EFFECT OF LEAF EXCISION TIME AND AGE, BA CONCENTRATION AND DARK TREATMENTS ON IN-VITRO SHOOT REGENERATION OF M.26 APPLE ROOTSTOCK

The effect of benzyladenine (BA) concentrations both during the last proliferating subculture before regeneration (0.0, 2.22 and 4.44 muM) and during organogenesis (11.1 and 22.2 muM), leaf excision time (15 and 30 d from the beginning of the subculture), leaf age and dark treatments on adventitious shoot regeneration of M.26 apple rootstock were evaluated. Leaves excised 30 d after the beginning of the last proliferating subculture and grown without BA in the medium gave the highest percentages of organogenesis, while the number of regenerated shoots per leaf did not differ significantly among the different BA x leaf excision time combinations. The highest BA concentration (22.2 muM) in the organogenetic medium produced the highest percentage of regenerating leaves, with no differences between the lengths and numbers of shoots per regenerating leaf. The first two unfurled apical leaves showed a greater regenerative ability than the third and fourth ones, whereas the lengths and numbers of regenerated shoots per leaf were similar. The highest leaf organogenetic rate was observed when darkness was imposed at the beginning of the last proliferating subculture and/or at the beginning of the organogenetic phase, but more regenerated shoots per leaf were obtained with darkness provided at the beginning or at the end of the last proliferating subculture, shoot lengths were similar in all the dark treatments. The great influence on organogenesis of all the treatments applied in the last proliferating subculture indicates the importance of this stage in preparing explants for shoot regeneration and thus the possibility of using inductive factors in this phase.