Regeneration and transformation of Eucalyptus camaldulensis

Reliable regeneration protocols for Eucalyptus camaldulensis using leaf explants from in vitro-grown plants have been developed. Out of the 24 clones tested 13 were regenerated and of these, 6 showed regeneration from more than 60% of the explants. Identical protocols were also successful in the regeneration of some clones of E. microtheca, E. ochrophloia, E. grandis and E. marginata, but at lower frequencies. Co-cultivation of E. camaldulensis leaf explants with Agrobacterium tumefaciens strains carrying a kanamycin resistance gene and the reporter gene β-glucuronidase (GUS), followed by selection on kanamycin at 9 mg l^–1, allowed the selection of transformed shoots that could be rooted on selective media. Transformation of the plants was verified by staining for the GUS enzyme in various plant tissues, NptII assays and by Southern blotting on isolated DNA using specific probes for both the GUS and selectable marker genes. Transformed tissue was obtained with 5 clones of E. camaldulensis tested and a number of A. tumefaciens strains. However, only 1 clone regenerated transformed whole plants reliably. Received: 14 October 1996 / Revision received: 18 February 1997 / Accepted: 1 April 1997     

Effects of flooding on Eucalyptus camaldulensis and Eucalyptus globulus seedlings

Summary Flooding for up to 40 days induced morphological changes and reduced growth of 6-week-old Eucalyptus camaldulensis and Eucalyptus globulus seedlings. However, the specific responses to flooding varied markedly between these species and with duration of flooding. Both species produced abundant adventitious roots that originated near the tap root and original lateral roots, but only E. camaldulensis produced adventitious roots on submerged portions of the stem. Flooding induced leaf epinasty and reduced total dry weight increment of seedling of both species but growth of E. globulus was reduced more. In both species dry weight increment of shoots was reduced more than dry weight increment of roots, reflecting compensatory growth of adventitious roots. Adaptation to flooding appeared to be greater in E. camaldulensis than in E. globulus. the importance of formation of adventitious roots in flooding tolerance is emphasized.Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison; CEPEC (Cacao Research Center), Bahia, Brazil; and BMBRAPA (Brazilian Research Institute), Brasilia, Brazil     

In vitro regeneration of Eucalyptus camaldulensis

An efficient in vitro regeneration protocol enables mass multiplication, genetic modification and germplasm conservation of desired plants. In vitro plant regeneration was achieved from nodal segments of 18-months-old superior genotypes of Eucalyptus camaldulensis trees through direct organogenesis (DO) and direct somatic embryogenesis (DSE) pathways. Initial bud break (BB) stage occurred via DO while shoot multiplication phase followed both DO and DSE pathways. Interestingly, both BB and shoot multiplication stages were achieved on shoot induction and multiplication (SIM) media composed of Murashige and Skoog (MS) basal medium supplemented with 2 mg l⁻¹ benzyl aminopurine (BAP) and 0.1 mg l⁻¹ naphthalene acetic acid (NAA). Best shoot elongation response was observed on half strength MS fortified with 0.5 mg l⁻¹ BAP, while root induction and elongation was superior in 1/2 MS + 1 mg l⁻¹ Indole butyric acid (IBA). Full strength MS fortified with cytokinins (BAP) and weak auxin (NAA) in the ratio of 20:1 favored direct regeneration pathways. Further, half strength MS supported shoot and root development. The absence of intervening callus phase in this protocol can help in minimizing the chance occurrence of somaclones. When compared to other compositions tried, hardening in 100 % coco peat resulted in maximum survival (80 %) of the in vitro raised plantlets. For mass multiplication, fortnight subculturing of a single nodal explants for eight passages on SIM medium resulted in 60–148 shoot initials. Repeated subculturing in SIM medium induced the formation of direct somatic embryos which in turn improved the turnover capacity and enabled large scale clonal multiplication of elite and desirable trees of E. camaldulensis. Following this protocol, it takes a minimum time period of four-months between in vitro explant inoculation to hardening stage. In the present study, DO and DSE pathway of plant regeneration was reported occurring simultaneously in the same nodal explants of E. camaldulensis.     

