Catherine A. Offord

Simultaneous encapsulation of seed and mycorrhizal fungi for long-term storage and propagation of terrestrial orchids

Ex situ conservation of threatened terrestrial orchids requires the simultaneous conservation of their mycorrhizal associations. A method for encapsulating both seed and fungi in alginate beads (known as encapsulation–dehydration) was applied to the storage and propagation of two endangered orchid species in NSW, Australia—Pterostylis saxicola D.L.Jones & M.A.Clem. and Diuris arenaria D.L.Jones. We tested the effect of storage duration and temperature on fungal recovery and germination potential in vitro, and recorded survival for seedlings subsequently transferred to potting mix. Storage at 23°C significantly reduced fungal recovery and germination for both species after only 3 months (P < 0.05), whereas storage at 4°C significantly reduced fungal recovery for P. saxicola after 6 months (P < 0.05). Storage for 6 months at −18 and −196°C had no significant effect on the fungal recovery and germination percentages of either species. All beads transferred directly from in vitro culture to potting mix resulted in the establishment of at least one seedling, and production of a healthy tuberoid, when transferred near the commencement of the natural growing season. The encapsulation–dehydration method may have a practical application for use in ex situ conservation of other terrestrial orchids, as well as their mycorrhizal fungi.

Conservation technologies for safeguarding and restoring threatened flora: case studies from Eastern Australia

This paper highlights recent advances and improved scientific understanding of conservation technologies through selected case studies on threatened plant species indigenous to Eastern Australia. This includes investigations into seed desiccation, storage responses and cryopreservation in rainforest species, particularly the socio-economically important Australian native Citrus spp., Davidsonia spp. (Davidson’s plum) and Syzygium spp. This work also (1) increases our understanding of ecological correlates of seed desiccation sensitivity for predictive use and (2) improves restoration practice through better understanding of seed storage and germination requirements. The use of in vitro conservation technologies in support of conservation actions for endangered species is outlined in case studies on Wollemia nobilis (Wollemi pine), epiphytic and terrestrial orchid species, and an endangered fern species.

In vitro propagation of Pimelea spicata R.Br (Thymelaeaceae), an endangered species of the Sydney region, Australia

Micropropagation was assessed as an ex situ conservation strategy for the endangered Australian plant Pimelea spicata (Thymelaeaceae). Although regeneration of this species was achieved, several physiological problems were observed and examined. Explants of P. spicata had a higher multiplication rate on MS medium, than on ½ MS, but there was a significantly higher percentage of necrotic shoot tips on the higher salt medium. Increasing calcium concentration and gas exchange exacerbated shoot-tip necrosis. A number of hyperhydrated shoots were produced in all treatments, the cause of which could not be determined, although less hyperhydicity was observed in the ½ MS treatment. Shoots, rooted in vitro on ½ MS in the absence of plant growth regulators, were successfully acclimatised to greenhouse conditions, while direct rooting of microshoots using IBA gel treatment proved unsuccessful. This is the first report of tissue culture propagation of this endangered species.

Micropropagation of Telopea speciosissima R. Br. (Proteaceae). 2: Rhizogenesis and acclimatisation to ex vitro conditions

Conditions affecting rhizogenesis in vitro and ex vitro and subsequent acclimatisation of Telopea speciosissima (waratah) were investigated. Clonal selections were successfully rooted in vitro in agar, on filter paper bridges or using crushed quartz-sand, the last substrate resulting in superior growth of roots. The in vitro substrates were impregnated with half-strength MS, 7.5 gl-1 sucrose and various concentrations of IBA. For the quartz-sand, an IBA concentration of 50 μM was optimal, 70% of microcuttings were rooted. No plantlets rooted in vitro were acclimatised to ex vitro conditions (using mist, fog or humidity tent regimes). Microcuttings (25–45 mm in length) were rooted ex vitro in a fog humidity regime (droplet size <10 μm) using an IBA powder dip (3 g IBA kg-1). Neither a mist nor a humidity-tent regime was suitable for rooting of waratah microshoots ex vitro. A peat and perlite mixture was superior to crushed quartz-sand or potting mix for the rooting of microshoots; this appeared to be related to the air-filled porosity (>20%) of the mixture, measured after the medium was saturated and then drained for 24h. Plantlets must be left under the high humidity regime until shoot growth resumes (four to eight weeks) otherwise plant mortality increase significantly. In vitro-produced leaves abscised between eight and 12 weeks after transfer to ex vitro conditions, indicating that these structures did not acclimatise ex vitro.

