Nanjappa Ashwath

Tissue Culture Studies of Tomato (Lycopersicon esculentum)

Tomato is a major vegetable crop that has achieved tremendous popularity over the last century. It is grown in almost every country of the world. Development of protocols for in vitro selection can provide new advances for the production of stress tolerant cultivars. Techniques have been optimised for the production of haploids and somatic hybrids. Attempts have also been made to transfer the higher regenerative ability of wild varieties to cultivated tomatoes. Although, some information is available on the morphogenesis of tomato, the techniques have not been developed to a level at which they can be utilised in large-scale multiplication of commercially important cultivars. The morphogenesis response seems to be highly dependent PGRs used in the media, which is again cultivar and genotypic specific. Somatic embryogenesis in tomato is still at its infancy, and efficient procedures for large-scale production via somatic embryogenesis are yet to be developed. Genetic stability of the tissue culture raised tomato plants also needs to be addressed. The use of a combination of molecular and conventional breeding techniques could be the option for the development of cultivars resistant to biotic and abiotic stresses. This paper reviews the advances made in various aspects of tissue culture in tomato. It also discusses the issues that still need to be addressed to utilise the full potential of plant tissue culture techniques in genetic improvement and mass propagation of tomato.

Mycorrhizas in the Kakadu region of tropical Australia

This research represents the first part of a study which aimed to characterize the role of mycorrhizal associations in undisturbed and disturbed habitats in the Alligator Rivers Region of the Northern Territory of Australia. This is a seasonally dry tropical region with a climate consisting of a long dry season and a monsoonal wet season. Intact soil cores were sampled from 22 sites in this region, representing eucalypt savanna woodland, wetland, rocky hill and rainforest habitats. Clover, sorghum and eucalypt seedlings were grown in these cores in bioassays to measure the inoculum potential of vesicular-arbuscular mycorrhizal (VAM) and ectomycorrhizal (ECM) fungi. Propagules of VAM fungi were concentrated in the surface horizon, and were not adversely affected by 6 months dry storage of soil. Bioassays detected VAM fungus propagules at all sites, but these were less numerous in three sites with sparse herbaceous vegetation (a shrub-dominated woodland site, a sandstone area and a disturbed gravel pit without topsoil), than in other woodland sites. Propagules of VAM fungi were particularly numerous in soil from a rainforest habitat, which had much denser plant cover than any of the savanna sites. Propagules of ECM fungi colonized eucalypt seedling roots in some cores from all sites, except two wetland areas and a disturbed area without eucalypt trees. Physical and chemical properties of soils varied between sites and some properties (texture, organic carbon, etc.) were correlated with the inoculum potential of VAM fungi.

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.

EFFECTS OF GENOTYPE, EXPLANT ORIENTATION, AND WOUNDING ON SHOOT REGENERATION IN TOMATO

SummaryEffects of genotype and explant orientation on shoot regeneration from cotyledonary explants of tomato were studied using 10 commercially important cultivars. The explant orientation affected shoot regeneration in all the tested genotypes. Cotyledons placed in abaxial (lower surface facing down) orientation consistently produced better shoot regenerative response and produced greater numbers and taller shoots compared to those inoculated in adaxial (upper surface facing down) orientation. Genotypic variation in terms of shoot regeneration response, number of shoots, and shoot height was apparent. Wounding of cotyledonary explants increased shoot regeneration response. However, shoot height was much lower in shoots regenerated from wounded explants compared to those that originated from intact cotyledons. Shoots produced from wounded cotyledons were abnormal in their form and structure.

Nitrogen relations of natural and disturbed plant communities in tropical Australia

Abstract Nitrogen relations of natural and disturbed tropical plant communities in northern Australia (Kakadu National Park) were studied. Plant and soil N characteristics suggested that differences in N source utilisation occur at community and species level. Leaf and xylem sap N concentrations of plants in different communities were correlated with the availability of inorganic soil N (NH+4 and NO−3). In general, rates of leaf NO−3 assimilation were low. Even in communities with a higher N status, including deciduous monsoon forest, disturbed wetland, and a revegetated mine waste rock dump, levels of leaf nitrate reductase, xylem and leaf NO−3 levels were considerably lower than those that have been reported for eutrophic communities. Although NO−3 assimilation in escarpment and eucalypt woodlands, and wetland, was generally low, within these communities there was a suite of species that exhibited a greater capacity for NO−3 assimilation. These “high- NO−3 species” were mainly annuals, resprouting herbs or deciduous trees that had leaves with high N contents. Ficus, a high-NO−3 species, was associated with soil exhibiting higher rates of net mineralisation and net nitrification. “Low-NO−3 species” were evergreen perennials with low leaf N concentrations. A third group of plants, which assimilated NO−3 (albeit at lower rates than the high-NO−3 species), and had high-N leaves, were leguminous species. Acacia species, common in woodlands, had the highest leaf N contents of all woody species. Acacia species appeared to have the greatest potential to utilise the entire spectrum of available N sources. This versatility in N source utilisation may be important in relation to their high tissue N status and comparatively short life cycle. Differences in N utilisation are discussed in the context of species life strategies and mycorrhizal associations.

