Naveen P. Bhatia

Studies on spatial distribution of nickel in leaves and stems of the metal hyperaccumulator Stackhousia tryonii using nuclear microprobe (micro-PIXE)and EDXS techniques

Stackhousia tryonii Bailey is one of the three nickel hyperaccumulators reported from Australia. It is a rare, herbaceous plant that accumulates (Ni) both in leaf and stem tissues. Localisation of Ni in leaf and stem tissues of S. tryonii was studied using two micro-analytical techniques, energy dispersive X-ray spectrometry (EDXS) and micro-proton-induced X-ray emission spectrometry (micro-PIXE). Dimethylglyoxime complexation of Ni was also visualised by bright- and dark-field microscopy, but this technique was considered to create artefacts in the distribution of Ni. Energy dispersive X-ray spectrometric analysis indicated that guard cells possessed a lower Ni concentration than epidermal cells, and that epidermal cells and vascular tissue contained higher levels of Ni than mesophyll, as reported for other Ni hyperaccumulators. The highest Ni concentration was recorded (PIXE quantitative point analysis) in the epidermal cells and vascular tissue (5400 μg g-1 DW), approximately double that recorded in palisade cells (2500 μg g-1 DW). However, concentrations were variable within these tissues, explaining, in part, the similarity between average Ni concentrations of these tissues (as estimated by region selection mode). Stem tissues showed a similar distribution pattern as leaves, with relatively low Ni concentration in the pith (central) region. The majority of Ni (73-85% for leaves; 80-92% for stem) was extracted from freeze-dried sections by water extraction, suggesting that this metal is present in a highly soluble and mobile form in the leaf and stem tissues of S. tryonii.

Growth responses and dependence of Acacia nilotica var. cupriciformis on the indigenous arbuscular mycorrhizal consortium of a marginal wasteland soil

Abstract The responses of Acacia nilotica L. var. cupriciformis to phosphorus application and inoculation with the indigenous consortium of arbuscular mycorrhizal (AM) fungi were evaluated in a nursery experiment using soil from a marginal wasteland. A positive growth response to mycorrhizal inoculation was observed at an Olsen-P level of 20 ppm in the presence of the natural population of AM fungi. There was growth stimulation by either inoculation or additional P at the highest soil P of 40 ppm. Colonization was negatively correlated to soil P but P content of both shoot and root were positively correlated. Inoculation with the indigenous AM consortium significantly increased the uptake of P at all levels of applied P. Acacia is moderately dependent upon the AM symbiosis and exhibited a maximal mycorrhizal dependence (MD) of 18.25% at 20 ppm Olsen-P level under the conditions studied. A sharp and considerable reduction in MD and dry matter yield observed at 40 ppm P suggests that the external P requirement for maximal production of biomass was met at approximately 20 ppm Olsen-P.

Heavy metal tolerance in common fern species

The effects of cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), lead (Pb) and zinc (Zn) on the growth and uptake of 10 fern species was investigated under a controlled environment in order to evaluate their suitability for phytoremediation. Fern species included Adiantum aethiopicum, Blechnum cartilagineum, Blechnum nudum, Calochlaena dubia, Dennstaedtia davallioides, Doodia aspera, Hypolepis muelleri, Nephrolepis cordifolia, Pellaea falcata and the arsenic (As) hyperaccumulating Pteris vittata. Ferns were exposed to four levels of metals at concentrations of 0, 50, 100 and 500 mg kg–1 for a period of 20 weeks. The response of ferns significantly varied among species and metals. In general, heavy-metal translocation was limited, with metals being absorbed and held in roots, suggesting an exclusion mechanism as part of the ferns’ tolerance to the applied metals. Similar metal-accumulation patterns were observed for all species in that accumulation generally increased with increasing metal treatments; in most cases a sharp increase in metal accumulation was observed between 100 and 500 mg kg–1 treatments, suggesting a breakdown in tolerance mechanisms and unrestricted metal transport. This was corroborated by enhanced visual toxicity symptoms and a reduction in survival rates among ferns when exposed to 500 mg kg–1 metal treatments; and to a lesser extent 100 mg kg–1 metal treatments. Of the species investigated, N. cordifolia and H. muelleri were identified as possible candidates in phytostabilisation of Cu, Pb, Ni or Zn contaminated soils. Similarly, D. davallioides appeared favourable for use in phytostabilisation of Cu and Zn contaminated soils. These species had high survival rates and accumulated high levels of the aforementioned metals relative to other ferns investigated. Ferns belonging to the family Blechnaceae (B. nudum, B. cartilagineum and D. aspera) and C. dubia (Family Dicksoniaceae) were least tolerant to most metals, had a low survival rate and were classified as being unsuitable for phytoremediation purposes. Metal tolerance was also observed in P. vittata when exposed to Cd, Cr and Cu; however, no hyperaccumulation was observed.

