Optimization of efficient direct organogenesis protocol for Punica granatum L. cv. Kandhari Kabuli from mature leaf explants

Abstract

Punica granatum L. is an important horticultural fruit crop with high medicinal and economic value. Its rising commercial demand necessitates the production of high-quality planting material. Here, we describe an efficient protocol for direct organogenesis in Punica granatum L. cv. Kandhari Kabuli from mature leaf explant. The optimized sterilization procedure for explant includes sequential treatment with 70% ethanol (0.75 min), 0.2% Bavistin (15 min), and 0.5% sodium hypochlorite (2 min), which resulted in 83% axenic cultures. The accumulation of phenolics was effectively controlled by subculturing of leaf explants three to four times at a regular interval of 24 h. The organogenic capability of leaf segments was investigated on full-strength Murashige and Skoog (MS) medium supplemented with different plant growth regulators (PGRs), including the cytokinins 6-benzylaminopurine (BAP) and thidiazuron (TDZ) alone or in combination with α-naphthaleneacetic acid (NAA). BAP promoted the greatest morphogenic response as compared to that from TDZ. However, the greatest frequency of shoot induction (43%) was achieved on MS medium supplemented with 10 μM BAP and 2.5 μM NAA under dark incubation for 2 wk. Furthermore, micro-shoot proliferation and elongation were achieved on multiplication medium consisting of MS medium supplemented with 9.0 μM BAP, 2.5 μM Kinetin (KN), and 0.5 μM gibberellic acid (GA3) up to the third subculturing. However, further subculturing resulted in vitrification. A hormone-free medium containing 300 mg L−1 activated charcoal (AC) was found to be effective to reduce vitrification and promote shoot multiplication. In vitro rooting was carried out on the ½ MS basal medium containing 500 mg L−1 AC using shoots from different subculture passages. Successive subculturing tends to have a positive effect on in vitro rooting and increased rooting up to 70.62%. Well-rooted plantlets were acclimatized successfully in the small plastic pots containing sterilized sand and later shifted to the soil. This optimized protocol can be routinely used for rapid large-scale propagation of pomegranate and is a prerequisite for trait improvement via genetic engineering.