Development of cryopreservation for Loxocarya cinerea - an endemic Australian plant species important for post-mining restoration

We report the development of a cryopreservation protocol for the endemic Western Australian plant species Loxocarya cinerea (Restionaceae). Shoot tips from two genotypes, SXH404 and SXH804, were cryopreserved using the droplet-vitrification technique. Control explants, which were cryoprotected, but not cooled, showed regeneration for both genotypes (SXH404, 22.1 ± 5.9%; SXH804, 67.7 ± 9.6%). Extension of incubation in PVS2 from 30 to 60 min did not lead to survival after cryopreservation. Thermal analysis using differential scanning calorimetry confirmed the beneficial effect of a loading phase but also revealed no or very little ice formation after cryoprotection of shoot tips in other treatments. Regeneration following cryopreservation was obtained for genotype SXH804 (4.3 ± 2.1%) but not for SXH404. Regenerated explants of L. cinerea SXH804 were morphologically identical to tissue-cultured plants. As an alternative to shoot tips, callus tissues of clone SXH404 were successfully cryopreserved (>66.7% post LN survival) using the same protocol

De Nederlandse geschiedenis is in de negentiende eeuw verzonnen. Interview met Maria Grever, Pieter Huistra, James Kennedy en Peter Raedts

De huidige strijd over de Nederlandse geschiedenis is niets nieuws. Het begon allemaal met gekrakeel van historici tweehonderd jaar geleden

'Deze moslima's zijn vooral voetballer'

Contains fulltext : 209633pub.pdf (publisher's version ) (Open Access)Meisjes die mee willen doen aan pleintjesvoetbal worden niet zomaar toegelaten door de jongens. Ze worden ook anders gecoacht.17 mei 201

'Deze moslima's zijn vooral voetballer'

Meisjes die mee willen doen aan pleintjesvoetbal worden niet zomaar toegelaten door de jongens. Ze worden ook anders gecoacht

Evaluation of the new vacuum infiltration vitrification (viv) cryopreservation technique for native Australian plant shoot tips

BACKGROUND: The application of a vacuum during the incubation in cryoprotective agents such as PVS2 allows for increased penetration, reducing total incubation times required before vitrification and post-cryopreservation regeneration is achieved. OBJECTIVE: This study compared a conventional droplet-vitrification protocol to the new vacuum infiltration vitrification protocol in four Australian plant species. MATERIALS AND METHODS: The new vacuum infiltration vitrification applied an 80 kPa vacuum during incubations in loading solution and PVS2. Infiltration of the cryoprotective agents into shoot tips was determined by differential scanning calorimetry measuring ice formation in the thermographs comparing a range of loading solution and PVS2 incubation times. RESULTS AND CONCLUSION: The application of the vacuum infiltration vitrification technique resulted in a significantly reduced PVS2 incubation time for cryogenic survival and regeneration for all four species, reducing the time needed to adequately protect shoot tips by half to a quarter when compared to a conventional droplet-vitrification technique

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 (180mM) 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

Current issues in plant cryopreservation and importance for ex situ conservation of threatened Australian native species

© 2019 CSIRO. An alarming proportion of Australia's unique plant biodiversity is under siege from a variety of environmental threats. Options for in situ conservation are becoming increasingly compromised as encroaching land use, climate change and introduced diseases are highly likely to erode sanctuaries regardless of best intentions. Ex situ conservation is currently limited to botanic garden living collections and seed banking, with in vitro and cryopreservation technologies still being developed to address ex situ conservation of species not amenable to conventional storage. Cryopreservation (storage in liquid nitrogen) has been used successfully for long-term biosecure storage of shoot tips of several species of threatened Australian plants. We present a case for building on this research and fostering further development and utilisation of cryopreservation as the best means of capturing critical germplasm collections of Australian species with special storage requirements (e.g. recalcitrant-seeded taxa and species with short-lived seeds) that currently cannot be preserved effectively by other means. This review highlights the major issues in cryopreservation that can limit survival including ice crystal damage and desiccation, toxicity of cryoprotective agents, membrane damage, oxidative stress and mitochondrial function. Progress in understanding and mitigating these stresses is vital for advancing cryopreservation for conservation purposes

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

Acclimation-induced changes in cell membrane composition and influence on cryotolerance of in vitro shoots of native plant species

Cell membranes are the primary sites of cryopreservation injury and measuring changes to membrane composition arising from cold acclimation may assist with providing a rationale for optimising cryopreservation methods. Shoot tips from two south-west Western Australian species, Grevillea scapigera and Loxocarya cinerea, and Arabidopsis thaliana (reference species) were subjected to cryopreservation using the droplet vitrification protocol. Two pre-conditioning regimes involving a constant temperature (23 °C, CT with a 12 h light/dark cycle) or an alternating temperature (AT) regime (20/10 °C with a 12 h light/dark cycle) were compared. Soluble sugars, sterols and phospholipids present in the shoot tips were analysed. Use of AT pre-conditioning (acclimation) resulted in a modest decrease in cryotolerance in A. thaliana, increased cryotolerance in G. scapigera, and increased survival in the non-frozen control explants of L. cinerea in comparison to CT pre-conditioning. Increased cryotolerance was accompanied by a higher total sugar sterol and phospholipid content, as well as an increase in strong hydrating phospholipid classes such as phosphatidylcholine. The double bond index of bound fatty acyl chains of phospholipids was greater after AT pre-conditioning, mostly due to a higher amount of monoenes in A. thaliana and trienes in G. scapigera and L. cinerea. These findings suggest that AT pre-conditioning treatments for in vitro plants can have a positive influence on cryotolerance for some plant species and this may be related to observed changes in the overall composition of cell membranes. However, alternative factors (e.g. oxidative stress) may be equally important with other species (e.g. L. cinerea)