Jennifer Crane

Cryopreservation of Recalcitrant (i.e. Desiccation-Sensitive) Seeds

cation and are often referred to as “recalcitrant” (Hong et al. 1998). Approximately 10–20% of angiosperm species produce seeds that acquire some, but not full, tolerance of desiccation during maturation (Dickie and Pritchard 2002). Incidence of recalcitrance does not distribute along phylogenetic clades, though some plant families include many species producing recalcitrant seeds (e.g., Fagaceae, Lauraceae, Sapindaceae, Meliaceae) while other families apparently lack species exhibiting this trait (e.g., Solanaceae, Asteraceae, Amaranthaceae). Life history traits of the plant, such as a long lived, perennial nature, and its habitat, such as aquatic or rainforest, are associated with seed recalcitrance, but not all plants with these characteristics produce recalcitrant seeds. The term “recalcitrant” is also used to describe seeds that are particularly difficult to germinate because they have deep dormancy or an unknown dormancy release mechanism. Though frustrating to work with, seeds with this dormancy physiology are amenable to

Survival of Zizania embryos in relation to water content, temperature and maturity status

The interactions between water content and temperature on freezing and desiccation damage were examined for Zizania (wild rice) embryos at several stages of maturity. The water content of excised embryos was manipulated by flash drying at 35°C or room temperature to between 2.5 g H 2 O/g dw and 0.05 g/g. Embryos were then exposed to temperatures ranging from 5 to −50°C. Viability following the drying and cooling treatments was assayed by leakage of electrolytes and germination in culture. Viability of embryos decreased when embryos were dried below a critical water content. The critical water content was greatest for the least mature embryos. Critical water contents were also temperature dependent and increased with decreasing temperature. Even though the critical water content varied with developmental status and temperature, the water activity corresponding to the critical water content appeared to be constant at 0.90. The most mature embryos survived temperatures as low as −50°C while the least mature embryos survived only to −18°C. These trends were predicted by ‘phase diagrams’ based on the physical properties of water in embryos at different stages of maturity (Vertucci et al. , 1994a). Our results confirm the earlier prediction that long term preservation of Zizania grains is possible at −20°C and the degree of success will be related to the maturity status of the embryos.

Massive cellular disruption occurs during early imbibition of Cuphea seeds containing crystallized triacylglycerols

The transition from anhydrobiotic to hydrated state occurs during early imbibition of seeds and is lethal if lipid reserves in seeds are crystalline. Low temperatures crystallize lipids during seed storage. We examine the nature of cellular damage observed in seeds of Cuphea wrightii and C. lanceolata that differ in triacylglycerol composition and phase behavior. Intracellular structure, observed using transmission electron microscopy, is profoundly and irreversibly perturbed if seeds with crystalline triacylglycerols are imbibed briefly. A brief heat treatment that melts triacylglycerols before imbibition prevents the loss of cell integrity; however, residual effects of cold treatments in C. wrightii cells are reflected by the apparent coalescence of protein and oil bodies. The timing and temperature dependence of cellular changes suggest that damage arises via a physical mechanism, perhaps as a result of shifts in hydrophobic and hydrophilic interactions when triacylglycerols undergo phase changes. Stabilizers of oil body structure such as oleosins that rely on a balance of physical forces may become ineffective when triacylglycerols crystallize. Recent observations linking poor oil body stability and poor seed storage behavior are potentially explained by the phase behavior of the storage lipids. These findings directly impact the feasibility of preserving genetic resources from some tropical and subtropical species.

Physical properties of water in Zizania embryos in relation to maturity status, water content and temperature.

Changes in the properties of water in excised embryos were measured during the late stages of grain development in two cultivars of Zizania palustris and a population of the endangered species Z. texana. The relationships between water content and water activity were determined from water sorption isotherms, measured at temperatures between 35 and 5°C and then derived for lower temperatures. The freezing and melting behaviour of water in embryos at different water contents was determined using differential scanning calorimetry. The moisture content of embryos at high water activities decreased with maturation, as did the moisture content at which freezing transitions were not observed. While the temperatures of freezing and melting transitions decreased as the moisture content of embryos decreased, there were no discernible differences among embryos at different developmental stages. The properties of water measured in maturing Zizania embryos approached those for orthodox seeds as determined from the strength of water sorption, the enthalpy of the melting transition and the moisture content at which water is unfreezable. From these data we conclude that the properties of water in recalcitrant Zizania embryos change with development to resemble those of embryos of desiccation-tolerant seeds, but that the seeds never achieve the orthodox condition. The effects of interactions between moisture content and temperature on desiccation damage, freezing damage and germination in Zizania are predicted, based on the physical properties of water reported here and the correspondence of these properties with physiological function reported for other species. The resulting ‘phase diagram’ defines possible combinations of moisture content and temperature for storage under equilibrium conditions.

Hydration of Cuphea seeds containing crystallised triacylglycerols

Seeds that exhibit intermediate storage behaviour seem to die under conventional -18°C storage conditions. Cuphea wrightii A. Gray, C. laminuligera Koehne, C. carthagenensis (Jacq.) J.F. Macbr. and C. aequipetala Cav are considered sensitive to low temperature storage. The seeds of these species have triacylglycerols (TAG) that are crystalline at -18°C and melt when the seeds are warmed to >35°C. In contrast, seeds of tolerant species, C. lanceolata W.T. Aiton and C. hookeriana Walp., have TAG that crystallise at temperatures below -18°C and are fluid at 22°C. Cuphea seeds imbided while TAG are crystalline fail to germinate and exhibit visual damage. However, germination proceeded normally when dry seeds were warmed adequately to melt any crystalline TAG before imbibition. Reduced germination and cellular disruption including loss of lipid body compartmentation and fragmented protein bodies develop in seeds with crystalline TAG equilibrated to >0.1 g H2O g-1 DW. This damage cannot be reversed, even when seeds are dried before the damage can be visually detected. Results from this work reveal that the seeds of some species with intermediate type physiologies can be successfully placed into conventional -18 and -80°C storage facilities.