Biophysical Characteristics of Successful Oilseed Embryo Cryoprotection and Cryopreservation Using Vacuum Infiltration Vitrification: An Innovation in Plant Cell Preservation

Heterogeneity in morphology, physiology and cellular chemistry of plant tissues can compromise successful cryoprotection and cryopreservation. Cryoprotection is a function of exposure time × temperature × permeability for the chosen protectant and diffusion pathway length, as determined by specimen geometry, to provide sufficient dehydration whilst avoiding excessive chemical toxicity. We have developed an innovative method of vacuum infiltration vitrification (VIV) at 381 mm (15 in) Hg (50 kPa) that ensures the rapid (5 min), uniform permeation of Plant Vitrification Solution 2 (PVS2) cryoprotectant into plant embryos and their successful cryopreservation, as judged by regrowth in vitro. This method was validated on zygotic embryos/embryonic axes of three species (Carica papaya, Passiflora edulis and Laurus nobilis) up to 1.6 mg dry mass and 5.6 mm in length, with varying physiology (desiccation tolerances) and 80 °C variation in lipid thermal profiles, i.e., visco-elasticity properties, as determined by differential scanning calorimetry. Comparisons between the melting features of cryoprotected embryos and embryo regrowth indicated an optimal internal PVS2 concentration of about 60% of full strength. The physiological vigour of surviving embryos was directly related to the proportion of survivors. Compared with conventional vitrification, VIV-cryopreservation offered a ∼ 10-fold reduction in PVS2 exposure times, higher embryo viability and regrowth and greater effectiveness at two pre-treatment temperatures (0 °C and 25 °C). VIV-cryopreservation may form the basis of a generic, high throughput technology for the ex situ conservation of plant genetic resources, aiding food security and protection of species from diverse habitats and at risk of extinction

Compromised root development constrains the establishment potential of native plants in unamended alkaline post-mining substrates

© 2018, Springer Nature Switzerland AG. Background and aims: Mined materials often require rehabilitation or ecological restoration through revegetation as part of mine closure and relinquishment practices, yet there is a widening gap between the expectations of recovery and what industry achieve. The edaphic conditions of post-mining substrates present a suite of potential limitations to plant growth and may constrain the establishment capability and development of native species. Methods: We assessed seedling emergence, relative growth rate and calculated standardised growth estimates using 10 measured root and shoot parameters for six locally-dominant native species from different families and nutrient-acquisition strategies in a range of representative mining restoration substrates (topsoil, tailings, capped tailings and waste rock), examining their suitability as pioneers for ecological restoration. Results: The establishment and growth of all six species in post-mining substrates were significantly compromised. Root development was significantly responsive to substrate, with measured root parameters on average 27% lower in capped tailings, 41% lower in waste rock and 67% lower for individuals grown in tailings compared with those grown in topsoil alone. Plant growth was compromised at different life cycle stages (seed germination, seedling establishment, early growth and development) and across a number of different traits, with primary edaphic constraints including high pH (>8.5) and insufficient available N. The highest-performing species on post-mining substrates was an N2-fixing legume, while lowest-performing species included those with ectomycorrhizal associations or no specific nutrient-acquisition strategy. Conclusions: Edaphic filters may be significant drivers of trajectory and success in rehabilitation and restoration projects at scales ranging from individuals (by limiting establishment or constraining growth and development) to communities (by causing species to assemble in a different manner than the desired reference community). If intractable edaphic parameters constraining plant establishment and early development such as extreme pH and a lack of available nutrients are not ameliorated, the restoration trajectory on post-mining landforms is likely unfavourable. Failure to adequately ameliorate post-mining substrates may represent a major liability for industry in meeting mine-closure requirements