Silver nitrate and aminoethoxyvinylglycine promote in vitro adventitious shoot regeneration of pomegranate ( Punica granatum L.)

A protocol is presented for direct adventitious shoot organogenesis and complete plant regeneration from seedling-derived explants of pomegranate ( Punica granatum L.), a tropical fruit tree. Murashige and Skoog (1962) (MS) medium enriched with 8.9 μmol/L benzyladenine (BA), 5.4 μmol/L naphthaleneacetic acid (NAA) and 10 percnt; coconut water (CW) induced adventitious shoot bud differentiation in axenic seedling-derived cotyledons as well as hypocotyl segments. The cotyledons were more responsive than the hypocotyls. Addition of ethylene inhibitors such as AgNO 3 (10-40 μmol/L) and aminoethoxyvinylglycine (AVG) (5-15 μmol/L) to the medium markedly enhanced regeneration frequency as well as number of shoots obtained per explant. The promotive effect of AVG and AgNO 3 on shoot organogenesis was observed only in cotyledon explants. The regeneration medium containing AgNO 3 (20 μmol/L) or AVG (10 μmol/L) induced adventitious shoot buds from 57 percnt; or 53 percnt; of the cotyledon explants respectively. These shoot buds developed into shoots upon transfer to a regeneration medium without AgNO 3 and AVG. The promotive effect of AVG on shoot regeneration was reversed by exogenous application of 20 μmol/L 2-chloroethylphosphonic acid (CEPA), an ethylene releasing compound. On the other hand, shoot regeneration stimulated by AgNO 3 was relatively less affected by CEPA. Regenerated shoots were rooted in half-strength MS medium (½ MS) containing 0.54 μmol/L NAA. The well rooted plantlets were acclimatized and eventually established in soil.