Response of durum wheat cultivars to immature embryo culture,callus induction and in vitro salt stress

Response of twenty eight cultivars of durum wheat (Triticum turgidum var. durum) to immature embryo culture, callus production and in vitro salt tolerance was evaluated. For assessment of cultivars to salt tolerance, growing morphogenic calli were exposed to different concentrations of NaCl (0, 0.3, 0.6, 0.9, 1.2, 1.5, 1.8 and 2.1% w/v) added to the culture medium during two subsequent subcultures (4 weeks each). Comparison of cultivars for callus induction from immature embryo was based on callus induction frequency and fresh weight growth of callus (FWG). While, for salt tolerance, the relative fresh weight growth (RFWG) and necrosis percent of callus were used. There were significant differences among cultivars for potential of regeneration from immature embryo, and ‘Shahivandi’ a native durum wheat cultivar originating from western Iran was superior among the cultivars tested. The FWG distinguished cultivars more than callus induction frequency did for callus induction evaluation. Hence, a range of FWG from 1.23 to 14.65 g was observed in ‘Mexical-75’ and ‘Omrabi-5’ cultivars, respectively. Growing calli derived from cultivars ‘PI 40100’ and ‘Dipper-6’ showed superiority for tolerating salinity under in vitro conditions. This revised version was published online in June 2006 with corrections to the Cover Date.     

Distribution of genetic variability in a durum wheat world collection

Summary A durum wheat world collection of 349 entries has been used to study the amount and distribution of genetic variability based on isoenzymatic characters involving a minimum of 13 loci. Genetic variability has been studied in a hierarchical fashion: between origins and within origins, further divided into between entries per origin and within entries. Factorial analysis of correspondences and chi-square distance were the basic statistical tools. The effect of domestication is deduced by comparing isozymic frequencies between wild emmer and durum wheat. It involves changes in frequencies mainly towards the accumulation of null alleles. The richest origins of genetic variation for durum wheat were Iran, Mexico, Ethiopia, Egypt and Afghanistan. Generally, between-entry variability was larger than the withinentry component. Exceptions were the accessions from Mexico, Greece, Argentina and Cyprus. The relationships between origins were greatly affected by their within-variability, the logic in the grouping is mostly along geographical or political lines. Egypt might be considered a microcenter of diversity for durum wheat within the Mediterranean center, although it is certainly related to Ethiopia (included in the Abisinic center). Mexico has become a new microcenter of diversity, quite likely man-made, and is distant from other centers of durum wheat diversity as far as gene frequency is concerned.The experimental part of this study was carried out at the Department of Genetics, Fac. Biologia, Universidad Complutense, Madrid, Spain     

Precise mapping of a locus affecting grain protein content in durum wheat

Grain protein content (GPC) is an important factor in pasta and breadmaking quality, and in human nutrition. It is also an important trait for wheat growers because premium prices are frequently paid for wheat with high GPC. A promising source for alleles to increase GPC was detected on chromosome 6B of Triticum turgidum var. dicoccoides accession FA-15-3 (DIC). Two previous quantitative trait locus (QTL) studies found that the positive effect of DIC-6B was associated to a single locus located between the centromere and the Nor-B2 locus on the short arm of chromosome 6B. Microsatellite markers Xgwm508 and Xgwm193 flanking the QTL region were used in this study to develop 20 new homozygous recombinant substitution lines (RSLs) with crossovers between these markers. These 20 RSLs, plus nine RSLs developed in previous studies were characterized with four new RFLP markers located within this chromosome segment. Grain protein content was determined in three field experiments organized as randomized complete block designs with ten replications each. The QTL peaks for protein content were located in the central region of a 2.7-cM interval between RFLP markers Xcdo365 and Xucw67 in the three experiments. Statistical analyses showed that almost all lines could be classified unequivocally within low- and high- protein groups, facilitating the mapping of this trait as a single Mendelian locus designated Gpc-6B1. The Gpc-6B1 locus was mapped 1.5-cM proximal to Xcdo365 and 1.2-cM distal to Xucw67. These new markers can be used to reduce the size of the DIC chromosome segment selected in marker-assisted selection programs. Markers Nor-B2 and Xucw66 flanking the previous two markers can be used to select against the DIC segment and reduce the linkage drag during the transfer of Gpc-6B1 into commercial bread and pasta wheat varieties. The precise mapping of the high GPC gene, the high frequency of recombinants recovered in the targeted region, and the recent development of a tetraploid BAC library including the Gpc-6B1 DIC allele are the first steps towards the map-based cloning of this gene.Communicated by J. Dvorak     

