Winter Forcing of Apple (Malus domestica Borkh.) Cuttings for Bud Induction to Enhance Explant Availability in Tissue Culture Applications

This study aimed to evaluate the effects of plant growth regulators, specifically benzyladenine (BA) and gibberellic acid (GA₃), on winter apple bud forcing, bud break characteristics, and subsequent tissue culture performance of six apple (Malus domestica Borkh.) cultivars. One year old semi-woody cuttings of Golden Delicious, Red Delicious, Barwary, Anna, Dwarf Apple and Granny Smith were subjected to cold pre-treatments (4°C) using BA and GA₃ at four combinations: 0:0, 0:10, 10:0, and 10:10 mg.l-1. Results showed that all the treatments significantly reduced the days to bud break, with the 10:10 mg.l-1combination yielding the fastest response (22 days), compared to 28.67 days in control. Bud break percentage and bud vigor were also significantly enhanced, particularly under the combined BA plus GA₃ treatment, reaching up to 100% bud break in Red Delicious and Anna. Bud vigor improved from weak (control) to strong under optimal treatment in most cultivars. The sustainability of flushed buds on MS medium was highest under the combined treatment, with Red Delicious, Barwary and Granny Smith showing 65–70% viability, while Dwarf Apple remained poorly responsive. To improve explant survival, antioxidants (citric and ascorbic acid) and activated charcoal (1.5 g.l-1) were tested. Activated charcoal significantly enhanced survival rates, reaching 100% in Granny Smith and 95% in Barwary, compared to 3.3% in the untreated control. Cultivar differences were evident throughout the study. In conclusion, combining BA and GA₃ with activated charcoal provides an effective strategy for year-round micropropagation of apple, with cultivar-specific responses warranting tailored protocols. Thus, this work will serve as a source for future apple micropropagation commercial projects.