Eric Bunn

Acetyl salicylic acid (Aspirin) and salicylic acid induce multiple stress tolerance in bean and tomato plants

The hypothesis that physiologically activeconcentrations of salicylic acid (SA) and itsderivatives can confer stress tolerance in plants wasevaluated using bean (Phaseolus vulgaris L.) andtomato (Lycopersicon esculentum L.). Plantsgrown from seeds imbibed in aqueous solutions (0.1--0.5 mM) of salicylic acid or acetyl salicylic acid(ASA) displayed enhanced tolerance to heat, chillingand drought stresses. Seedlings acquired similarstress tolerance when SA or ASA treatments wereapplied as soil drenches. The fact that seedimbibition with SA or ASA confers stress tolerance inplants is more consistent with a signaling role ofthese molecules, leading to the expression oftolerance rather than a direct effect. Induction ofmultiple stress tolerance in plants by exogenousapplication of SA and its derivatives may have asignificant practical application in agriculture,horticulture and forestry.

Biodiversity conservation and conservation biotechnology tools

This special issue is dedicated to the in vitro tools and methods used to conserve the genetic diversity of rare and threatened plant species from around the world. Species that are on the brink of extinction because of the rapid loss of genetic diversity and habitat come mainly from resource-poor areas of the world and from global biodiversity hotspots and island countries. These species are unique because they are endemic, and only a few small populations or sometimes only a few individuals remain in the wild. Therefore, the challenges to support conservation by in vitro measures are many and varied. The editors of this invited issue solicited papers from experts from Asia, Africa, Europe, Australia, and North and South America. This compilation of articles describes the efforts in these diverse regions toward saving plants from extinction, and details the direct application of in vitro and cryopreservation methods. In addition, these contributions provide guidance on propagation of rare plants, including techniques for large-scale propagation, storage, and reintroduction. The in vitro techniques for conserving plant biodiversity include shoot apical or axillary-meristem-based micropropagation, somatic embryogenesis, cell culture technologies and embryo rescue techniques, as well as a range of in vitro cold storage and cryopreservation protocols, and they are discussed in depth in this issue.

Benzoic acid may act as the functional group in salicylic acid and derivatives in the induction of multiple stress tolerance in plants

Benzoic acid, sulfosalicylic acid and methyl salicylic acid wereevaluated for their regulatory role in inducing multiple stress tolerance inbean (Phaseolus vulgaris cv Brown Beauty) and tomato(Lycopersicum esculentum cv Romano) plants. All threemolecules were effective in inducing tolerance to heat, drought and chillingstress similar to that reported previously for salicylic and acetylsalicylicacids. Benzoic acid is effective at lower concentrations than salicylic acid orits derivatives. The benzoic acid structural portion is common to all fivemolecules and is the most likely basic functional molecular structure impartingstress tolerance in plants.

Smoke-saturated water promotes somatic embryogenesis in geranium

The effect of smoke saturated-water (SSW) on somatic embryogenesis was studied using geranium hypocotyl culture as a model system. Treatment of explants with 10% SSW or the inclusion of SSW with thidiazuron, a compound which induces somatic embryogenesis, enhanced the embryogenic potential of the geranium hypocotyl culture. Prolonged exposure to SSW was detrimental to embryogenesis. The SSW treatment also accelerated the rate of embryo development suggesting a growth regulatory role of SSW.

Genetic fidelity and viability of Anigozanthos viridis following tissue culture, cold storage and cryopreservation

Abstract The effects of long-term storage conditions on the viability and genetic fidelity of plant somatic tissues are poorly known. In this study, the effects of three storage methods (tissue culture, cold storage and cryostorage) on genetic fidelity and shoot apex viability were evaluated for Anigozanthos viridis subspp. terraspectans (Haemodoraceae), a threatened plant from south west Australia. Genetic fidelity was assessed following 12 months of storage using the PCR-based multi-locus DNA fingerprinting technique Amplified Fragment Length Polymorphism (AFLP). Shoot apex viability was evaluated at 0, 3, 6 and 12 months for cryogenically stored material. The AFLP technique generated a total of 95 fragments for three primer pairs, and no differences were detected across treatments. Post-cryostorage viability was high (mean=85%) and not significantly different across storage times. These results show that genetic fidelity and shoot apex viability (for cryopreserved material) were maintained following tissue culture, cold storage and cryostorage of A. viridis subspp. terraspectans for up to 12 months.

