Optimizing Shoot Proliferation and Plant Regeneration of Punica Granatum

The effect of cultivar, explant type and developmental stage, macro- and micronutrient composition of culture medium and growth regulator concentration on plant regeneration of Punica granatum was studied. Leaves, nodal segments and shoot apical meristems were obtained from field-grown trees and surface-sterilized by constant agitation in 25% commercial bleach for 5 min followed by 3 rinses of 5 min each in sterile distilled water.  Shoot apical meristems (5.0 mm long) were cultured on MS and C₂D medium containing 4.0, 8.0 or 12 µM BAP. Leaves were cultured on MS medium containing 2.0, 4.0 or 6.0 µM BAP and 1.0 or 2.0 µM NAA. Severe necrosis of explants was observed after 3 days of incubation on culture medium resulting in tissue death and loss of cultures. Necrosis was significantly reduced by etiolating shoots and leaves with a 7-day-dark treatment.  Apical meristems excised from rapidly growing etiolated shoots exhibited maximum survival (40%) and shoot proliferation. Indirect organogenesis was observed from unopened leaf explants (1.5 - 5.0 mm long) cultured on MS medium containing 4 µM BAP and 1.0 µM NAA. Among the various media and growth regulator concentrations tested, maximum shoot proliferation was observed after 9 weeks of culture on C₂D medium containing 4.0 µM BAP. Among the various cultivars tested, the best response was observed in ‘Wonderful’, followed by ‘Entek Habi Saveh’, ‘Kala Bala Mirusal’ and ‘Salavatski’. Plant regeneration was obtained by rooting proliferating shoots on C₂D medium containing 0.4 µM NAA. The developed protocols are currently being refined and will be suitable for large-scale micropropagation of pomegranate cultivars.