High frequency axillary shoot proliferation and plant regeneration from cotyledonary nodes of pomegranate (Punica granatum L.) Soumendra K. Naik, Sitakanta Pattnaik, Pradeep K. Chand * Plant Tissue and Cell Culture Facility, Post-Graduate Department of Botany, Utkal University, Bhubaneswar 751004, Orissa, India Accepted 2 December 1999 Abstract 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. Keywords: Axillary proliferation; Cotyledonary node; In vitro regeneration; Fruit tree; Punica granatum 1. Introduction The use of tissue culture techniques for fruit tree propagation has increased considerably over the past few decades. Reliable and ef®cient protocols for plant Scientia Horticulturae 85 (2000) 261±270 Abbreviations: BA, N 6 -benzyladenine; Kn, Kinetin; MS, Murashige and Skoog (1962) medium; NAA, Naphthaleneacetic acid * Corresponding author. Tel.:91-674-581529; fax:91-674-509037. 0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved. PII:S0304-4238(99)00149-1