Fumigant toxicity of Eucalyptus intertexta, Eucalyptus sargentii and Eucalyptus camaldulensis against stored-product beetles

Abstract:  Three essential oils extracted from the species of the family Myrtaceae found in Kashan (centre regions of Iran) were shown to have potent fumigant toxicity against three major stored-product insects: Callosobruchus maculatus (Fab.), Sitophilus oryzae (L.) and Tribolium castaneum (Herbst). These were the essential oils from Eucalyptus intertexta R.T. Baker, Eucalyptus sargentii Maiden and Eucalyptus camaldulensis Dehnh. The mortality of 1- to 7-day-old adults of the insect pests increased with concentration from 37 to 926  μ l/l air and with exposure time from 3 to 24 h. The LC₅₀ values to the selected essential oils were between 2.55 and 3.97  μ l/l air for C. maculatus , 6.93 and 12.91  μ l/l for S. oryzae and 11.59 and 33.50  μ l/l air for T. castaneum . Fumigant effects of these essential oils were considered to warrant further research into their potential for commercial use.     

Characterization of two HKT1 homologues from Eucalyptus camaldulensis that display intrinsic osmosensing capability

Plants have multiple potassium (K(+)) uptake and efflux mechanisms that are expressed throughout plant tissues to fulfill different physiological functions. Several different classes of K(+) channels and carriers have been identified at the molecular level in plants. K(+) transporters of the HKT1 superfamily have been cloned from wheat (Triticum aestivum), Arabidopsis, and Eucalyptus camaldulensis. The functional characteristics as well as the primary structure of these transporters are diverse with orthologues found in bacterial and fungal genomes. In this report, we provide a detailed characterization of the functional characteristics, as expressed in Xenopus laevis oocytes, of two cDNAs isolated from E. camaldulensis that encode proteins belonging to the HKT1 superfamily of K(+)/Na(+) transporters. The transport of K(+) in EcHKT-expressing oocytes is enhanced by Na(+), but K(+) was also transported in the absence of Na(+). Na(+) is transported in the absence of K(+) as has been demonstrated for HKT1 and AtHKT1. Overall, the E. camaldulensis transporters show some similarities and differences in ionic selectivity to HKT1 and AtHKT1. One striking difference between HKT1 and EcHKT is the sensitivity to changes in the external osmolarity of the solution. Hypotonic solutions increased EcHKT induced currents in oocytes by 100% as compared with no increased current in HKT1 expressing or uninjected oocytes. These osmotically sensitive currents were not enhanced by voltage and may mediate water flux. The physiological function of these osmotically induced increases in currents may be related to the ecological niches that E. camaldulensis inhabits, which are periodically flooded. Therefore, the osmosensing function of EcHKT may provide this species with a competitive advantage in maintaining K(+) homeostasis under certain conditions.     

Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants

An efficient system for Agrobacterium-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants was developed. Transformation was accomplished by cocultivation of hypocotyl segments with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase and β-glucuronidase (GUS) genes. A modified Gamborg's B5 medium used in this study was effective for both callus induction and regeneration of transgenic shoots. This medium could also effectively maintain the organogenic capability of callus for more than a year. Culturing transgenic shoots in Murashige and Skoog medium supplemented with 0.1 mg ⋅ l^–1 benzylaminopurine prior to root induction in rooting medium markedly increased the rootability of shoots that were recalcitrant to rooting. Histochemical assay revealed the expression of the GUS gene in leaf, stem, and root tissues of transgenic plants. Insertion of the GUS gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis, further confirming the integration and expression of T-DNA in these plants. Received: 1 August 1997 / Revision received: 11 December 1997 / Accepted: 24 January 1998     

Hydrological and land use determinants of Eucalyptus camaldulensis occurrence in floodplain wetlands

