Ecophysiology of nickel hyperaccumulation in Stackhousia tryonii Bailey

Selective accumulation of certain metals (elements) to exceptionally high concentrations in plants is intriguing. Approximately 425 species of so-called metal hyperaccumulators are currently known, of which about 75% hyperaccumulate nickel. Stackhousia tryonii Bailey (Stackhousiaceae) - a rare, herbaceous, serpentine-endelnic species - is one of the three nickel hyperaccumulators reported from Australia. This thesis reports research aimed at two broad aspects: propagation and ecophysiology of Ni hyperaccumulation in S. tryonii. Protocols were developed for seed germination, vegetative propagation and micropropagation and with the view to producing sufficient plants for use in the current study. Four-year-old S. tryonii seeds had poor germination (< 25%). However, this species was relatively easy to propagate via stem cuttings and micropropagation methods, as it possessed very high regenerative capacity (one explant produced up to 18 shoots within 4 weeks). Micropropagated shoots also responded well to ex vitro rooting, and were successfully hardened under controlled conditions. These propagation protocols could be useful to underpin conservation programs and mine site revegetation. The examination of natural populations of S. tryonii for arbuscular mycorrhizal colonisation suggested that S. tryonii is a favourable host. A moderately high colonisation (29-39%) of roots by arbuscular mycorrhizal fungi suggested a possible role of these fungi in improved nutrition of S. tryonii in typically nutrient-poor serpentine soils. A positive relationship between root colonisation and leaf Ni concentration suggested that mycorrhizal fungi might be involved in increased influx of Ni into the roots, which is readily transported and localised in the tissues. Spore density was very low (3-4 spores 100 g-¹dry soil, for two depths) in the associated serpentine soils and the dominant mycorrhizal species were: Glomus albidum, aggregatum, G. intraradices and G. tenebrosum. Based on five key soil characteristics (viz. pH, Ca, Mg, Ni and P), the study sites were segregated into four groups using hierarchical cluster analysis. Considerable variation existed in tissue Ni (and other elements) concentrations, both within and between populations and followed the order: leaf> root> stem. Localisation and spatial distribution of nickel, within both vegetative (leaf and stem) and reproductive (fruit) tissues were investigated using two microanalytical techniques [viz., micro-proton-induced x-ray emission spectrometry (micro-PIXE; nuclear microprobe) and scanning electron microscope with energy-dispersive x-ray spectroscopy (SEM-EDXS)]. In leaf and stem tissues, Ni was localised within epidermal and sub-epidermal tissues, palisade/mesophyll tissues, vascular bundles and/or pith. In contrast, in fruits, this metal was partitioned to the fruit wall (pericarp), while endospermic and cotyledonary tissues contained very little Ni. Accumulation of higher levels of Ni within the pericarp does not appear to inhibit seed germination in S. tryonii. To elucidate physiological mechanisms o fNi detoxification in S. tryonii, organic acids (leaf tissue) and free amino acids (xylem sap) were quantified using HPLC. Nickel concentration in the leaf tissues increased from 3695 g g-¹to 13,717 g g-¹with soil nickel supplementation, of which > 60% was extracted with dilute acid (0.025 M HCI). Oxalic, citric and malic acids were detected and quantified in the leaf tissue. Malic acid was the dominant organic acid, and based on a Ni to malic acid ratio (between 0.2:1 and 1:1), malic acid appears to play a major role in detoxification/transport and storage of Ni in S. tryonii. The total amino acid concentrations in the xylem sap decreased with nickel treatment. Glutamine was the major amino acid in both the low- and high- nickel treated plants. A role of amino acids in nickel complexation and transport in S. tryonii could not be established. The possibility of hyperaccumulated Ni acting as an osmoticum under waterstress (drought) in serpentine soils was also investigated. Drought severely affected the growth and overall biomass of the plants. However, survival of plants at the lowest levels of soil moisture (i. e. 20% of field capacity) suggested that it possesses an efficient water regulation mechanism. The results indicated possible involvement of Ni in osmotic adjustment under drought stress.