A Simple but Effective Combination of pH Indicators for Plant Tissue Culture.

The use of pH indicators provides a simple, semi-quantitative visual method for quickly assessing pH changes in tissue culture media; however, pH indicators are rarely used in routine plant tissue culture media. In this study, chlorophenol red, bromocresol purple, and bromocresol green were tested to assess their functionality in the growth medium for plant shoot cultures. In addition, a combination of bromocresol green and bromocresol purple was tested to determine if they would widen the observable colour change to better assess pH changes in the medium. Varying the ratio of bromocresol green to bromocresol purple alters the pH at which the colour changes from blue to green to yellow, with a 1:3 ratio providing a useful pH range of 5-6.5, while a 1:1 ratio provides a useful pH range of 4.5-6. All the pH indicators showed no toxic side effects for the plant species tested in this study and were able to be autoclaved to ensure media sterility. The addition of these pH indicators to quickly assess media pH in large tissue culture collections can aid in routine maintenance. These pH indicators can be used as a 'traffic light' system, with blue indicating a high pH, green a normal pH, and yellow a low pH in the media.

How does metabolic rate in plant shoot tips change after cryopreservation?

Cryopreservation allows the long-term storage of plant germplasm, but can cause damage to plant tissues, which must be repaired for survival to occur. This repair process is fuelled by the metabolic function of mitochondria; however, little is known about how metabolic function is affected by the cryopreservation process in plants. We compared metabolic rates of shoot tips of two Australian native species, Androcalva perlaria and Anigozanthos viridis. Overall, cryopreservation resulted in a significant reduction in the metabolic rates of shoot tips from both species, even in tissues that regenerated after cryopreservation. Metabolic rate did not increase within 48 h after of thawing, even in shoot tips which later regenerated. When examined in isolation, both pre-treatment on desiccation medium and exposure to cryoprotective agents significantly decreased metabolic rates in regenerating shoot tips of A. viridis, however both caused a significant increase in shoot tips of A. perlaria, suggesting diversity of response to cryopreservation stresses across species. Measurements of shoot tip metabolic rate during cryopreservation will inform investigations into cellular energy production and provide critical information on the state of shoot health after exposure to different cryoprotective treatments, which could play a useful role in guiding protocol optimisation for threatened species to maximise post-cryopreservation regeneration.

Myrtaceae in Australia: Use of Cryobiotechnologies for the Conservation of a Significant Plant Family under Threat.

The Myrtaceae is a very large and diverse family containing a number of economically and ecologically valuable species. In Australia, the family contains approximately 1700 species from 70 genera and is structurally and floristically dominant in many diverse ecosystems. In addition to threats from habitat fragmentation and increasing rates of natural disasters, infection by myrtle rust caused by Austropuccinia psidii is of significant concern to Australian Myrtaceae species. Repeated infections of new growth have caused host death and suppressed host populations by preventing seed set. Although most Myrtaceae species demonstrate orthodox seed storage behavior, exceptional species such as those with desiccation sensitive seed or from myrtle rust-suppressed populations require alternate conservation strategies such as those offered by cryobiotechnology. Targeting seven key Australian genera, we reviewed the available literature for examples of cryobiotechnology utilized for conservation of Myrtaceae. While there were only limited examples of successful cryopreservation for a few genera in this family, successful cryopreservation of both shoot tips and embryonic axes suggest that cryobiotechnology provides a viable alternative for the conservation of exceptional species and a potential safe storage method for the many Myrtaceae species under threat from A. psidii.

Review: The case for studying mitochondrial function during plant cryopreservation.

Cryopreservation has several advantages over other ex situ conservation methods, and indeed is the only viable storage method for the long term conservation of most plant species. However, despite many advances in this field, it is increasingly clear that some species are ill-equipped to overcome the intense stress imposed by the cryopreservation process, making protocol development incredibly difficult using traditional trial and error methods. Cryobiotechnology approaches have been recently recognised as a strategic way forward, utilising intimate understanding of biological systems to inform development of more effective cryopreservation protocols. Mitochondrial function is a model candidate for a cryobiotechnological approach, as it underpins not only energy provision, but also several other key determinants of germplasm outcome, including stress response, reduction-oxidation status, and programmed cell death. Extensive research in animal cell and tissue cryopreservation has established a clear link between mitochondrial health and cryopreservation survival, but also indicates that mitochondria are routinely subject to damage from multiple aspects of the cryopreservation process. Evidence is already emerging that mitochondrial dysfunction may also occur in plant cryopreservation, and this research can be greatly expanded by using considered applications of innovative technologies. A range of mitochondria-targeted prophylactic and therapeutic interventions already exist with potential to improve cryopreservation outcomes through mitochondrial function.

