Cryopreservation of embryogenic cultures of Picea mariana (black spruce) using vitrification

This study reports on the first use of a vitrification procedure for the successful cryopreservation of embryogenic cultures of a coniferous species. Using Picea mariana embryogenic cultures, we obtained the highest survival by first preculturing embryogenic masses on semi-solid medium containing 0.8 M sorbitol for 48 h followed by incubation in PVS2 cryoprotective vitrification solution at 0°C for 30 min and direct immersion in liquid nitrogen. The replacement of sorbitol with 1.6 M glycerol also resulted in high survival. When sorbitol and glycerol were used in preculture treatments, 9 of 11 embryogenic lines survived liquid nitrogen treatments. We also demonstrated that 100% post-liquid nitrogen survival of mature somatic embryos could be obtained without pretreatments. A brief desiccation pretreatment, however, increased uniformity of somatic embryo germination

Seed ageing of four Western Australian species in relation to storage environment and seed antioxidant activity

The influence of the storage environment on seed viability and antioxidant potential was examined for four species native to Western Australia: Acacia bivenosa DC., Anigozanthos manglesii D. Don, Banksia ashbyi E.G. Baker, and Mesomelaena tetragona (R. Br.) Benth. Seeds were stored at four water contents (at c. 5%, 11-15%, 20-23% and 50% relative humidity) at each of five temperatures (-196, -18, 5, 23 and 50°C), and seed germination and seedling vigour monitored over an 18-month period. Deterioration was apparent in all species (except A. bivenosa) stored at 50°C, with 11% RH maximizing longevity for B. ashbyi and M. tetragona seeds, and 5% or 11% RH preventing deterioration for A. manglesii seeds. Seed viability generally remained high for all species stored at 23°C or less. Notably, however, germination and seedling vigour of A. manglesii and M. tetragona seeds gradually declined when stored at -18°C, suggesting that storage at this temperature was detrimental. The antioxidant activity of lipid extracts of seeds after 18 months storage at 5, 23 and 50°C was also examined to determine whether the seed viability decline was associated with a loss of antioxidants. Antioxidant activity varied between storage treatments and was not related to seed viability

Water sorption characteristics of seeds of four Western Australian species

The relationship between storage temperature, relative humidity and seed water content was investigated for four species native to Western Australia: Acacia bivenosa DC., Anigozanthos manglesii D. Don., Banksia ashbyi E.G. Baker and Mesomelaena tetragona (R.Br.) Benth. Water sorption isotherms were constructed at 5, 23 and 50°C and the enthalpy of water sorption was calculated by van\u27t Hoff analysis. Seeds of three species, A. manglesii, B. ashbyi and M. tetragona, showed a sigmoidal relationship between seed water content and relative humidity. Intact seeds of Acacia bivenosa maintained a constant water content at temperatures of 23°C or less due to the impermeable seed coat; however, isotherms of scarified seeds were similar in shape to those of the other species at all temperatures. The enthalpy of water sorption ranged from -19 kJ mol-1 for M. tetragona seeds to -29 kJ mol-1 for B. ashbyi seeds and was dependent on water content. However, all species had a maximum sorption strength at 2-6% water content and three regions of water-binding strength were evident. Each of these species has water sorption characteristics consistent with orthodox storage behaviour and the results of this study provide a framework for improving seed storage methods for the highly diverse Western Australian flora

Advances in understanding the fundamental aspects required for successful cryopreservation of Australian flora

Australia is host to an amazing diversity of species, many of which require conservation efforts. In vitro culture provides a tool for not only conserving these threatened species but allows for their propagation from limited starting material. Cryopreservation provides the greatest long-term storage option for in vitro cultures and as a conservation tool for other germplasm. However, while cryopreservation has proven capable of delivering viable long-term storage with some plant taxa, the process of deriving protocols is still largely an incremental process. The key to faster and more intuitive optimising of cryopreservation protocols lies with continuing to develop a better understanding of key factors, including issues with plant physiology (such as genetic stability, the composition of the proteome and metabolome, cell membrane characteristics, and antioxidant defences) and how the stresses imposed by cryopreservation (such as the excision damage, desiccation, cryoprotective agent toxicity, ice crystal damage, and cooling to cryogenic temperatures) interact and contribute to the cryocapability of a species. This review focuses on the advances that have been made towards understanding cryogenic stress and how this has led to improved cryopreservation protocols, in the context of cryopreserving Australian flora