The role of oxidative stress in successful cryopreservation of south-west Western Australian plant species

The southwest Western Australian Floristic region contains high plant diversity in need of conservation. Cryopreservation, the storage of plants in liquid nitrogen (-196°C), is the safest long-term conservation method available. However, there are many factors that can influence the plants ability to survive cryopreservation. Oxidative stress, which can damage DNA, proteins, and lipids, was shown to be a major factor affecting cryopreservation. Plants that mitigated excessive oxidative stress showed improved post-cryogenic survival

Cold-induced changes affect survival after exposure to vitrification solution during cryopreservation in the south-west Australian Mediterranean climate species Lomandra sonderi (Asparagaceae)

There is limited knowledge of the effects of exposure to low temperatures in the unique Mediterranean climate plant species of Western Australia. We have thus investigated the effect of low temperature on cryogenic tolerance in Lomandra sonderi, an endemic perennial species of southwest Western Australia. Lomandra sonderi plants were preconditioned in tissue culture at constant 23 °C (12 h light/dark cycle) or alternating 20/-1 °C (16 h light and 8 h dark cycle). Shoot tips from both conditions were analysed for their phospholipid, sterol and soluble sugar compositions. Shoot tips were also cryoexposed via a droplet-vitrification protocol. Survival in both preconditioning regimes for cryoexposed and non-cryoexposed samples was the same, but plants from the 20/-1 °C regime displayed an improved tolerance to the overall cryopreservation process in both cryoexposed and non-cryoexposed samples, thereby eliminating exposure to liquid nitrogen as a primary cause of reduced post-cryogenic viability. Preconditioning of in vitro shoots of L. sonderi at 20/-1 °C induced significant increases in phosphatidylcholine (from 7.30 ± 3.46 to 22.2 ± 7.80 ng mg-1 FW) and increases in several soluble sugars (fructose, galactose, glucose, sucrose) compared to shoots incubated at 23 °C—changes consistent with known cold acclimation responses in plant species generally—but sterol content remained largely unchanged. Analysis of electrolyte leakage in shoot tips from both preconditioning regimes generated a significantly lower LT50 value in the 20/-1 °C samples (-5.45 ± 0.53 °C) over the 23 °C samples (-2.5 ± 0.08 °C). Increased tolerance to cryoexposure in L. sonderi appears to lie mainly with acclimation-induced changes in membrane composition and promotion of membrane stability and hence increased resistance to freeze damage

Cryopreserving plants for long-term conservation

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Cryo-mesh: a simple alternative cryopreservation protocol

BACKGROUND: The continued development of new cryopreservation protocols has improved post-cryogenic success rates for a wide variety of plant species. Methods like the cryo-plate have proven beneficial in simplifying the cryopreservation procedure. OBJECTIVE: This study assessed the practicality of a stainless steel mesh strip (cryo-mesh) for cryopreserving shoot tips from Anigozanthos viridis. MATERIALS AND METHODS: Shoot tips of A. viridis (Kangaroo Paw) were precultured on 0.4 M sucrose medium for 48 h. Precultured shoot tips were coated in a 2% alginate solution and placed onto the cryo-mesh (a 25 x 7 mm, 0.4 mm aperture, 0.224 mm diameter wire stainless steel mesh strip). The alginate was set for 20 min in a loading solution containing 100 mM CaCl2, anchoring the shoot tips to the cryo-mesh. The cryo-mesh was then transferred to PVS2 on ice for 20, 30 or 40 min prior to plunging the cryo-mesh into liquid nitrogen. The cryo-mesh protocol was compared to the droplet-vitrification protocol. RESULTS: A maximum of 83% post-cryogenic regeneration was achieved with the cryo-mesh when exposed to PVS2 for 30 min. No significant difference in post-cryogenic regeneration was observed between the cryo-mesh and droplet-vitrification protocols. CONCLUSION: Anigozanthos viridis shoot tips were successfully cryopreserved utilising the new cryo-mesh. The cryo-mesh thus provides a simple and successful alternative for cryopreservation

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

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.</jats:p

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&ndash;6.5, while a 1:1 ratio provides a useful pH range of 4.5&ndash;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 &lsquo;traffic light&rsquo; system, with blue indicating a high pH, green a normal pH, and yellow a low pH in the media

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