Exploring the use of DMSO and ascorbic acid to promote shoot development by excised embryonic axes of recalcitrant seeds.

Seeds of Trichilia dregeana, T. emetica and Protorhus longifolia are recalcitrant (desiccation-sensitive), hence cryopreservation is the only ex situ means feasible for long-term conservation of these germplasm. For cryopreservation of these species, the excised embryonic axis is the explant of choice due to their small size and higher tolerance to desiccation. However, for many species with seeds having fleshy cotyledons, shoot development fails to occur after excision, which has been attributed to a reactive oxygen species (ROS) burst during excision wounding. This is a critical limiting step in developing cryopreservation protocols for such species. In embryos of T. dregeana, T. emetica and P. longifolia, the cotyledonary insertions are in close proximity to the shoot apical meristem and oxidative stress upon excision of the axis from cotyledons has been consistently associated with shoot tip necrosis, which precludes shoot development. This study tested the effects of dimethyl sulphoxide (DMSO) pre-culture prior to complete removal of the cotyledons, and post-excision soaking in DMSO or in the antioxidant, ascorbic acid, on shoot development by axes of T. dregeana and P. longifolia. These treatments had a significant (P < 0.05) positive effect on shoot production with a 6 h DMSO pre-culture combined with a DMSO post-excision soak being optimal for promoting shoot production in 70 percent of the axes of T. dregeana and 60 percent of those of P. longifolia. Embryonic axes of T. emetica responded best to a 6 h DMSO pre-culture alone, with 55 percent of axes producing shoots. It was further shown that two different post-harvest developmental stages of T. dregeana axes differed significantly initially (P < 0.05) in their response to DMSO and ascorbic acid treatments.