In vitro cloning of apple (Malus domestica Borkh) employing forced shoot tip cultures of M9 rootstock

For micropropagation of adult apple (Malus domestica Borkh) rootstock M9, explants were harvested from pre-chilled (4±3°C) dormant cuttings forced in growth chamber. Primary explants were established by using a slightly modified version of culture initiation procedure developed earlier. Multiple shoots, raised by axillary branching of surviving explants, were obtained from established cultures in two cultural pathways: (i) repeat transfer of primary explants on the MS supplemented with BAP (2.22 μM), IBA (0.49 μM) and Kn (2.2 μM), and (ii) repeat subculture of harvested microshoots and basal portion of sectored proliferating clumps on the same but suitably PGR amended medium, in which cytokinins concentration was reduced by half. A 4-fold shoot multiplication was achieved during each sub culture of 3±1 week's duration that repeatedly produced crop of proliferated shoots without loss of vigour. Nodal segments (5-15 mm), obtained from in vitro raised microshoots were also used to initiate a new cycle of proliferating cultures. Isolated cloned microshoots (15-20 mm) with apical bud were cultured on MS basal medium supplemented with IBA (14.70 μM) for 8±3 d to initiate root. The microshoots were re-implanted in PGR free half strength basal MS medium with full complement of organics for 4±1 week for root development. The transferred in vitro hardened plantlets to polyvinyl cups or polybags, under carefully controlled descending RH regime of 95% to 70±5% over a period of 5±1 week, resulted in 80% ex vitro survival. The present protocol highlighted a novel strategy of micropropagation of apple rootstock M9 using three-step culture initiation procedure of forced primary explants.