Mycorrhizal dependency and growth responses of Acacia nilotica and Albizzia lebbeck to inoculation by indigenous AM fungi as influenced by available soil P levels in a semi-arid Alfisol wasteland

A series of available phosphorus (Olsen) levels ranging from 10 to 40 ppm were achieved in a semi-arid soil. The influence of the levels of phosphorus on the symbiotic interaction between two subtropical tree species, Acacia nilotica and Albizzia lebbeck, and a mixed inoculum of indigenous arbuscular mycorrhizal (AM) fungi was evaluated in a greenhouse study. The extent to which the plant species depended on AM fungi for dry matter production decreased as the levels of soil P increased, but the degree of this decrease differed in the two species tested. Acacia nilotica colonized by AM fungi showed a significant increase in shoot P and dry matter at a soil P level of 10 ppm whereas in Albizzia lebbeck, such increase occurred at 20 ppm. Mycorrhizal inoculation response disappeared beyond soil P levels of 25 ppm in Acacia nilotica and 30 ppm in Albizzia lebbeck. Levels of soil P greater than 25 ppm suppressed AM fungus colonization in both species. Soil P levels of 30 and 40 ppm and 40 ppm caused negative mycorrhizal dependencies (MD) in Acacia nilotica and Albizzia lebbeck respectively. Values of MD for both species were negatively correlated with soil P levels. Based on the MD values, regression equations were developed to predict MD for given levels of available P.

In vitro propagation of Stackhousia tryonii Bailey (Stackhousiaceae): a rare and serpentine-endemic species of central Queensland, Australia

Stackhousia tryonii Bailey, a rare species whichhyperaccumulates nickel and with a potential to be exploited inphytoremediation/phytomining, is difficult to propagate via seeds. This studyinvestigated the development of a micropropagation protocol for the productionof large stocks of S. tryonii. Disinfested shoot tips andnodal buds were precultured on Gamborgs (B5) basal medium toobtain aseptic shoots for the optimisation of the protocol. 6-Benzyl aminopurine(BAP) at 1.0 mg l−1 produced the highest number ofshoots per explant in B5 medium. Comparison betweenB5 and MS media showed similar responses, but with marked influenceof BAP concentration on shoot numbers. Transfer of shoots from MS(multiplication) medium to MS medium supplemented with indole-3-acetic acid(IAA) and indole-3-butyric acid (IBA), individually or in combination, indicatedthat a combination of IAA and IBA (0.75 mg l−1each) is required to produce roots on young shoots (75%) compared to IBA(15–45%) or IAA (0–10%) alone. This study demonstrated that by usingthis protocol, a high multiplication rate (up to 18 shoots per explant) could be produced within 4 weeks, andthey can be readily hardened (98% survival) in a glasshouse by transplantingthem into a potting mixture of sand and perlite (4:1).

Ex Vitro Rooting of Micropropagated Shoots of Stackhousia Tryonii

Micropropagated shoots of Stackhousia tryonii were exposed (individually or in combination) to indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), and 1-naphthalene acetic acid (NAA) at concentrations 1, 2 or 4 g dm−3 with the view to induce rooting under ex vitro conditions. The treated microshoots were grown in a mist room for four weeks and assessed for survival, rooting percentage, number of roots and root length. The results showed that IBA at 2 g dm−3 was most effective in inducing roots. Mixing of two or more auxins markedly reduced rooting percentage indicating antagonistic effects. The results demonstrated the potential of combining ex vitro rooting and hardening in one step, with view to reducing costs of multiplying plants via micropropagation.

