Bryn Funnekotter

Current Issues in Plant Cryopreservation

Anja Kaczmarczyk1,2, Bryn Funnekotter1,2, Akshay Menon1,2, Pui Ye Phang1,2, Arwa Al-Hanbali1,2, Eric Bunn2,3 and Ricardo L. Mancera1 1Curtin Health Innovation Research Institute, Western Australian Biomedical Research Institute, Curtin University, Perth 2Botanic Gardens and Parks Authority, Fraser Avenue, West Perth 3School of Plant Biology, Faculty of Natural and Agricultural Sciences, University of Western Australia Australia

Influence of abiotic stress preconditioning on antioxidant enzymes in shoot tips of Lomandra sonderi (Asparagaceae) prior to cryostorage

Lomandra sonderi (F.Muell.) Ewart (Asparagaceae) is endemic to the south-west Western Australian jarrah (Eucalyptus marginata Donn ex Sm.) forest region, and is a difficult to propagate species important to post-mining restoration. Micropropagation is the only way to currently produce plants of this species for restoration. This study describes investigations into optimising cryopreservation for efficient long-term germplasm storage. In order to investigate the effect of preconditioning on post-cryogenic survival of shoot tips, in vitro grown plants were exposed to a range of light-, temperature- and osmotic-induced preconditioning treatments under culture room conditions for 3 weeks. Room temperature (24°C) preconditioning resulted in the greatest post-cryogenic survival, followed by low light (17 µmol m–2 s–1) preconditioning. Alternating temperature (25/5°C), high temperature (35°C), high sucrose (180 mM) and high light (93 µmol m–2 s–1) preconditioning treatments all led to significantly and progressively lower post-cryogenic shoot tip survival than room temperature preconditioning. Antioxidant activity of superoxide dismutase in preconditioned shoot tips showed a positive correlation to post-cryogenic survival overall, whereas the activities of glutathione reductase, glutathione peroxidase and catalase showed little correlation. Analysis throughout the cryopreservation protocol showed that the activity of glutathione reductase decreased significantly after cryopreservation, whilst the activity of glutathione peroxidase and catalase did not change.

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.