Clonal in vitro multiplication of grey mangrove and assessment of genetic fidelity using single primer amplification reaction (SPAR) methods

An efficient protocol for clonal multiplication of an important mangrove, Avicennia marina, was developed through in vitro culture of nodal segments obtained from a mature plant. The nodal explant induced multiple shoots when cultured on the Murashige and Skoog (MS) basal medium supplemented with varying concentrations and combinations of 6-benzyladenine (BA) and -naphthalene acetic acid. The highest response in terms of per cent regeneration (73%), average number of shoots/explant (3.25 +/- 0.25) and maximum shoot length (5.2 +/- 0.27 cm) was obtained on the MS medium supplemented with BA 5.0 mol/L + NAA 1.0 mol/L + 3 g/L activated charcoal after 8 weeks of culturing. The regenerated shoots were rooted well in the MS medium supplemented with 1.0 mol/L indole-3-butyric acid with an average of 2.9 +/- 0.24 roots per microshoot. The rooted plantlets were successfully transferred to pots containing normal garden soil with 70% survival rate. The genetic stability of the regenerated plants was evaluated using single primer amplification reaction (SPAR) methods viz., random amplified polymorphic DNA, directed amplification of minisatellite DNA and intersimple sequence repeat polymorphism. The SPAR analysis revealed monomorphic banding patterns in all in vitro regenerated plantlets of A. marina and similar with that of the mother tree confirming their genetic uniformity and clonal fidelity.