Pushed to the limit: consequences of climate change for the Araucariaceae: a relictual rain forest family

BACKGROUND AND AIMS Under predicted climate change scenarios, increased temperatures are likely to predispose trees to leaf and other tissue damage, resulting in plant death and contraction of already narrow distribution ranges in many relictual species. The effects of predicted upward temperatures may be further exacerbated by changes in rainfall patterns and damage caused by frosts on trees that have been insufficiently cold-hardened. The Araucariaceae is a relictual family and the seven species found in Australia have limited natural distributions characterized by low frost intensity and frequency, and warm summer temperatures. The temperature limits for these species were determined in order to help understand how such species will fare in a changing climate. METHODS Experiments were conducted using samples from representative trees of the Araucariaceae species occurring in Australia, Agathis (A. atropurpurea, A. microstachya and A. robusta), Arauacaria (A. bidwilli, A. cunninghamii and A. heterophylla) and Wollemia nobilis. Samples were collected from plants grown in a common garden environment. Lower and higher temperature limits were determined by subjecting detached winter-hardened leaves to temperatures from 0 to -17 °C and summer-exposed leaves to 25 to 63 °C, then measuring the efficiency of photosystem II (F(v)/F(m)) and visually rating leaf damage. The exotherm, a sharp rise in temperature indicating the point of ice nucleation within the cells of the leaf, was measured on detached leaves of winter-hardened and summer temperature-exposed leaves. KEY RESULTS Lower temperature limits (indicated by FT(50), the temperature at which PSII efficiency is 50 %, and LT(50) the temperature at which 50 % visual leaf damage occurred) were approx. -5·5 to -7·5 °C for A. atropurpurea, A. microstachya and A. heterophylla, approx. -7 to -9 °C for A. robusta, A. bidwillii and A. cunninghamii, and -10·5 to -11 °C for W. nobilis. High temperature damage began at 47·5 °C for W. nobilis, and occurred in the range 48·5-52 °C for A. bidwillii and A. cunninghamii, and in the range 50·5-53·5 °C for A. robusta, A. microstachya and A. heterophylla. Winter-hardened leaves had ice nucleation temperatures of -5·5 °C or lower, with W. nobilis the lowest at -6·8 °C. All species had significantly higher ice nucleation temperatures in summer, with A. atropurpurea and A. heterophylla forming ice in the leaf at temperatures >3 °C higher in summer than in winter. Wollemia nobilis had lower FT(50) and LT(50) values than its ice nucleation temperature, indicating that the species has a degree of ice tolerance. CONCLUSIONS While lower temperature limits in the Australian Araucariaceae are generally unlikely to affect their survival in wild populations during normal winters, unseasonal frosts may have devastating effects on tree survival. Extreme high temperatures are not common in the areas of natural occurrence, but upward temperature shifts, in combination with localized radiant heating, may increase the heat experienced within a canopy by at least 10 °C and impact on tree survival, and may contribute to range contraction. Heat stress may explain why many landscape plantings of W. nobilis have failed in hotter areas of Australia.

Growing up or growing out? How soil pH and light affect seedling growth of a relictual rainforest tree

Our aim was to uncover the drivers of seedling growth in a rare rainforest conifer. Wollemia nobilis is limited to canyons, characterized by deeply shaded understories and acid soils. In a glasshouse experiment, we grew seedlings at a range of pH and light levels. Growth increased with increasing light, and was higher at low pH, regardless of light. Number of stems, however, was greatest in lower light. Wollemia nobilis seedlings may vary their architecture - growing up when light is high, and growing out when light is lower. Nevertheless, low light is likely the key limitation of W. nobilis growth in the wild.