Seed storage behaviour of tropical members of the aquatic basal angiosperm genus Nymphaea L. (Nymphaeaceae).

Eighteen native species of Nymphaea (waterlilies) inhabit a range of freshwater wetlands in northern Australia, which are threatened by increased development and the potential impacts of climate change. To investigate conservation seed banking of these vulnerable species, we aimed to characterize their seed storage physiology by determining (i) seed desiccation tolerance and (ii) the effects of moisture content and storage temperature on seed germination and viability. Seeds of N. immutabilis, N. lukei, N. macrosperma and N. violacea (including multiple collections of three species) were placed in experimental storage at a range of temperatures (25°C, 5°C, -20°C and -190°C) following pre-equilibration at different RHs (15%, 30%, 50%, 70% or 95%). Seeds were also experimentally aged at 60% RH and 45°C to assess comparative longevity. We found seeds of all species to be desiccation tolerant. However, the responses of seeds to experimental storage conditions were complex and variable between species and collections of the same species, and seeds of many species/collections were short-lived across many of the storage treatments. In many cases decreasing storage temperature did not increase longevity. Additional protocol development is necessary before we can have confidence that ex situ seed banking is a viable long-term germplasm conservation strategy for Nymphaea.

Monitoring of oxidative status in three native Australian species during cold acclimation and cryopreservation.

KEY MESSAGE: Three wild species exhibited a significant reduction in antioxidants throughout the cryopreservation protocol, whilst the half-cell reduction potential became more oxidised. Antioxidant content recuperated in recovering shoot tips. Cryopreservation is the most efficient and cost-effective long-term storage solution for the conservation of a wide range of plant species and material. Changes in the levels of antioxidants during the process of cryopreservation are known to reduce post-cryogenic survival due to oxidative stress. Low-molecular-weight thiols (cysteine, γ-glutamylcysteine, and glutathione) and ascorbic acid, which represent the two major water-soluble antioxidants in plants, were analysed at specific stages during cryopreservation of shoot tip material of three native Australian plant species [Anigozanthos viridis (Haemodoraceae), Lomandra sonderi (Asparagaceae), and Loxocarya cinerea (Restionaceae)] to quantify the oxidative stress experienced during cryopreservation. Post-cryogenic regeneration of shoot tips was greatest in A. viridis (78%) followed by L. sonderi (50%), whilst L. cinerea did not show any post-cryogenic regeneration. The application of a 3-week cold (5 °C) preconditioning regime, commonly used to increase post-cryogenic survival, resulted in significantly lower post-cryogenic regeneration for A. viridis (33%), but had little effect on the other two species. Total antioxidant concentration in shoot material decreased significantly with each step throughout the cryopreservation process, particularly in the cryoprotection and washing stages. Antioxidant levels in shoot tips then increased during the subsequent 7-day post-cryopreservation recovery period, with the greatest increase measured in A. viridis. Concentrations of thiols and their corresponding disulphides were used to calculate the corresponding half-cell reduction potentials, whereby the ability of these plant species to maintain a strong reducing environment in shoot tissues throughout the cryopreservation protocol was found to correlate with post-cryogenic survival.

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.

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.

Cryopreservation of Lomandra sonderi (Asparagaceae) shoot tips using droplet-vitrification.

A cryopreservation protocol was developed for Lomandra sonderi (Asparagaceae), an endemic plant of southwest Western Australia used for mine site restoration. Thermal analysis of L. sonderi shoot tips using differential scanning calorimetry was used to detect the formation of ice in shoot tips and consequently allowed optimisation of the time of incubation in plant vitrification solution 2 (PVS2), which attempted to minimise phytotoxicity of, and excessive dehydration by, its cryoprotective components. Sugar pretreatments did not improve survival. Use of a loading solution containing 2 M glycerol and 0.4 M sucrose prior to incubation in PVS2 improved survival of control shoot tips. Preconditioning at 20+/-1 degree C day/night alternating temperature with a 16 h photoperiod or at a constant 5 degree C temperature with a 12 h photoperiod both significantly improved both control shoot tip survival and post-cryopreservation survival. Shoot tips that recovered from liquid nitrogen immersion were successfully re-established as actively growing in vitro plantlets.