Thermal behaviour of lipids in short-lived seeds of Australian rainforest species

Authors: SOMMERVILLE, KAREN - Royal Botanic Garden Sydney; Hill, Lisa; OFFORD, CATHERINE - Royal Botanic Garden Sydney; Walters, Christina

Submitted to: Annals of Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/4/2025
Publication Date: N/A
Citation: N/A

Interpretive Summary: There is a class of seeds that survive drying but not freezer storage. We typically think that death upon exposure to low temperature results from water freezing, but water does not freeze in these seeds because the seeds are so dry. We, therefore explored the concept of lipids freezing (i.e. crystallizing) during conventional genebanking conditions of -20C. We used differential scanning calorimetry to assess crystal nucleation and growth of seed oils. We observed extensive themal activity (i.e., crystalization, melting, and recrystalization) in the -20C temperature range used to genebank most seeds, which we believe is related to damage in this class of seeds. We can circumvent this damage by cryogenically storing seeds (at liquid nitrogen temperatures), which would require a shift in thinking and protocol for standard genebanks, including NLGRP. Most plant seed banks do not have cryogenic facilities so they are unsuitable for preserving genetic resources of seeds originating from tropical and subtropical areas. This is important to the US nut crop industry as well as US commercial interests in breeding crop seeds that produce tropical oils.

Technical Abstract: Studies on the storage behaviour of desiccation-tolerant Australian rainforest seeds demonstrated that around a quarter were short-lived in storage. We sought to understand structural changes that might occur in seed cells upon storage at -20°C, particularly from lipid fractions, that could contribute to and aid in predicting such a short lifespan. We used differential scanning calorimetry (DSC) to examine energy exothermic and endothermic transitions during freezing and thawing in dry seed samples of 23 Australian rainforest species. Seed samples and extracted triacylglycerols (TAGs) were cooled to -150°C and rewarmed to 50°C at 10°C min-1; slower and faster rates of cooling/warming were used for a subset of species to examine lipid crystallisation and melting kinetics. Thermograms were analysed for temperature and enthalpy of observed peaks, and these were compared with expected values to detect anomalies. Extracted lipids were further analysed using gas chromatography (GC) to characterize fatty acid composition. The thermal profiles of six species were used to design experiments comparing the impact of storage at -20°C to storage at temperatures outside the range of observed thermal transitions. Thermal activity was detected in all samples within the narrow temperature range of -30 and -10°C; activity at broader temperature ranges was also detected depending on species, cooling protocol and fatty acid composition. A profound interaction between DSC parameters and time at low temperature, as well as fatty acid composition, led to the hypothesis that TAG crystallisation rates contribute to low temperature sensitivity. We confirmed that damage from TAG crystallisation could be avoided by storing seeds short-term at temperatures above TAG crystallisation and melting events; storage at cryogenic temperatures (where further structural changes to TAG crystals would be inhibited) improved survival over storage at -20°C but requires further optimisation to maintain pre-storage germination potential. We conclude that the crystallisation and melting of TAGs during storage impact seed longevity. Seed thermal profiles and rate of TAG crystallisation may serve as predictive tools for sensitivity to storage at -20°C.