Hydrological and land use changes can affect species in human altered landscapes. Typically the impacts of hydrological and land use changes are examined separately, with hydrological determinants used to explain the distribution of species in water dependent and aquatic habitats and land use factors used to examine terrestrial species. However, given the connectedness of aquatic and terrestrial habitats, stressors originating in one domain may be important in the other. To explore the importance of integrating both hydrological and land use factors, we tested the relative contribution of hydrological factors and land use context as determinants of the dominant riparian tree species, Eucalyptus camaldulensis Dehn. throughout wetlands of the Condamine catchment, southern Queensland, Australia. The occurrence of E. camaldulensis was modelled against hydrological and land use factors using generalized linear models (GLMs) and validated using internal bootstrapping procedures. Validated models which included both hydrological (distance from weir, wetland–river connectivity and groundwater depth) and land use factors (agricultural land cover and grazing intensity) performed better than those developed using only hydrological factors. The study results highlight the importance of an integrated perspective which considers both hydrological and land use factors in order to understand occurrence patterns of riparian and floodplain tree species in a range of settings. This approach could be especially important when assessing changes to hydrology and land use which may be triggered by climatic changes.     

Species discrimination, population structure and linkage disequilibrium in Eucalyptus camaldulensis and Eucalyptus tereticornis using SSR markers

Eucalyptus camaldulensis and E. tereticornis are closely related species commonly cultivated for pulp wood in many tropical countries including India. Understanding the genetic structure and linkage disequilibrium (LD) existing in these species is essential for the improvement of industrially important traits. Our goal was to evaluate the use of simple sequence repeat (SSR) loci for species discrimination, population structure and LD analysis in these species. Investigations were carried out with the most common alleles in 93 accessions belonging to these two species using 62 SSR markers through cross amplification. The polymorphic information content (PIC) ranged from 0.44 to 0.93 and 0.36 to 0.93 in E. camaldulensis and E. tereticornis respectively. A clear delineation between the two species was evident based on the analysis of population structure and species-specific alleles. Significant genotypic LD was found in E. camaldulensis, wherein out of 135 significant pairs, 17 pairs showed r(2)≥0.1. Similarly, in E. tereticornis, out of 136 significant pairs, 18 pairs showed r(2)≥0.1. The extent of LD decayed rapidly showing the significance of association analyses in eucalypts with higher resolution markers. The availability of whole genome sequence for E. grandis and the synteny and co-linearity in the genome of eucalypts, will allow genome-wide genotyping using microsatellites or single nucleotide polymorphims.     

Hormonal control of proliferation in meristematic agglomerates of Eucalyptus camaldulensis Dehn

Summary Eucalyptus camaldulensis can be micropropagated through so-called meristematic agglomerates (MAs). MAs (4–6 mm diameter) are dense shoot clusters initiated by the outgrowth of numerous successive buds. Their reddish nature is associated with an increase in their endogeneous cytokinin level during the exponential phase of growth. A simultaneous decrease in the auxin level favors a high cytokinin/auxin ratio. A low level of polyamines occurs at the time of the lowest level of auxins. Slow hormone release by activated charcoal plays a role in this very prolific organogenesis.     

Nuclear RFLP variation in Eucalyptus camaldulensis Dehnh. from northern Australia

Eucalyptus camaldulensis Dehnh. is the most widely planted eucalypt in the tropics. Natural populations are riparian and sampling strategies for breeding programmes have assumed that gene flow among drainage basins is limited. RFLP variation, within and among 31 populations from river systems across northern Australia, was analysed to test this hypothesis. To allow comparisons within and between river systems, trees were sampled from up to three populations per river system. Allele frequencies were correlated with longitude for more than half the 33 RFLP loci surveyed. Genetic identity was greatest between populations in closest geographic proximity, irrespective of river system, suggesting that sampling strategies for breeding programmes should be based on geographic distance rather than river system. The level of genetic variation was similar throughout the geographic range examined (mean H(E) = 0.49). However, there was evidence of a barrier to gene flow between populations in the east and west of the species range. The RFLP data support morphological evidence of hybridisation between E. camaldulensis and E. tereticornis Sm. in several populations in northeast Queensland and the genetic divergence of E. camaldulensis subsp. simulata Brooker and Kleinig.     

