Callus-mediated plant regeneration in Isodon amethystoides using young seedling leaves as starting materials

An efficient indirect organogenesis protocol was developed for use in Isodon amethystoides. Seven-day-old leaves from seedlings and mature plants were used as starting materials for callus induction and plant regeneration. Ascorbic acid and activated carbon were applied to alleviate callus browning during the differentiation process. The polyphenol content and polyphenol oxidase (PPO) and peroxidase (POD) activities were compared between calluses induced from both explants. The optimal medium for callus induction from both young seedling and mature plant materials was determined as Murashige and Skoog (MS) basal salts fortified with 4.4 µM 6-benzylaminopurine (BAP) and 2.7 µM 1-naphthylacetic acid (NAA) and solidified with 0.7% agar or 0.2% phytagel. This medium achieved a callus induction rate of > 90%. Young seedling-derived calluses had a regeneration rate of 83.3% on MS medium fortified with 0.44 µM BAP, 4.6 µM kinetin, 2.7 µM NAA, and 0.14 mM ascorbic acid and solidified with 0.2% phytagel. However, the mature plant-derived calluses browned and died eventually, even in the presence of browning inhibitors. In addition, the seedling derived-calluses presented no significant change in biochemical parameters (P > 0.05), while mature plant-derived calluses showed significantly varied polyphenol content and PPO activity during the differentiation process (P < 0.05). Our findings suggested that the causes for callus browning in this plant species during differentiation culture are associated with an increase in PPO activity and a decrease in total polyphenol content.