Production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture

The objective of this work was to produce doubled haploid plants from durum wheat through the induction of androgenesis. A microspore culture technique was developed and used to produce fertile doubled haploid plants of agronomic interest. Five cultivars, one selected line, plus a collection of 20 F₁ crosses between different genotypes of high breeding value were used. Studies on several factors such as pre-treatments and media components were carried out in order to develop a protocol to regenerate green haploid plantlets. Anthers were pre-treated in 0.7 M mannitol. Microspores, from anther maceration, were plated on a C₁₇ induction culture medium with ovary co-culture. The optimum regeneration medium J25–8 was used. From 35 microspore isolations, 407 green plantlets were obtained. With this technique mature embryos were obtained. Green plants were regenerated from all genotypes used and approximately 67% of them were spontaneously doubled haploids. Some haploids and a very few polyploids plants were obtained. From the 407 plants, 275 were completely fertile and gave enough seeds to be assayed in the field. This protocol could be used complementary to or instead of the intergeneric crossing with maize as an economically feasible method to obtain doubled haploids from most durum wheat genotypes.     

Evaluation of five strategies for obtaining a core subset from a large genetic resource collection of durum wheat

The use of plant genetic resources contained in a large collection may be enhanced by specifying subsamples, called core samples. Five strategies for selecting a core sample from a collection of 3000 durum wheat accessions were applied and evaluated using four qualitative and eight quantitative spike characters. Each of the following strategies generated about 500 accessions for the core sample: random, random-systematic according to chronology of entry of the accessions into the collection, stratified by countryof-origin, stratified by log frequency by country-of-origin, and stratified by canonical variables. The first three strategies produced samples representative of the whole collection, but the remaining two produced the desired effect of increasing frequencies from less-represented countries-of-origin for several characters. The stratified canonical sample increased phenotypic variances. The quality of core samples is dependent upon good passport and evaluation data to partition the collection. The multivariate approach is extremely useful, but requires considerable data from the whole collection. Ecogeographic origin may be used in the absence of evaluation data on several characters to select useful core samples.     

Cadmium uptake and bioaccumulation in selected cultivars of durum wheat and flax as affected by soil type

Accumulation of cadmium (Cd) in crop plants is of great concern due to the potential for food chain contamination through the soil-root interface. Although Cd uptake varies considerably with plant species, the processes which determine the accumulation of Cd in plant tissues are affected by soil factors. The influence of soil type on Cd uptake by durum wheat (Triticum turgidum var. durum L.) and flax (Linum usitatissimum L.) was studied in a pot experiment under environmentally controlled growth chamber conditions. Four cultivars/lines of durum wheat (Kyle, Sceptre, DT 627, and DT 637) and three cultivars/lines of flax (Flanders, AC Emerson, and YSED 2) were grown in two Saskatchewan soils: an Orthic Gray Luvisol (low background Cd concentration; total/ABDTPA extractable Cd: 0.12/0.03 mg kg^-1, respectively) and a Dark Brown Chernozem (relatively high background Cd concentration; total/ABDTPA Cd: 0.34/0.17 mg kg^-1 respectively). Plant roots, stems, newly developed heads, and grain/seeds were analyzed for Cd concentration at three stages of plant growth: two and seven weeks after germination, and at plant maturity. The results showed that Cd bioaccumulation and distribution within the plants were strongly affected by both soil type and plant cultivar/line. The Cd concentration in roots leaves and stems varied at different stages of plant growth. However, all cultivars of both plant species grown in the Chernozemic soil accumulated more Cd in grain/seeds than plants grown in the Orthic Gray Luvisol soil. The different Cd accumulation pattern also corresponded to the levels of ABDTPA extractable and metal-organic complex bound soil Cd found in both soils. Large differences were found in grain Cd among the durum wheat cultivars grown in the same soil type, suggesting the importance of rhizosphere processes in Cd bioaccumulation and/or Cd transport processes within the plant. Distribution of Cd in parts of mature plants showed that durum grain contained up to 21 and 36% of the total amount of Cd taken up by the plants for the Orthic Gray Luvisol and Chernozemic soils, respectively. These results indicate the importance of studying Cd speciation, bioaccumulation and cycling in the environment for the management of agricultural soils and crops.     

