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.

石榴是一种重要的园艺水果，具有很高的药用和经济价值。其日益增长的商业需求使得必须生产高质量的种植材料。在这里，我们描述了在石榴中直接器官发生的有效协议L.简历 来自成熟叶片外植体的Kandhari Kabuli。用于外植体的最佳灭菌程序包括依次用70％乙醇（0.75分钟），0.2％Bavistin（15分钟）和0.5％次氯酸钠（2分钟）处理，从而导致83％的树莓培养。通过以24小时的固定间隔将叶片外植体传代培养三到四次，可以有效地控制酚类化合物的积累。在补充了不同植物生长调节剂（PGR）的全强度Murashige和Skoog（MS）培养基上研究了叶段的器官发生能力，其中包括单独或与α-联合使用的细胞分裂素6-苄基氨基嘌呤（BAP）和噻噻隆（TDZ）。萘乙酸（NAA）。与TDZ相比，BAP促进了最大的形态发生反应。然而，在黑暗培养2周的条件下，添加10μMBAP和2.5μMNAA的MS培养基达到了最高的芽诱导频率（43％）。此外，在由添加了9.0μMBAP，2.5μMKinetin（KN）和0.5μM赤霉素（GA）的MS培养基组成的繁殖培养基上，微芽增殖和伸长得以实现₃）直到第三次传代。然而，进一步的传代培养导致玻璃化。发现含有300 mg L ^-1活性炭（AC）的无激素培养基可有效减少玻璃化并促进枝条繁殖。体外生根物含有500个毫克的L 1/2 MS培养基上进行^-1  AC使用枝条来自不同传代培养传代。连续传代培养对体外趋于产生积极影响生根，生根增加至70.62％。根深蒂固的小植株已在装有灭菌沙子的小塑料盆中成功地适应了环境，后来转移到土壤中。这种优化的协议可常规用于石榴的快速大规模繁殖，并且是通过基因工程改良性状的前提。