Silver nitrate and aminoethoxyvinylglycine promotein 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 AgNO3 (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 AgNO3 on shoot organogenesis was observed only in cotyledon explants. The regeneration medium containing AgNO3 (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 AgNO3 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 AgNO3 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.