Microsatellite analysis reveals a progressive widening of the genetic basis in the elite durum wheat germplasm

It has been argued that the level of genetic diversity in the modern durum wheat (Triticum turgidum L. var. durum) elite germplasm may have declined due to the high selection pressure applied in breeding programs. In this study, 58 accessions covering a wide spectrum of genetic diversity of the cultivated durum wheat gene pool were characterized with 70 microsatellite loci (or simple sequence repeats, SSRs). On average, SSRs detected 5.6 different allelic variants per locus, with a mean diversity index (DI) equal to 0.56, thus revealing a diversity content comparable to those previously observed with SSRs in other small-grain cereal gene pools. The mean genetic similarity value was equal to 0.44. A highly diagnostic SSR set has been identified. A high variation in allele size was detected among SSR loci, suggesting a different suitability of these loci for estimating genetic diversity. The B genome was characterized by an overall polymorphism significantly higher than that of the A genome. Genetic diversity is organised in well-distinct sub-groups identified by the corresponding foundation-genotypes. A large portion (92.7%) of the molecular variation detected within the group of 45 modern cvs was accounted for by SSR alleles tracing back to ten foundation-genotypes; among those, the most recent CIMMYT-derived founders were genetically distant from the old Mediterranean ones. On the other hand, rare alleles were abundant, suggesting that a large number of genetic introgressions contributed to the foundation of the well-diversified germplasm herein considered. The profiles of recently released varieties indicate that the level of genetic diversity present in the modern durum wheat germplasm has actually increased over time.Communicated by F. Salamini     

The inheritance of tetraploid wheat seed peroxidases

Summary Embryo and endosperm peroxidases from dry mature seeds of three subspecies of tetraploid wheat (Triticum turgidum L.) were subjected to genetic analysis. The inheritance of eight isozymes (embryo isozymes a₂, d₁, d₂, e and f; and endosperm isozymes b, d and 4) were studied in F₂'s obtained from different wheat accessions. Simple monogenic inheritance producing three banded: one null segregation and two epistatic segregations (97 and 151) were found. In the case of isozymes b, d and 4, monogenic or epistatic segregation depended on the F₂ analyzed. Segregation data indicated that at least 9 different loci would determine the peroxidase isozymes of tetraploid wheat seed, all the loci studied containing null alleles. Furthermore, several loci determining embryo peroxidases were noticed to be mutually linked. All these data are discussed in context of the inheritance of seed peroxidases in hexaploid wheat and rye.     

The addition of Dasypyrum villosum (L.) Candargy chromosomes to durum wheat (Triticum durum Desf.)

Summary Six monosomic addition lines were produced in which different Dasypyrum villosum (L.) Candargy chromosomes were added to the chromosome complement of Triticum durum Desf. cv. Creso. Each added alien chromosome was found to have a specific effect on plant morphology and fertility. Transmission rate varied widely (from 7.5 to 27.7%) among the six univalent chromosomes. Different monotelosomic addition plants derived by a relatively high frequency of chromosome misdivision were isolated. The addition lines should be useful for studying Dasypyrum chromosome homoeology and the introduction of alien variation into durum and common wheats.Research supported by a grant from the Italian Research Council for Finalized Project IPRA. Sub-project Plant Breeding, Paper No. 1095     

Leaf development and phenology of Triticum aestivum and T. durum under different moisture regimes

Wheat grain yield production in the rain-fed areas is limited by water deficits during crop growth. A greenhouse experiment was conducted during spring 1992 at ICARDA, Tel Hadya, Syria, with eight genotypes representing two Triticum species (Triticum turgidum var. durum and Triticum aestivum L.) under four soil-moisture regimes (95%, 75%, 55%, and 35% field capacity) to study the effect of water deficit on leaf development. The phyllochron was similar in the two species across the watering regimes. The range in variation in phyllochron among the genotypes was similar in the two species. Phyllochron response to water stress among genotypes was distinct in the driest regime in both species. Cham 6 (T. aestivum) and Gallareta (T. turgidum var. durum) had similar phyllochron across all moisture regimes whereas in other genotypes phyllochron was higher in the dries regime. Leaf area decreased with increasing moisture stress. Triticum turgidum var. durum genotypes were later in flowering as they had, on average, one leaf more than Triticum aestivum genotypes with similar leaf appearance rates.     