Phytocapping: An Alternative Technology for the Sustainable Management of Landfill Sites

Landfill remains the predominant means of waste disposal throughout the globe. Numerous landfills exist in developed and underdeveloped countries, engineered with contrasting degrees of effectiveness. Modern landfill closure in developed countries involves the conventional capping of waste with materials such as compacted clay or geosynthetic clay liners, typically overlain with other soil materials. Conventional capping technologies are now accepted to be increasingly ineffective in reducing percolation into waste. Cost-effective alternative systems are of increasing interest, including the use of plants to control and limit water entry into waste, otherwise known as “Phytocapping”. Phytocapping reduces percolation through three main mechanisms: (a) canopy interception of rainfall, (b) storage of moisture in the soil layers, and (c) evapotranspiration (i.e., hydraulic lift) of stored water. Phytocapping has been shown to be at least as effective as clay capping in reducing percolation through landfill cover materials, provided site specific conditions are factored into design, and providing many additional benefits, including increased cap stability, reduced erosion of capping materials, reduction of wind-blown dust, enhanced biological diversity, increased opportunity to establish commercial plants, carbon sequestration, and enhanced methane oxidation from microbial communities. Phytocapping has been suggested as having potential in phytoremediation of landfill leachate. The most common phytocapping approach to date is the construction of vegetation assemblages for the purposes of creating natural vegetation nodes. Phytocapping technology can be enhanced by appropriate selection of soil amendments such as biosolids, biochar, compost, or other materials. Appropriate selection of plant species and soil amendment products can enhance methane oxidation in capping soils. There is considerable potential for the use of high biomass energy plants but further work is needed in choosing appropriate plant species that will serve both purposes of site water balance as well as commercial (e.g., timber, bioenergy) and biodiversity needs of the community.

Monitoring plant physiological characteristics to evaluate mine site revegetation: A case study from the wet-dry tropics of northern Australia

Biologically driven markers or monitors were used to evaluate plant and ecosystem health of uranium-mining affected sites. Plant water, nitrogen (N) and phosphorus (P) status were used to measure physiological characteristics of tree and shrub species at sites perturbed by mining activities (waste rock dumps: WRD 1, WRD 2; mine wastewater irrigated woodland) and of species at undisturbed woodland (tropical savanna). Plant water status was evaluated by measuring leaf relative water content (RWC) and carbon isotope discrimination (δ13C). Leaf RWC varied significantly (P<0.0001) between wet and dry season in species at the woodland sites with higher RWC in the wet season compared to the dry season. No seasonal differences were observed in RWC in species at the WRDs. Leaf δ13C was similar in species at woodland sites and WRD 2 (−28.8 to −28.1‰) but was significantly (P<0.05) lower in species at WRD 1 (−27.6‰). This suggests that species at WRD 1 had a lower water availability and/or lower water use compared to species at all other sites. WRD substrate had an up to 4-orders of magnitude greater availability of inorganic phosphate (Pi) compared to woodland soil as determined using in situ ion exchange resin. Pi concentrations in xylem sap of species at WRDs were 2- to 3-fold higher compared to species at woodland sites. Plant nitrate reductase (NR) activity was low in most species at woodland and WRD 1. In contrast, Eucalyptus and Acacia species had high NR activities of up to 300–700 pkat g-1 fw at WRD 2 indicating that these species had greater nitrate use than species at all other sites. Nitrate availability in the top five cm of the profile, as determined using in situ ion exchange resins, increased at all sites in the wet season, but no significant differences were observed between sites using this method. However, traditional soil analysis revealed that WRD substrate had a 2-times higher nitrate content (0 to 1000 mm depth) compared to woodland soil. Thus, it is likely that plants at WRD2 accessed nitrate from deeper parts of the profile. Proline, an indicator of plant stress, was found in appreciable quantities in leaves of herbaceous species but not in woody species. Soil and leaf δ15N were measured to investigate N-cycling and the contribution of diazotrophic N2 fixation to plant N nutrition. Soil δ15N values were highest and most variable at WRD 2 (6.2‰) compared to all other sites (irrigated woodland 3.1‰, undisturbed woodland 2.5‰, WRD 1 0.9‰). This may indicate that N-turnover and nitrification was greatest at WRD 2 leading to greater 15N enrichment of soil N. At all sites, Acacia species were nodulated and putatively fixing N2. With the exception of WRD 2 where leaf δ15N of Acacia species averaged 0.9‰, Acacia species had 15N depleted values characteristic of species that receive N derived from N2 fixation (−0.8 to −0.6‰).Eucalyptus species at the woodland also had 15N depleted values (average −0.4‰) but 15N enriched values (0.3 to 1.8‰) at the three mining affected sites. The results show that for the plants studied foliar δ15N could not be used as an unequivocal measure of plant N sources. The results suggest that biomonitoring of plant and ecosystem health has potential in evaluating performance of mine site revegetation.

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).

In vitro spore germination of the fern Schizaea dichotoma

Spores of Schizaea dichotoma were disinfested using sodium hypochlorite (NaOCl), streptomycin, or a combination of both streptomycin and NaOCl. Only the spores treated with streptomycin germinated. The highest germination (34%) was recorded in MS1/4 C compared to MS1/2, BB, or MM medium. The spore germination was not affected by the presence or absence of light or mycorrhizal fungi (Glomus clarum). The germinated spores developed up to five cells and failed to develop into a mature gametophyte.

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.