Role of aluminum-binding ligands in aluminum resistance of Eucalyptus camaldulensis and Melaleuca cajuputi

We investigated the roles of Al-binding ligands in Al exclusion from roots and in internal Al detoxification in roots as Al resistance mechanisms in two Al-resistant Myrtaceae trees, Eucalyptus camaldulensis Dehnh. and Melaleuca cajuputi Powell. The amounts of ligands secreted from roots and contained in root tips of these species were compared with those of an Al-sensitive species, Melaleuca bracteata F. Muell., after the roots were exposed to 0 or 1 mM AlCl₃ solution. Secretion of well-known ligands (citrate, oxalate, and malate) from roots under Al treatment was low in all species. However, in E. camaldulensis, the Al-binding capacity of root exudates under Al treatment was considerable and was higher than that in M. bracteata. Gel filtration chromatography revealed that a low-molecular-weight Al-binding ligand was secreted from roots in response to Al only in E. camaldulensis. On the other hand, the Al-binding capacity of cell sap in root tips under Al treatment was similar for the resistant and sensitive species. These results suggest that Al exclusion by secretion of the unknown low-molecular-weight Al-binding ligand from roots contributes to the Al resistance of E. camaldulensis, whereas M. cajuputi has developed Al-resistance mechanisms other than secretion of ligands from roots or concentration of internal ligands in root tips.     

Characterization of citrate transport through the plasma membrane in a carrot mutant cell line with enhanced citrate excretion

The superior ability of citrate excretion in a carrot (Daucus carota L.) mutant cell line, namely IPG (insoluble phosphate grower) [Takita et al. (1999a) Plant Cell Physiol. 40: 489] cells has been characterized in terms of citrate transport at the plasma membrane. IPG cells released about a 20-fold increase in citrate in comparison with malate, while the concentration of malate was only 35% lower than that of citrate in the cell sap. Citrate excretion was sensitive to anion channel blockers, such as niflumic acid and anthracene-9-carboxylic acid. These results indicate that IPG cells release citrate through the plasma membrane using citrate specific anion channels. The rate of citrate release from IPG cells was not affected by the concentration of aluminum (0 and 50 micro M), soluble P(i) (0 or 2 mM) and the pH (4.5-5.6) of the medium, suggesting that anion channels would not be regulated by such external conditions. Citrate excretion correlated with the H(+) efflux, possibly from the action of H(+)-ATPase on the plasma membrane. The activity of plasma membrane H(+)-ATPase was about three times higher in IPG cells than in wild-type cells, and might be involved in the high citrate excretion ability.     

A tissue culture method for rapid clonal propagation of mature trees of Eucalyptus torelliana and Eucalyptus camaldulensis

Multiple shoots were induced from nodal segments of mature Eucalyptus torelliana F. Muell. and E. camaldulensis Dehnh trees on Murashige and Skoog's medium supplemented with Kn, BAP, Cal.Pan and Bio. Incubation in semi-solid media at 15°C with continuous illumination followed by growth in agitated liquid media was essential for shoot induction and development in primary explants of E. camaldulensis. For culture of E. gorelliana, growth in agitated liquid media alone was sufficient. Rooting could be induced in shoot cultures of E. torelliana by treatment with NAA whereas treatment with a mixture of IAA, IBA, IPA and NAA was essential for E. camaldulensis. After auxin treatment, transfer to a charcoal-containing medium was necessary. Rooted plantlets could be successfully transferred to pots and field. By this method it is estimated that about 50,000 plantlets of E. torelliana and 20,000 of E. camaldulensis can be produced, in a year, from a single nodal segment of a mature tree.Abbreviations Kn Kinetin - BAP 6-Benzylaminopurine - Ca.Pan Calcium Pantothenate - Bio Biotin - NAA -Naphthalene acetic acid - IAA Indole-3-acetic acid - IBA Indole-3-butyric acid - IPA Indole-3-propionic acid Communication No. 3315     

Iron Enhances Aluminum-induced Leaf Necrosis and Plant Growth Inhibition in Eucalyptus camaldulensis

