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.

In vitro plant regeneration system for common bean (Phaseolus vulgaris): effect of N6-benzylaminopurine and adenine sulphate

“…In this sense, browning hampers the establishment of tissue culture in Aloe (Abrie and van Staden, 2001), Dioscorea alata (Bhat and Chandel, 1991), Musa spp. (Titov et al 2006) and Pinus sylvestris (Laukkanen et al 1999). Moreover, some plant growth regulators such as BAP, kinetin, TDZ, IAA alone or in combination with 2,4-D were reported to increase browning in Aloe species (Natali et al 1990;Meyer and van Staden, 1991).…”

“…Browning is caused by the oxidation of phenolic compounds, released from the cut ends of the explants, by polyphenoloxidases, peroxidases or air (Bhat and Chandel, 1991;Laukkanen et al 1999). In this sense, browning hampers the establishment of tissue culture in Aloe (Abrie and van Staden, 2001), Dioscorea alata (Bhat and Chandel, 1991), Musa spp.…”

Effects of Ascorbic Acid in Controlling Lethal Browning in <i>in Vitro</i> Culture of <i>Brahylaena huillensis</i> Using Nodal Segments

“…[34,35] showed that accumulation of these compounds leads to browning and possibly death of the explants. Moreover, oxidized phenolic compounds may inhibit enzyme activity and result in the darkening of the culture medium and subsequent lethal browning of explants [36,37]. Thus, the study revealed that for successful control of oxidative lethal browning the concentration of antioxidant ascorbic acid is of great importance.…”

In vitro regeneration ability of diploid and autotetraploid plants of Cichorium intybus L.

“…It is possible that the increased content of total phenolic compounds in tetraploid origin calluses resulted in the loss of their organogenic potential. Phenolic compounds, especially oxidized phenolics, may contribute to the inhibition of enzymatic activities, resulting in the darkening of the culture medium and subsequent lethal browning of explants [32,33]. It has been reported that oxidized phenolics generally affect in vitro proliferation and regeneration negatively [34].…”