High frequency somatic embryogenesis from leaf tissue of Populus spp.

Abstract

A high frequency somatic embryogenesis protocol was developed for biotechnological manipulations of hybrid poplar in which embryogenic callus was derived from non-embryonic explants. Somatic embryos of Populus alba × P. grandidentata (NC-5339) were differentiated directly on leaf explants and indirectly from embryogenic cell suspension callus initiated from leaf tissue. Leaf explants were induced to form embryogenic callus in darkness on Murashige and Skoog (MS) medium with 5 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 mg/l benzyladenine (BA), and 30 gg/l sucrose. The auxin to cytokinin ratio (5 mg/l 2,4-D to 0.05 mg/l zeatin) was optimized for globular-shaped embryo initiation from cell suspension callus. Heart-shaped embryo development occurred after removal of 2,4-D from the culture medium, and development progressed to embryo maturity on the same medium. Late stage torpedo-shaped embryos germinated following a 1-week subculture on MS medium containing 5 mg/l IAA and 0.05 mg/l BA, and germinated embryos were successfully transferred ex vitro to greenhouse growth conditions.