Diversity and selective dominance of vesicular-arbuscular mycorrhizal fungi

VAM fungal species which form an effective link between plants and the ecosystem through symbiotic interactions, thus protecting the environmental quality, show diversity/dominance with relation to the type of ecosystem, edapho-climatic conditions, seasonal variations, host genotype, plant cover etc. which are known to be the critical determinants. In natural ecosystems, because of the greater degree of variability (in terms of critical determination) the diversity of VAM fungal species is more pronounced compared to disturbed ecosystems. The severity and duration of disturbance are other important deciding factors in the potential long-term dominance of VAM in ecosystems; disturbed for one reason or the other. Also, in severely disturbed ecosystems, the phenomenon of dependency and facultativeness influences the succession patterns. Moreover, as temperature has a distinct influence on rate and intensity of infection, a considerable amount of diversity with respect to representative species of VAM fungi was found in two of the major climatic regimes viz. tropical and temperatre. Species of the genera Glomus showed a wide range of adaptability to a variety of ecosystems, irrespective of kind of the disturbances.

Influence of Drought Stress on the Nickel-Hyperaccumulating Shrub Hybanthus floribundus (Lindl.) F.Muell. subsp. floribundus

Hyperaccumulation of nickel (Ni) in certain plants may play a role in drought resistance under water stress. This article tests the influence of water stress on the Ni-hyperaccumulating shrub Hybanthus floribundus Lindl. F.Muell. subsp. floribundus. Plants grown in 1000 mg kg−1 Ni-amended Clastic Rudosol were exposed to five levels of soil water potentials (−33 [field capacity], −60, −400, −600, and −1000 kPa) for 12 wk. Water stress did not induce significant changes in growth rate, relative water content, rates of gas exchange, or carbon isotope discrimination. Water use efficiency (WUE) values were approximately threefold lower in plants at water potentials <−400 kPa than they were in those at water potentials of −33 kPa. Low WUE values suggest that this species possesses an efficient water conservation mechanism that enables its survival in competitive water-limited environments. A 38% decline in water potential and a 68% decline in osmotic potential occurred between −1000- and −33-kPa water potentials (), indicating that osmotic adjustment (OA) may have provided turgor maintenance in response to increasing water stress. However, Ni concentration in plants did not significantly increase in response to decreasing water potentials and is therefore unlikely to play a role in OA.

The role of low molecular weight ligands in nickel hyperaccumulation in Hybanthus floribundus subspecies floribundus

The mechanisms responsible for nickel (Ni) hyperaccumulation in Hybanthus floribundus (Lindl.) F.Muell. subspecies floribundus are obscure. In this study, organic acids and free amino acids (AAs) were quantified in 0.025 M HCl H. floribundus subsp. floribundus shoot extracts using HPLC and ultra performance liquid chromatography (UPLC). In a 20 week pot experiment, plants exposed to five levels of Ni (0–3000 mg kg–1 Ni) accumulated up to 3200 mg Ni kg–1 dry weight in shoots, and the shoot : root Ni concentration ratios were >1.4. Concentration of organic acids followed the order malic acid > citric acid > oxalic acid. Citric acid concentration significantly increased upon Ni exposure, with concentrations between 2.3- and 5.9-fold higher in Ni treated plants that in control plants. Molar ratios of Ni to citric acid ranged from 1.3 : 1 to 1.7 : 1 equivalent to >60% of the accumulated Ni. Malic acid concentration also increased upon exposure to applied Ni. However, concentrations were statistically at par across 0–3000 mg kg–1 Ni treatments, suggesting that the production of malic acid is a constitutive property of the subspecies. Total AA concentrations were stimulated upon exposure to external Ni treatment, with glutamine, alanine and aspartic acids being the predominant acids. These AAs accounted for up to 64% of the total free AA concentration in control plants and up to 75% for the 2000 mg kg–1 Ni treatment plants. These results suggest that citric acid in addition to the aforementioned AAs are synthesised in H. floribundus subsp. floribundus plants following exposure to elevated concentrations of Ni and may act as potential ligands for detoxification and possibly storage of accumulated Ni.