Efficient and rapid <Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated genetic transformation of durum wheat (<Emphasis Type="Italic">Triticum turgidum L. var. durum)</Emphasis> using additional virulence genes

Genetic transformation of wheat, using biolistics or Agrobacterium, underpins a range of specific research methods for identifying genes and studying their function in planta. Transgenic approaches to study and modify traits in durum wheat have lagged behind those for bread wheat. Here we report the use of Agrobacterium strain AGL1, with additional vir genes housed in a helper plasmid, to transform and regenerate the durum wheat variety Ofanto. The use of the basic pSoup helper plasmid with no additional vir genes failed to generate transformants, whereas the presence of either virG⁵⁴² or the 15 kb Komari fragment containing virB, virC and virG⁵⁴² produced transformation efficiencies of between 0.6 and 9.7%. Of the 42 transgenic plants made, all but one (which set very few seeds) appeared morphologically normal and produced between 100 and 300 viable seeds. The transgene copy number and the segregation ratios were found to be very similar to those previously reported for bread wheat. We believe that this is the first report describing successful genetic transformation of tetraploid durum wheat (Triticum turgidum L. var. durum) mediated by Agrobacterium tumefaciens using immature embryos as the explant.     

The use of the electrolyte leakage method for assessing cell membrane stability as a water stress tolerance test in durum wheat

This work was carried out to adapt the electrolyte leakage technique todurum wheat and then to evaluate its relevance in the assessment of the cellmembrane stability as a mechanism of water stress tolerance in this species.Themethod currently used is based on in vitro desiccation ofleaf tissues by a solution of polyethylene glycol (PEG) and a subsequentmeasurement of electrolyte leakage into deionised water. It consists of threesuccessive steps: (1) a washing treatment to remove solutes from both leafsurfaces and cells damaged by cutting; (2) a stress period during which theleaftissues are plunged in a PEG-solution and (3) a rehydration period during whichafter-effects of the stress are evaluated. During the washing period, the majorpart of electrolytes was removed within 15 min. Varying the stressconditions influenced both the percent and the kinetics of electrolyte leakageduring rehydration. Electrolyte leakage exhibited a characteristic patternreflecting the condition of cellular membranes (repair and hardening). Inpractice, we recommend a 15-minute washing time, a10-hour stress period and 4 h of rehydration. Theextent of the cell membrane damage not only correlated well with the growthresponses of wheat seedlings belonging to various cultivars to withholdingwaterbut also with the recognised field performances of these cultivars. Therelativeproportion of endogenous ions lost in the effusate during the rehydration stepmay vary strongly according to the element analysed and the precise nutritionalstatus of the plant should therefore be considered. However, an increase ininorganic ion leakage does not fully explain the recorded PEG-induced increasein electrical conductivity (EC) during the subsequent rehydration step andorganic ions are probably also involved in such an increase.     

Effect of phosphorus supply to the surface roots of wheat on root extension and rhizosphere chemistry in an acidic subsoil

