Secondary Somatic Embryogenesis in Hevea brasiliensis (Mull. Arg.): An Alternative Process for Long-term Somatic Embryogenesis

A procedure for long-term somatic embryogenesis called Maintained Somatic Embryogenesis (MSE) was previously described in Hevea brasiliensis to obtain embryogenic callus lines directly from integument explants. However the frequency with which embryogenic friable callus lines are directly established from the inner integument of immature fruit is low and restricted to the clones PB 260 and RRIM 703. In this study, the ability of somatic embryos to develop friable callus lines with proliferating and embryogenic capacities was highlighted. Histological analyses revealed dedifferentiation of the epidermal and perivascular cells, giving rise to the formation of friable embryogenic callus on the periphery of somatic embryo explants. Cell proliferation induced by subsequent subcultures of calli on maintenance medium resulted in the establishment of embryogenic callus lines. This new procedure is called indirect Secondary Somatic Embryogenesis (SSE). The potential of embryo-derived embryogenic callus lines was assessed in comparison with the integument-derived embryogenic callus line produced using the MSE procedure. Embryogenic callus lines were established with the clones PB 260 and RRIM 703 for the new process with a higher frequency than those obtained with the MSE procedure. In addition, the number of subcultures needed to establish callus lines was shortened. Secondary somatic embryogenesis also enabled the production of friable callus lines for the PB 217 clone, which was recalcitrant to the previous process. This alternative procedure opens the way for applications of long-term embryogenesis to many other clones for which primary somatic embryogenesis is successful.