Effects of non-freezing low temperatures were studied on somatic embryo (SE) maturation in interior spruce (Picea glauca (Moench) Voss x engelmannii Parry ex. Engelm.). Embryo maturation cultures of two genotypes, cell lines ISP11 and ISP160, were kept at 5 degrees C, 10 degrees C, 15 degrees C or 20 degrees C, as well as in different temperature combinations for a culture period of 8 weeks or longer. Constant temperature treatment during maturation of 5 degrees C and 10 degrees C arrested SE development and inhibited maturation while 15 degrees C and 20 degrees C promoted maturation. An initial pretreatment of 4 weeks at 20 degrees C was required to achieve morphologically normal mature embryos. These were subjected to 5 degrees C, 10 degrees C, 15 degrees C or 20 degrees C during embryo maturation to test whether low temperature affected the acquisition of cold tolerance in mature embryos. Low temperature treatments altered cold tolerance: freezing damage tests based on electrolyte leakage showed that SEs matured at low temperatures possessed significantly higher freezing tolerance than SEs matured at 20 degrees C. It was also shown that such clonal embryos have a broad phenotypic plasticity regarding this trait. An ultrastructural study showed that accumulation of lipids, starches and proteins was influenced by temperature. Prolonged cold (5 degrees C) inhibited build up of storage reserves. In all other treatments lipids were generally abundant and often associated with protein bodies. At low temperatures, protein bodies were small in size and starch grains showed odd shapes, as well as evidence of degradation. Embryo conversion was significantly better at low temperatures. There were genotype differences, in particular, a decline in conversion due to longer maturation periods.