The combined effects of excess Fe and Al on Eucalyptus camaldulensis Dehnh. were studied by investigating time course and visible symptoms of leaf necrosis, plant biomass, the status of some antioxidants and pigments and nutrient concentrations. Seedlings were grown hydroponically in nutrient solutions containing 0 or 500 μM AlCl₃, each with a FeSO₄ range of 1, 12 and 120 μM at pH 4.2. Leaf necrosis and plant growth inhibition were induced by Al and enhanced by the increase in Fe concentration. The process from the first appearance of necrotic spots to leaf death (shedding) of a leaf proceeded from a few days to about 20 days after the leaf had fully expanded. Either 120 μM Fe without Al or Al reduced plant growth to a similar extent but 120 μM Fe without Al did not cause leaf necrosis. In leaves, excess Fe (12 and 120 μM) without Al reduced concentration of ASC and GSH, while concentration of Fe, DHA and GSSG and DHA:ASC and GSSG:GSH ratios tended to increase with the increase in Fe concentration in treatment solution with or without Al. At 1 μM Fe, Al increased concentration of DHA and DHA:ASC and GSSG:GSH ratios. Catalase activity in leaves reduced with the increase in leaf Al concentration. At 1 μM Fe, Al greatly reduced concentrations of Fe and chlorophylls in leaves but increase two times Fe concentration in stems. These suggest that the enhancement effects of Fe on Al-induced leaf necrosis and plant growth inhibition can be discussed in context of the excess Fe itself weakens antioxidant capability of ASC–GSH cycle in leaves and greatly reduces plant growth; and the increase in Fe accumulation in stems is involved in Al-induced leaf chlorosis.     

Leaf dynamics and insect herbivory in a Eucalyptus camaldulensis forest under moisture stress

Abstract The influence of soil moisture content on leaf dynamics and insect herbivory was examined between September 1991 and March 1992 in a river red gum (Eucalyptus camaldulensis) forest in southern central New South Wales. Long-term observations of leaves were made in trees standing either within intermittently flooded waterways or at an average of 37. 5m from the edge of the waterways. The mean soil moisture content was significantly (P≤0.05) greater in the waterways than in the non-flooded areas. Trees in the higher soil moisture regime produced significantly larger basal area increments and increased canopy leaf area. This increase in canopy leaf area was achieved, in part, through a significant increase in leaf longevity and mean leaf size. Although a greater number of leaves was initiated and abscissed per shoot from the non-flooded trees, more leaves were collected from litter traps beneath the denser canopies of the flooded trees. Consumption of foliage by insects on the trees subjected to flooding compared to the non-flooded trees was not significantly different. However, the relative impact of insect herbivory was significantly greater on the non-flooded trees. Leaf chewing was the most common form of damage by insects, particularly Chryso-melidae and Curculionidae. No species was present in outbreak during this study. Leaf survival decreased as the per cent area eaten per leaf increased. In addition, irrespective of the level of herbivory, leaf abscission tended to be higher in E. camaldulensis under moisture deficit. The influence of soil moisture content on the balance between river red gum growth and insect herbivory is discussed.     

Growth and mineral accumulation in Eucalyptus camaldulensis seedlings irrigated with mixed industrial effluents

