Tissue browning of in vitro cultures of Scots pine: Role of peroxidase and polyphenol oxidase

Abstract

Callus cultures from shoot tips of mature Scots pine (Pinus sylvestris L.) were characterized by rapid browning and an inability to regenerate. The peroxidase (POD) and polyphenol oxidase (PPO) activities and relationship to browning in such cultures were compared with embryogenic and non‐embryogenic cultures of Scots pine, started from immature embryos of three different pine clones. The browning in callus cultures derived from pine buds was visible approximately after 2 weeks of culture, and continued thereafter until the callus was dark brown and poorly growing. The non‐embryogenic cultures induced from immature embryos showed either light yellow coloring or browning, whereas the embryogenic cultures showed browning. POD activity increased during the first 4 weeks in callus tissue initiated from pine buds, and was significantly higher than in pine buds or cultures derived from immature embryos. The ability of cultures initiated from pine buds to oxidize catechol was notably high compared with cultures initiated from immature embryos, regardless of the time of measurement. Addition of catalase revealed that both POD and PPO were able to use catechol as substrate. An antibody raised against broad bean (Vicia faba) chloroplast PPO was used to recognize PPO. One polypeptide with a molecular mass of 50 kDa was detected in all pine samples on SDS‐PAGE and non‐denaturing PAGE. Another polypeptide with a molecular mass of 70 kDa was shown exclusively in the light‐yellow non‐embryogenic cultures. The results suggest that especially the high POD activities in callus tissues started from mature trees cause rapid and early browning and possibly subsequent cell death.