Cloning of mature pomegranate (Punica granatum) cv. Jalore seedless via in vitro shoot production and ex vitro rooting

Abstract

A novel approach of in vitro shoot amplification and ex vitro rooting for cloning of mature plants of Punica granatum cv. Jalore seedless/soft-seeded has been defined. Surface-sterilized nodal shoots were cultured for axillary meristem activation, bud breaking and shoot amplification. Multiple shoots differentiated by bud breaking from 82.8% of the explants on Murashige and Skoog (Physiol Plant 15:473–497, 1962) MS medium with 13.32 µM 6-benzylaminopurine (BAP). These were amplified by subculturing of fresh shoots and by repeated transfer of mother explants on MS medium + BAP or Kinetin (Kin)/Furfurylaminopurine (FAP) in combination with Indole acetic acid (IAA) or α-naphthalene acetic acid (NAA). MS medium with BAP 2.22 µM + IAA 0.57 µM was found to be most suitable for shoot (14.2 ± 1.03 shoots per culture vessel) multiplication. On half-strength MS medium with 9.84 µM of Indole butyric acid (IBA) and 16.65 µM of activated charcoal (AC), 72.9% of the micro-shoots rooted. Alternatively, base(s) of the micro-shoots treated with 1476 µM of IBA for 300 s. and transplanted on autoclaved soilrite (moistened with one-fourth strength of MS macro-salts) in glass bottles (420 ml; 70 mm diameter × 130 mm height). More than 85% of the IBA-treated shoots rooted ex vitro in a greenhouse. Ex vitro rooting of cloned shoots is a new approach for propagation of pomegranate. The process described is different and superior to all the described tissue culture methods for cloning of pomegranate. This is faster, cost-effective and saves resources enabling acclimatization/hardening with ease while minimizing microbial contamination, thus ensuring quick field transfer of hardened plantlets. This can be applied for mass and clonal propagation of selected genotypes and also for long-term conservation of germplasm of P. granatum.