Christina W. Vertucci

Homoiohydrous (recalcitrant) seeds: Developmental status, desiccation sensitivity and the state of water in axes of Landolphia kirkii Dyer

The desiccation sensitivity in relation to the stage of development was investigated in embryonic axes from the homoiohydrous (recalcitrant) seeds of Landolphia kirkii. Electrolyte leakage, used to assess membrane damage after flash (very rapid) drying, indicated that axes from immature (non-germinable) seeds were the most desiccation-tolerant, followed by those from mature seeds, while axes from seeds germinated for increasing times were progressively more desiccation-sensitive. Differential scanning calorimetry was used to study the relationship between desiccation sensitivity and the properties of water in the tissues. Axes from immature seeds had a lower content of non-freezable water than that of any other developmental stage and a higher enthalpy of melting of freezable water. For mature and immature axes electrolyte leakage increased at the point of loss of freezable water. At other developmental stages the water content at which electrolyte leakage increased markedly correlated with the other properties of the water, such as the change in the shape of the melting endotherm and the onset temperature. Ultrastructural studies of axes at the various developmental stages showed changes in the degree and pattern of vacuolation, the presence and quantities of lipid and starch, and the degree of endomembrane development. The results are discussed in relation to current hypotheses on the basis of desiccation tolerance.

Cryopreservation of desiccation-sensitive axes of Camellia sinensis in relation to dehydration, freezing rate and the thermal properties of tissue water

Summary The inter-relationships between water content, rate of freezing to −196 °C, thermal properties of water, and survival were studied in excised embryonic axes of tea seeds. Three freezing rates were used: 10°C min−1, about 200°C min−1 and rapid freezing (attained by plunging samples into nitrogen slush). Differential scanning calorimetry yielded three categories of melting endotherms: those without endothermic peaks (no freezable water), those with a broad endothermic peak with onset temperature varying with water content, and those with an additional sharp peak at about −2°C. When axes were cooled slowly, the sharp peak was present at high water contents (> 1.8 g H2O g−1 dw) and was diminished as axes were dried. The sharp peak was not apparent when axes were subjected to rapid freezing. Axes at moisture contents and subjected to freezing rates such that the sharp peak was present in the melting endotherms did not survive in tissue culture. Axes rapidly dried to water contents between 1.1 and 1.6 g H2O g−1 dry mass prior to rapid freezing showed 100 % survival in tissue culture. Ultrastructural studies of freeze fractured replicas showed considerable freezing damage at intermediate freezing rates, but good preservation of subcellular detail under rapid freezing conditions. It is suggested that the sharp peak observed on melting endotherms represents the melting of pure water arising from ice crystals formed during freezing. If axes with a sufficiently high water content are cooled at a freezing rate that prevents the formation of large ice crystals, damage is minimized and survival enhanced. If axes are dried to water content close to the level of non-freezable water, the additional stress of freezing is deleterious.

Effects of developmental status and dehydration rate on characteristics of water and desiccation-sensitivity in recalcitrant seeds of Camellia sinensis

The effect of rate of dehydration was assessed for embryonic axes from mature seeds of Camellia sinensis and the desiccation sensitivity of axes of different developmental stages was estimated using electrolyte leakage. Rapidly (flash) dried excised axes suffered desiccation damage at lower water contents (0.4 g H 2 O (g DW) −1 ) than axes dried more slowly in the whole seed (0.9 g H 2 O (g DW) −1 ). It is possible that flash drying of isolated axes imposes a stasis on deteriorative reactions that does not occur during slower dehydration. Differential scanning calorimetry (DSC) of the axes indicated that the enthalpy of the melting and the amount of non-freezable water were similar, irrespective of the drying rate. Very immature axes that had completed morphogenesis and histodifferentiation only were more sensitive to desiccation (damage at 0.7 g H 2 O (g DW) −1 ) than mature axes or axes that were in the growth and reserve accumulation phase (damage at 0.4 g H 2 O (g DW) −1 ). As axes developed from maturity to germination, their threshold desiccation sensitivity increased to a higher level (1.3−1.4 g H 2 O (g DW) −1 ). For the very immature axes, enthalpy of the melting of tissue water was much lower, and the level of non-freezable water considerably higher, than for any other developmental stage studied. There were no marked correlations between desiccation sensitivity and thermal properties of water. Desiccation sensitivity appears to be related more to the degree of metabolic activity evidenced by ultrastructural characteristics than to the physical properties of water.