Production of reactive oxygen species in excised, desiccated and cryopreserved explants of Trichilia dregeana Sond

The tropical tree Trichilia dregeana Sond. has recalcitrant seeds that cannot be stored by conventional seed banking methods that include drying and storage at low temperatures, or by using cryopreservation protocols that have been successfully applied to recalcitrant-seeded species such as Castanea sativa. We recently showed that in C. sativa both excision of the embryonic axes and subsequent dehydration cause transitory (5 min) bursts of extracellular superoxide (O2−) production. Here we show that both excision and partial dehydration of the embryonic axes from seeds of T. dregeana cause large, prolonged extracellular bursts of O2−. Furthermore, during rehydration after cryopreservation, another burst of O2− occurs with slightly different kinetics. Compared with C. sativa, rates of O2− production in T. dregeana are approximately twice as great and decline much more slowly, suggesting that excessive radical formation may be responsible for poor survival of the axes following cryopreservation. Fractionating the cell wall proteins of embryonic axes and cotyledons in conjunction with electrophoretic analyses of the fractions showed that most O2− was produced by two peroxidases with molecular masses of c. 50 and 80 kD that were loosely bound to the cell walls of the embryonic axes. Future successful cryopreservation of T. dregeana would appear to depend on manipulations of O2− production, and the discovery of peroxidases as the enzymes responsible described here may help in the development of more effective protocols.