Somatic embryogenesis, embyogenic cell suspension from ocimum sanctum (l.) leaf callus cultures and their rosmarinic acid accumulation

Abstract

The accumulation of rosmarinic acid (RA) in Ocimum sanctum (L.) somatic embryogenic callus and its cell suspension cultures was investigated. For callus induction, leaves, stems and inflorescence explants excised from field-grown plants were cultured on Murashige and Skoog (MS) medium containing 2,4-D (1 mg l-1) combined with kinetin (0.1-0.5 mg l-1), 2,4-D (0.5-2.5 mg l-1) individually, IAA (0.5-2.5 mg l -l) individually, and NAA (0.5-2.5 mg l -l) individually. Due to better proliferation ability and high RA content of leaf callus, it was chosen to induce somatic embryogenesis by supplementing with different concentration and combination of BAP + NAA + kinetin. Among the different combination tested, BAP (1 mg l -l) + NAA (1 mg l -l) + kinetin (0.5 mg l -l) exhibited a maximum embryogenic potential (73%) and number of globular embryo (34.3) per callus. In order to study the RA production, the proliferation of embryogenic suspension culture was tested by transferring embyogenic callus at stationary growth phase from MS solid to liquid medium containing the same composition and hormonal concentration. After five subcultures, the suspensions were maintained continuously for six weeks without subculturing. Suspension cultures showed progressive callus growth and RA accumulation throughout the experimental period. The highest RA (82.1 mg l -1) content and biomass growth (11.6 g l -1) was observed in the cultures supplemented with BAP (1 mg l -l) + NAA (1 mg l -l) + kinetin (0.5 mg l -l). It was 40 fold higher than the leaf callus.