Effects of mixed industrial effluents on growth, dry matter accumulation and mineral nutrient in Eucalyptus camaldulensis seedlings were studied. The objective was to evaluate the adaptability of E. camaldulensis to effluent, tolerance to excess/deficiency of mineral elements and ultimately to determine suitable combinations of industrial/municipal effluent for their use in biomass production in dry areas. Different irrigation treatments were: T(1): good water; T(2): municipal effluent; T(3): textile effluent; T(4): steel effluent; T(5): textile effluent+municipal effluent in 1:1 ratio; T(6): steel effluent+municipal effluent in 1:2 ratio; T(7): steel+textile+municipal effluent in 1:2:2 ratio; and T(8): steel+textile effluent in 1:2 ratio. High concentrations of metal ions and low concentrations of Ca, Mg, K, Na, N and P in soil and seedlings of T(4) resulted in mortality of the seedlings within a few days. Addition of the textile/municipal effluent increased the survival time of the seedlings for two to three months in T(6), T(7) and T(8) treatments. Among the remaining treatments, the seedlings of T(2) attained 131 cm height, 1.97 cm collar diameter, 19 total branches and produced 158 g seedling(-1) of dry biomass at the age of 10 months. The seedling of T(3) produced the least growth and biomass. Growth equivalent to that of the seedlings of T(1) treatment was achieved when municipal effluent was mixed with textile effluent (T(5)). There was a decrease in soil pH, EC, SOC, NH(4)-N, NO(3)-N, PO(4)-P and basic cations and increase in the concentration of Cu, Fe, Mn and Zn with T(4) treatment. The reverse trend was observed in T(3) where a high concentration of Na might have reduced Mg and micronutrient concentration in seedlings potentially affecting root and leaf growth. Mixing of effluents may be useful in tree irrigation to increase biomass productivity, which is evidenced by improved growth in T(5) and survival in T(6), T(7) and T(8) treatments. Further, reduction of toxic concentration of metal ions in effluents may be helpful for a long-term field application.     

Activated carbon from Eucalyptus camaldulensis Dehn bark using phosphoric acid activation

The powdered activated carbon prepared by phosphoric acid activation was significantly affected by the carbonization temperature and the weight ratio between raw material and phosphoric acid. With an activation time of 1h and an impregnation ratio of 1:1, the activated carbons with better adsorption capacity were obtained at 500 degrees C. A reduction in the adsorption capacity of the carbon product at higher acid content than this was observed, possibly due to the collapse of the micropore structure. The properties of the resulting activated carbon were: bulk density 0.251gcm(-3), ash content 4.88%, yield 26.2%, iodine adsorption 1043mgg(-1), methylene blue adsorption number 427mgg(-1), and BET surface area 1239m(2)g(-1).     

Changes in Eucalyptus camaldulensis essential oil composition as response to drought preconditioning

Abstract

Water deficit, a common constraint in forestry, is the main cause of plant stress during plantation. The survival and growth of seedlings is also compromised by herbivory during establishment. The potential of nursery preconditioning to enhance survival chances of future trees by reducing palatability or attracting beneficial insects as a result of changes in chemical defences may be an answer to overcome this situation. Changes in essential oil production and composition were evaluated by GC and GC-MS in Eucalytus camaldulensis seedlings submitted to drought during four weeks at the last stage of nursery period (20 weeks). Significant changes in essential oil relative composition were found. Seedlings (young leaves) submitted to drought developed a terpenoid blend, which has been previously associated to mature leaves and related to their higher resistance towards herbivory. Total amount of non-oxygenated terpenes decreased by 44%, whereas some oxygenated sesquiterpenes (globulol, epiglobulol and ledol) were doubled, and 1,8-cineole content was enhanced by 28.3%.     

Characterisation of two distinct HKT1-like potassium transporters from Eucalyptus camaldulensis

Potassium is an essential macronutrient in higher plants. It plays an important physiological role in stoma movements, osmoregulation, enzyme activation and cell expansion. The demand for potassium can be substantial, especially when the plant concerned is a Eucalyptus tree in excess of 50 m tall. We have isolated two cDNAs, EcHKT1 and EcHKT2, from Eucalyptus camaldulensis (river red gum) which are expressed in leaves, stems and roots. These encode potassium transporter polypeptides with homology to the wheat K⁺-Na⁺ symporter, HKT1. EcHKT1 and EcHKT2 both complemented the K⁺-limited growth of an Escherichia coli K⁺-uptake-deficient triple mutant. EcHKT1 and EcHKT2also mediated Na⁺ and K⁺ uptake when expressed in Xenopus oocytes. A comparison of the EcHKT1 and EcHKT2 sequences and their transport properties indicated that these cDNAs represent two K⁺ transporters with distinct functional characteristics. The functional and structural conservation between these two E. camaldulensis genes and the wheat HKT1 suggests that they play an important, albeit elusive, physiological role.