High frequency axillary shoot proliferation and plant regeneration from cotyledonary nodes of pomegranate (Punica granatum L.)

A complete protocol is presented for in vitro regeneration of pomegranate (Punica granatum L.), a tropical fruit tree, using cotyledonary nodes derived from axenic seedlings. Shoot development was induced from cotyledonary nodes on Murashige and Skoog (1962) (MS) medium supplemented with 2.3±23.0 mM benzyladenine (BA) or kinetin (Kn). Both type and concentration of cytokinin signi®cantly in ̄uenced shoot proliferation. The maximum number of shoots (9.8 shoots/explant) was developed on a medium containing 9.0 mM BA. Shoot culture was established by repeatedly subculturing the original cotyledonary node on a fresh batch of the same medium after each harvest of the newly formed shoots. In vitro raised shoots were cut into nodal segments and cultured on a fresh medium for further multiplication. Thus, from a single cotyledonary node about 30±35 shoots were obtained in 60 days. Shoots formed in vitro were rooted on half-strength MS supplemented with 0.054±5.4 mM naphthaleneacetic acid (NAA). However, a medium containing 0.54 mM NAA resulted in the highest per cent rooting of shoots and signi®cantly higher number of roots than other concentrations. Plantlets were successfully acclimated and established in soil. # 2000 Elsevier Science B.V. All rights reserved.