Effect of thidiazuron on multiple shoot induction and plant regeneration from cotyledonary nodes of chestnut

The effect of thidiazuron (TDZ) on the shoot production capacity of preconditioned explants of Castanea sativa Mill. was evaluated. Embryonic axes of chestnut were aseptically germinated for 14 d in Murashige and Skoog (1962) basal medium supplemented with 0.1 mg l-1 TDZ or 1 mg l-1 benzyladenine (BA) (preconditioning medium), and hypocotyl, epicotyl and cotyledonary node explants were then cultured on shoot-induction medium (basal medium containing 0.01 mg l-1 naphthaleneacetic acid in combination with different TDZ concentrations). After four weeks of culture the explants were transferred for a further eight weeks to media with low BA concentrations and no TDZ. Shoots were produced only by cotyledonary node explants which contain preformed meristematic tissue. Best shoot production, in terms of the proportion of explants producing shoots and shoot productivity per explant, was achieved by BA-preconditioned explants exposed to 1-2 mg l-1 TDZ. Fewer TDZ-preconditioned explants produced shoots, and more produced only callus. An histological study showed preconditioned explants to have an altered meristematic axillary region, with meristematic cell proliferation giving rise to expanded, flattened apical meristems and reduced leaf primordium differentiation. Upon shoot-induction treatment the proliferating meristems originated multiple shoot buds without an intervening callus phase. Shoot elongation was achieved by culturing the original explants in Gresshoff and Doy (1972) medium supplemented with 0.05 mg l-1 BA. Shoots longer than 10 mm were subcultured separately to initiate clonal shoot cultures that were proliferated following the outgrowth of axillary shoots. Rooting frequencies greater than 75% were achieved by 16 of the 21 clones evaluated. Rooting capacity was not influenced by either the preconditioning medium or the shoot induction medium, but considerable variation was observed between clones (genotypes).This research was partially supported by CICYT, Spain (AGL2000-1073).Peer reviewe