Stereochemical arrangement of hydroxyl groups in sugar and polyalcohol molecules as an important factor in effective cryopreservation

The efficacy of several sugars and polyalcohols in preculture medium was investigated using Anigozanthos viridis ssp terraspectans Hopper (Haemodoraceae), a threatened plant species endemic to the south west of Western Australia. A vitrification protocol involving preculturing of shoot apices for 3 days on different concentrations of sugars and polyalcohols, followed by incubation in plant vitrification solution 2 (PVS2) for 25 min, prior to immersion in liquid nitrogen (LN) and warming resulted in shoot tip survival ranging from 34 to 84%. High levels of survival were obtained with polyalcohols, compared to sucrose, glucose, trehalose and raffinose when used at the same molarity (0.4 M) or at the equivalent concentration of total hydroxyl (OH) groups present in molecules. In both cases glycerol proved more effective. When polyalcohols (ribitol and erythritol) with similar stereochemical arrangement of OH groups as glycerol were examined, at the same molarity (0.4 M) and with equivalent OH numbers, higher survival was achieved when the total number of OH groups present was the same as glycerol. Additionally, when the structural isomers mannitol/sorbitol and ribitol/xylitol were compared at the same molarity (0.4 M), the isomer with the higher number of OH groups along the same side resulted in significantly higher levels of post-LN survival. We propose that the mode of action of polyalcohols is based not on molarity, but on the total number of OH groups present in the medium. Furthermore, based on these results we propose that the orientation of OH groups is a determining factor in effective cryopreservation.

The contribution of in vitro technology and cryogenic storage to conservation of indigenous plants

In vitro culture has enabled a variety of recalcitrant and threatened plant taxa to be micropropagated in the absence of viable conventional propagation methods. Cryogenic storage research has provided alternative protocols for efficient long-term germplasm storage for many plant species. Recent advances in tissue-culture methods such as somatic embryogenesis have enabled the production of >20 000 somatic embryos of a recalcitrant native Australian rush in a few months, far higher than other in vitro methods for these types of plants. Cryogenic protocols are reported for >30 species of Australian vascular plants, seed and numerous mycorrhizal fungi (mainly orchid spp.), greatly extending the range and type of material that can be stored through the application of cryogenic methods. The role of in vitro and cryogenic research initiatives in botanic gardens for plant biodiversity conservation and restoration is discussed, using examples of successful ex situ conservation through tissue-culture and cryogenic-storage research.

Cryopreservation of threatened native Australian species—what have we learned and where to from here?

Cryogenic storage techniques have been developed and adopted for more than 100 (mainly agricultural) plant species worldwide, and within Australia, at least 30 critically endangered plants have been stored long term using cryogenic approaches. Nevertheless, there are many species that are very difficult to store using current procedures, and organizations involved in plant germplasm conservation (such as botanic gardens, agricultural institutions, etc.) that utilise cryogenic storage techniques are in some respects at a crossroads in their endeavours to cheaply and effectively store a wide selection of species and genotypes for conservation and agricultural/horticultural purposes. For taxa that are not amenable to current cryogenic approaches, new ways of developing cryogenic storage techniques need to be investigated, including research into the ways in which cell membranes interact and change when cooled to cryogenic temperatures (−196°C in liquid nitrogen) in the presence of various cryoprotective agents. This review highlights the current state of cryogenic research both within Australia and internationally, provides a case study on threatened plant species and also describes several new research initiatives that aim to provide answers to why some native species are quite amenable to widely utilised cryogenic approaches whilst others are currently non-responsive. New approaches aim to integrate laboratory and membrane modelling paradigms to provide guidelines for the development of new cryopreservation protocols and to assess the robustness of theoretical models in predicting optimum cryogenic conditions.

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

Micropropagation of the critically endangered Western Australian species, Symonanthus bancroftii (F. Muell.) L. Haegi (Solanaceae)

A micropropagation protocol was developed for the conservation of the critically endangered Western Australian shrub,Symonanthus bancroftii. It was necessary to screen antioxidant treatments to prevent the occurrence of lethal browning of explants upon excision. Potassium citrate and citric acid (0.1% w/v in a 4:1 ratio) prevented oxidative browning and was superior to the untreated control or other antioxidant treatments tested. Half strength Murashige and Skoog (MS) medium containing 0.5 μM kinetin and 0.25 μM benzyladenine produced three-fold multiplication compared to 1.75×, 1.5×, 1.8× and 1× multiplication for 2.5 μM kinetin + 0.25 μM benzyladenine, 0.5 μM kinetin + 5 μM gibberellic acid, 1 μM kinetin + 3 μM gibberellic acid and half strength MS with no plant growth regulators, over 4 weeks. Root production was achieved with indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA) at 0.5/0.5 μM (31% rooting) and 1.0/1.0 μM (36% rooting), after four weeks. Paclobutrazol (PBZ) at 0, 3.4 (1 mg 1−1), 10.2 (3 mg 1−1), or 17 μM (5 mg 1−1) improved tolerance to desiccation after transfer ofin vitro rooted shoots to soil. PBZ at 10.2 μM increased survival to 90% compared to 50% for those plantlets not treated with PBZ. The acclimatisation period from the glasshouse to the shadehouse was 1 week for plantlets treated with PBZ compared to 4 weeks for plantlets without any PBZ. PBZ at 3.4 μM increased the number of roots per shoot compared to untreated controls.