Regeneration of shoots via direct somatic embryogenesis from the leaf surface of Scaevola taccada (Gaertn.) roxb. – a climate resilient species of coastal areas

Abstract

Scaevola taccada (Gaertn.) Roxb. (family Goodeniaceae) is a large littoral shrub of great economic and ecological importance. Plantlets were regenerated from the in vitro raised leaf explants via direct somatic embryogenesis in this study. Nodes were cultured on Murashige and Skoog's (MS) medium augmented with 1.5 mg L −1 6-benzylaminopurine (BAP) and the in vitro raised leaves used as secondary explants to induce somatic embryogenesis directly from the leaf surface on the MS medium supplemented with two kinds of cytokinins, namely BAP and N6-furfuryladenine (Kinetin). The highest percentage of explants (92%) responded with globular somatic embryos after 4 weeks of incubation on 2.0 mg L −1 BAP. The anatomical observations of somatic embryos (SE) revealed that continuous cell divisions lead to the formation of proembryos and followed by globular somatic embryos. The bipolar structures with the shoot apical meristems formation observed after 4 weeks of cultures. Light microscopic evaluations showed that direct somatic embryogenesis from leaf explants were nonsynchronous. Culturing of four weeks old somatic embryos (SE) on MS medium fortified with 0.25 mg L −1 BAP and 0.15 mg L −1 IAA induced substantial changes in formation of multiple shoots. It was observed that full strength MS medium retarded the germination frequency of SE, and tip browning and vitrified shoots were detected. The reduced concentrations of ammonium nitrate salt (half) in the MS medium increased number of healthy shoots (36 shoots per culture vessel). Similarly, increased quantity of calcium chloride (CaCl2) in the medium (1.5 times) alleviated tip browning of the shoots. The shoots were successfully rooted in vitro on half strength MS medium + 2.0 mg L −1 IBA with 12.6 roots per shoot. The rooted shoots were hardened in the soilrite® in the greenhouse and finally transferred to the field. After 3 months, 100% survival rate was recorded under in vivo conditions. The developed protocol could be used for genetic transformation systems in S. taccada as leaves might be the better choice of explants.