Cryostorage of Germplasm of Tropical Recalcitrant-Seeded Species: Approaches and Problems

Premise of research. Storage of hydrated recalcitrant seeds is possible only in the short (and, occasionally, medium) term, as these metabolically active, desiccation-sensitive propagules either germinate and/or are overrun by proliferating fungi. However, cryostorage of the germplasm of recalcitrant-seeded species—generally as embryonic axes excised from the seeds—in or over liquid nitrogen (LN) affords the means of long-term conservation. Axis cryopreservation protocols all incorporate a series of common steps, but often these need fine-tuning per species. This notwithstanding, successful cryopreservation—ultimately assessed by seedling development—is generally more easily achieved for temperate recalcitrant-seeded species than for tropical/subtropical types.Methodology. Cryopreservation protocols involve axis excision, elimination of microfloral inoculum, rapid (flash) drying alone or preceded by cryoprotectant treatment, and immersion in LN. After cryostorage, warming and rehydration are pivotal steps. Effects of each manipulation on viability need to be ascertained by in vitro axis culture; thus, optimization of the germination medium is an a priori requirement.Pivotal results. Each procedural step imposes oxidative stress, manifested by elevated levels of reactive oxygen species, the consequences of which are cumulative and often lethal. To minimize oxidative stress, ameliorative measures must be developed, including use of noninjurious decontaminants, rapidly achieved flash drying, determination of the best cooling rate, and provision of nontoxic antioxidants. The nature of the medium in which axes are warmed and then rehydrated is also critical. We describe a process we call cathodic amelioration, in which the distilled water that is generally used is replaced with the cathodic fraction of an electrolyzed dilute salt solution; this is a powerful reductant and is markedly improving the prospects of successful cryopreservation.Conclusions. All the procedures impose oxidative stress—a major factor contributing to failure of seedling production after axis cryopreservation. For success, it is imperative that this be counteracted by introduction of benign ameliorative measures, such as cathodic protection.