens strain LBA4404 containing the vector pTOK233 that harbors genes for beta-glucuronidase and hygromycin resistance. Four lines of Phalaenopsis orchid (T0, T5, T10 and Hikaru) were tested on three multiplication media for the production of PLBs. Two types of explants, intact and transversely bisected PLBs, were checked for efficiency of multiplication, and it was found that transversely bisected PLBs were much better than intact ones. The effect of hygromycin concentrations on rooting of PLBs was also established. Initial recovery of the PLBs following co-cultivation was allowed without selection for 6 weeks. The first selection was carried out on regeneration medium containing 3 mg/l hygromycin for 2 months. New PLBs, as well as shoots and roots, were formed from original PLBs when they were regenerated under hygromycin selection. Leaves, roots, original and some newly formed PLBs were taken from hygromycin-resistant rooted plantlets and then subjected to GUS assay. Newly formed PLBs from GUS-positive plantlets were then regenerated on the above-mentioned regeneration medium but containing a lower concentration of hygromycin (1.5 mg/l). When the PLBs regenerated into plantlets, original PLBs were subjected to GUS assay, and again newly formed PLBs were regenerated and selected as described above. The procedures were repeated monthly for four cycles and it was found that a long selection period with the low hygromycin concentration yielded stable transformants. Histochemical GUS assay indicated successful transformation, which was further confirmed by PCR analysis and Southern hybridization of transformants. (C) 2002 Elsevier Science B.V. All rights reserved.