In the present study, a protocol for Agrobacterium tumefaciens-mediated transformation has been optimized for Woodfordia fruticosa (L.) Kurz. Precultured axenic leaf segments were cocultivated with A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301 with b-glucuronidase (uidA) containing intron as the reporter gene and hygromycin phosphotransferase (hpt) as a selectable marker gene. After 3 days of co-cultivation, leaf segments were cultured on MS medium containing Thidiazuron (TDZ 4.54 lM) and Indole-3-acetic acid IAA (1.14 lM) + 20 mg/ l hygromycin + 200 mg/l cefotaxime (PTSM 1 ) for 4 weeks (includes a single subculture onto the same medium at a 2 week interval). They were subsequently cultured for 3 weeks on MS medium containing Thidiazuron (TDZ 4.54 lM) and Indole-3-acetic acid IAA (1.14 lM) + 25 mg/l hygromycin + 100 mg/l cefotaxime (PTSM 2 ) medium for further development and shoot elongation. The hygromycin resistant shoots were rooted on a rooting medium (PTRM) containing half strength MS medium + 4.90 lM IBA + 25 mg/l hygromycin. A highest transformation efficiency of 44.5% with a mean number of 2.6 transgenic shoots per explant was achieved. Successful transformation was confirmed by the histochemical GUS activity of the regenerated shoots, PCR and RT-PCR analysis using respective primers. Southern blot analysis revealed that the hpt gene integrated into the genome of transgenic W. fruticosa. Establishment of genetic transformation protocol may facilitate the improvement of this medicinal plant in terms of enhancement of secondary metabolites.Ó 2015 Production and hosting by Elsevier B.V. on behalf

An efficient in vitro leaf regeneration protocol has been established in Woodfordia fruticosa L. (Lathyraceae), a threatened woody shrub. The leaf segments derived from in vitro-grown plants were cultured on Murashige and Skoog's medium supplemented with Thidiazuron (2.27, 4.54, 6.81,and 9.08 µM) and 6- Benzyladenine (4.4, 8.90, 13.30, and 17.70 µM) alone or in combination with Indole-3-acetic acid (1.14 and 2.28 µM). Maximum number of shoots (15.60 ± 1.15) with highest shoot length (2.90 ± 0.24) were regenerated directly from the leaf explants with a combination of TDZ (4.54 µM) and IAA (2.28 µM), whereas the intervening callus phase was observed in the media supplemented with TDZ or BA alone. The in vitro-regenerated shoots were rooted (100%) on half-strength MS salts fortified with 4.90 µM IBA. The regenerated plantlets hardened in the greenhouse showed an 85% survival rate. ISSR PCR of in vitro-regenerated plantlets reveals their genetic fidelity with the mother plant.