Rate of dehydration, state of subcellular organisation and nature of cryoprotection are critical factors contributing to the variable success of cryopreservation: studies on recalcitrant zygotic embryos of Haemanthus montanus

Abstract

Effects of sequential procedures required for cryopreservation of embryos excised from the recalcitrant seeds of Haemanthus montanus were assessed ultrastructurally and in conjunction with respiratory activity and the rate of protein synthesis. Fresh material (water content, 5.05 ± 0.92 g g⁻¹ dry mass) afforded ultrastructural evidence of considerable metabolic activity, borne out by respiratory rates. Neither exposure to glycerol nor sucrose as penetrating and non-penetrating cryoprotectants, respectively, brought about degradative changes, although increased vacuolation and autophagy accompanied both, while respiratory and protein synthetic activity were not adversely affected. Glycerol-cryoprotected embryos flash dried to water contents >0.4 g g⁻¹ showed organised ultrastructural features and considerable autophagy consistent with metabolic activity, and although respiratory activity was lower, protein synthesis rate was enhanced relative to fresh material. However, at water contents <0.4 g g⁻¹, embryo tissue presented a mosaic of cells of variable density and ultrastructural status, but trends in rates of respiration and protein synthesis remained similar. Flash drying after sucrose exposure was accompanied by considerable ultrastructural abnormality particularly at water contents <0.4 g g⁻¹, lysis of individual and groups of cells and considerable depression of respiration, but not of protein synthesis. Success, assessed as ≥50% axes forming seedlings after cryogen exposure, was obtained only when glycerol-cryoprotected embryos at water contents >0.4 g g⁻¹—in which the degree of vacuolation remained moderate—were rapidly cooled. The outcomes of this study are considered particularly in terms of the stresses imposed by prolonged, relatively slow dehydration and ultimate water contents, on embryos showing considerable metabolic activity.