Submitted to: Cryo-Letters
Publication Type: Peer reviewed journal
Publication Acceptance Date: 11/1/2003
Publication Date: 12/1/2004
Citation: Wesley-Smith, J., C. Walters, P. Berjak and N.W. Pammenter. 2004. The influence of water content, cooling and warming rate upon survival of embryonic axes of Poncirus trifolata (L.). CryoLetters 25:129-138. Interpretive Summary: In a previous paper, we showed that we could achieve cooling rates to liquid nitrogen temperatures of hundreds of degrees (C) per sec for trifoliate orange embryos by manipulating cooling protocols and thermal mass. This ability allows us to test the hypothesis that as cytoplasmic viscosity increases (by reducing the water content of embryos), the stringency of a requirement for rapid cooling can be relaxed. This hypothesis is supported by data showing that as embryos are progressively dried, the cooling rate required for survival is reduced. Survival and normal development were noted in greater than 85% of embryos given appropriate cooling protocols, demonstrating that routine cryostorage of citrus germplasm is currently possible.
Technical Abstract: 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 Poncirus trifoliata seeds. Reducing water contents from 1.7 and 0.26 g H2O/g dry weight is believed to increase cytoplasmic viscosity. Cooling to -196C was done at rates averaging between 0.17 and 1300C/s, and warming rates averaged 600 or 1.35C/s. 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 1300C/s for axes containing 0.26 g H2O/g dry weight, narrowing with increasing hydration to an apparent optimum at about 414C/s in axes containing 0.8 g H2O/g dry. At 1.7 g H2O/g dry, axes cooled at 0.17 C/s 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.