Rapid micropropagation of Calabrian pine from primary and secondary buds on shoot explants

Axillary shoot production was achieved in 6 weeks using excised shoot explants of Pinusbrutia Ten. on a modified Schenk and Hildebrandt medium containing cytokinin. Primary shoots arose from existing axillary buds and secondary buds arose from bases of the primary shoots. Their production could be increased by regulating the cytokinin level and by surgical removal of apical buds from the cultured explants. However, the best performance was achieved with a low level of 6-benzylaminopurine (3 × 10−6 M) or a mixture of 6-benzylaminopurine and kinetin (10−6 M of each). Subsequent transfer to a cytokinin-free medium resulted on average in the production of 43 shoots per cultured explant and up to 67 shoots per clone within 12 weeks. When the primary shoots, which had already produced one crop of secondary shoots, were maintained under conditions favourable for shoot production, a doubling in number was obtained within 4–6 weeks. To encourage further elongation, newly formed shoots were incubated for 2 weeks on a cytokinin-free medium to which 1% activated charcoal was added. The time taken with this method was much shorter than with other published methods and is, therefore, likely to be important for the vegetative propagation and multiplication of selected seedlings of this species.