The influence of water content, cooling and warming rate upon survival of embryonic axes of Poncirus trifoliata (L.)

The present study investigated the relative contributions of water content and non-equilibrium cooling and warming rates to the survival of cryopreserved axes of recalcitrant P. trifoliata seeds. Reducing water contents from 1.7 and 0.26 gH₂O g^-1 dry mass (g g^-1) is believed to increase cytoplasmic viscosity. Cooling to-196°C was done at rates averaging between 0.17 and 1300°C s^-1, and warming at 600 or 1.35°C s^-1. Survival was assessed after 4 weeks in vitro . Rapid warming resulted in higher survival and normal development of axes at all water contents. The effects of cooling rate were dependent on the water content of axes. Cooling rates resulting in >70% normal development ranged between 0.17 and about 1300°C s^-1 for axes at a water content of 0.26 g g^-1, narrowing with increasing hydration to an apparent optimum at about 686°C s^-1 in axes at 0.8 g g^-1. At 1.7 g g^-1, axes cooled at 0.17°C s^-1 yielded nearly 40% normal development, whereas faster cooling was deleterious. Results are interpreted in the context of the effect of water content on cytoplasmic viscosity and the rate of intracellular ice formation. At low water contents, the high intracellular viscosity slows ice crystallization making survival independent of cooling rate. At higher water contents, the reduced viscosity requires faster cooling to prevent ice crystal damage. The ability to cool rapidly with increasing hydration is balanced with an increasing limitation to dissipate heat fast enough to prevent severe damage.