Seed ecology of the invasive woody plant African Olive (Olea europaea subsp. cuspidata): implications for management and restoration

Knowledege of the seed ecology of invasive exotic species, including soil seedbank dynamics, is essential to understanding key factors in successful invasion and in identifying management opportunities. African Olive, Olea europaea L. subsp. cuspidata, is an exotic invasive woody plant in Hawaii, Norfolk Island and eastern Australia, and is now well established in the Cumberland Plain region of western Sydney, Australia. In the present study, the key aspects of the seed ecology of African Olive were determined for populations in western Sydney. Extracted seed germinated at a wide range of temperatures, consistent with tolerance of a wide range of climatic conditions. A seed-burial experiment indicated a slow decrease in viability down to 70.3% during the first year, followed by a rapid decline down to 14.7% in the second year. Probit analysis indicated that under field conditions, seed persistence in the soil was ~29 months (2.4 years). In situ germination was low (3.3%) and did not occur until the mechanical constriction of the endocarp was released through decomposition. The woody seed endocarp was found to be permeable to water, indicating that physical dormancy was not imposed by providing a barrier to water uptake. Within its invasive range, African Olive produces abundant seed. However, the rapid loss of viability of soil-stored seed results in a narrow window of opportunity for germination. The short persistence of seed in the soil may provide an opportunity for managers to achieve control of African Olive once mature plants are removed.

Micropropagation of Telopea speciosissima R. Br. (Proteaceae). 1: Explant establishment and proliferation

Axillary bud explants of 11 selected mature waratah clones were established in vitro on a modified Murashige & Skoog medium. Adequate proliferation of axillary shoots was achieved by optimisation of the growth regulator status of the culture medium. For the majority of clones, a three to six times rate of proliferation was achieved with 1.25 μM BA and 1.0 μM GA3 without the occurrence of abnormalities. The white flowering clone did not respond favourably to the addition of GA3 to the medium.

Rising temperature may negate the stimulatory effect of rising CO2 on growth and physiology of Wollemi pine (Wollemia nobilis)

Rising atmospheric [CO2] is associated with increased air temperature, and this warming may drive many rare plant species to extinction. However, to date, studies on the interactive effects of rising [CO2] and warming have focussed on just a few widely distributed plant species. Wollemi pine (Wollemia nobilis W.G.Jones, K.D.Hill, & J.M.Allen), formerly widespread in Australia, was reduced to a remnant population of fewer than 100 genetically indistinguishable individuals. Here, we examined the interactive effects of three [CO2] (290, 400 and 650ppm) and two temperature (ambient, ambient+4°C) treatments on clonally-propagated Wollemi pine grown for 17 months in glasshouses under well-watered and fertilised conditions. In general, the effects of rising [CO2] and temperature on growth and physiology were not interactive. Rising [CO2] increased shoot growth, light-saturated net photosynthetic rates (Asat) and net carbon gain. Higher net carbon gain was due to increased maximum apparent quantum yield and reduced non-photorespiratory respiration in the light, which also reduced the light compensation point. In contrast, increasing temperature reduced stem growth and Asat. Compensatory changes in mesophyll conductance and stomatal regulation suggest a narrow functional range of optimal water and CO2 flux co-regulation. These results suggest Asat and growth of the surviving genotype of Wollemi pine may continue to increase with rising [CO2], but increasing temperatures may offset these effects, and challenges to physiological and morphological controls over water and carbon trade-offs may push the remnant wild population of Wollemi pine towards extinction.

Establishing a Wild, Ex Situ Population of a Critically Endangered Shade-Tolerant Rainforest Conifer: A Translocation Experiment.

Translocation can reduce extinction risk by increasing population size and geographic range, and is increasingly being used in the management of rare and threatened plant species. A critical determinant of successful plant establishment is light environment. Wollemia nobilis (Wollemi pine) is a critically endangered conifer, with a wild population of 83 mature trees and a highly restricted distribution of less than 10 km2. We used under-planting to establish a population of W. nobilis in a new rainforest site. Because its optimal establishment conditions were unknown, we conducted an experimental translocation, planting in a range of different light conditions from deeply shaded to high light gaps. Two years after the experimental translocation, 85% of plants had survived. There were two distinct responses: very high survival (94%) but very low growth, and lower survival (69%) and higher growth, associated with initial plant condition. Overall survival of translocated W. nobilis was strongly increased in planting sites with higher light, in contrast to previous studies demonstrating long-term survival of wild W. nobilis juveniles in deep shade. Translocation by under-planting may be useful in establishing new populations of shade-tolerant plant species, not least by utilizing the range of light conditions that occur in forest understories.