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Mark P. Bridgen, Masood Z. Hadi, and Madeleine Spencer-Barreto

A laboratory exercise on direct and indirect organogenesis from leaf explants is presented for students of plant tissue culture or plant propagation. Torenia fournieri, the wishbone flower, is used for this laboratory exercise because the in vitro production of adventitious shoots from Torenia is easy to control, seeds are easy to obtain, and plants are easy to grow. Direct shoot organogenesis results from leaf explants without an intervening callus phase, and indirect shoot organogenesis is possible after 4 to 6 weeks of callus production from leaf explants. The basal medium for all forms of organogenesis contains Murashige and Skoog (MS) salts and vitamins, 30 g sucrose/liter, and 7 g agar/liter at pH 5.7. To obtain direct shoot organogenesis, leaf explants should be placed on the MS basal medium with 1.1 μM (0.25 mg·liter-1) 6-benzylaminopurine (BAP) and 0.25 μM (0.05 mg·liter-1) indole-3-butyric acid (IBA). If leaf explants are placed on MS medium with 2.3 μM (0.5 mg·liter-1) 2,4-dichlorophenoxyacetic acid (2,4-D), callus formation will occur. Callus can be subcultured onto a MS medium with 8.88 μM BAP (2.0 mg·liter-1) plus 2.5 μM IBA (0.5 mg·liter-1) for indirect shoot organogenesis to occur.