Seedlings of two cultivars of wheat (Triticum aestivum L.) differing in tolerance to aluminium (Al) were grown using a split-root sand/soil culture technique. Each culture tube was divided horizontally into a surface (0–150 mm) compartment and a subsurface (150–250 mm) compartment separated by a root-permeable paraffin wax barrier. Thus phosphorus (P) supplied to surface roots could not percolate or diffuse into the soil in the subsurface compartment. The soil in the subsurface compartment was divided into ‘rhizosphere’ and ‘non-rhizosphere’ zones using a porous (5 μm) membrane. Root growth of both cultivars into the subsurface zone was enhanced by increased P supply to surface roots, but did not conform to known relationships between root growth and soil pH, extractable-Al, or pH, Al or P concentrations in soil solution. Concentrations of Al in soil solution in the rhizosphere were greater than those in solution in the bulk soil. Concentrations of Al reactive with pyrocatechol violet (30s-RRAI) in the rhizosphere soil solution were generally greater than those in non-rhizosphere soil. With the Al-sensitive cultivar, root dry weight and length increased as concentrations of RRAl in the rhizosphere soil solution increased. Increased concentrations of Al in rhizosphere soil solutions were not related to the presence of organic ligands in solution. The effect of P in promoting root penetration into the acidic subsurface stratum was not related to differential attainment of maturity by the plant shoots, but appeared to be related to the effect of P in enhancing the rate of root growth. Thus, suboptimal supply of P to the surface roots of a plant, even at levels sufficient to preclude development of nutritional (P) stress symptoms, may seriously reduce tolerance to Al, and hence diminish the ability of roots to penetrate into acidic subsoils.     

Genetically controlled differences in the effects of chlormequat on tetraploid wheat (Triticum turgidum)

Two crosses between Triticum turgidum wheat lines differing in their response to chlormequat (CCC) were tested. In the F₂ population of one cross, which was segregating for the Rht1 dwarfing allele, each plant was cloned by separation of two tillers, one of which was treated with CCC. The tall (rht1/rht1) and the intermediate (Rht1/rht1) genotypes showed a greater response to CCC than the semi-dwarf (Rht1/Rht1) genotype, as expressed by culm length and date of ear emergence. The F₃ families of another cross and their two semi-dwarf parents were grown in a three-replicated field test in paris of rows, one of which was treated with CCC. In one of the parents and in 1/4 of the F₃ families CCC induced a wide-angled tiller growth, suggesting a monogenic control of this growth habit in response to CCC.Based on an M.Sc. thesis presented by the senior author to the Faculty of Agriculture of The Hebrew University of Jerusalem.     

Electrostatic surface properties of plasmalemma vesicles from oat and wheat roots. Ion binding and screening investigated by 9-aminoacridine fluorescence

Right-side-out and sealed plasmalemma vesicles were isolated from roots of spring wheat (Triticum aestivum L. cv. Drabant) and oat (Avena sativa L. cv. Brighton) by two-phase partition in a medium containing sucrose (0.25 mol l^-1). Oat root plasmalemma vesicles were discovered to contain a strongly fluorescent compound with an emission maximum at 418 nm. The surface potential of the membranes was monitored by 9-aminoacridine fluorescence and the effect of protein concentration, mannitol versus sucrose, absence of osmoticum, concentrations of salt, and titrations with chelators investigated. It is concluded that i) protein concentrations of less than 50 g ml^-1 for oat and 100 g ml^-1 for wheat plasmalemma vesicles should be used to avoid serious problems with non-linearity of response of 9-aminoacridine fluorescence, ii) mannitol can be used instead of sucrose as the osmoticum, iii) the vesicles were ruptured in the absence of osmoticum allowing us to monitor both sides of the membranes, iv) plasmalemma vesicles from oat roots are more negative than vesicles from wheat roots, and v) oat and wheat root plasmalemma vesicles are isolated with about the same amounts of bound Ca²⁺ and Mg²⁺. These bound divalent cations may not, however, reflect the in-vivo conditions since the tissues were homogenised in the presence of ethylenediaminetetraacetic acid.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - c1/2 value concentration at which half of the maximum effect is observed - Mops 3-(N-morpholino)propanesulfonic acid     

Improvements in the production of doubled haploids in durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) through isolated microspore culture

The objective of this study was to produce durum wheat doubled haploid (DH) plants through the induction of microspore embryogenesis. The microspore culture technique was improved to maximize production of green plants per spike using three commercial cultivars. Studies on factors such as induction media composition, induction media support and the stage and growth of donor plants were carried out in order to develop an efficient protocol to regenerate green and fertile DH plants. Microspores were plated on a C₁₇ induction culture medium with ovary co-culture and a supplement of glutathione plus glutamine; 300 g/l Ficoll Type-400 was incorporated to the induction medium support. Donor plants were fertilized with a combination of macro and microelements. With the cultivars ‘Ciccio’ and ‘Claudio’ an average of 36.5 and 148.5 fertile plants were produced, respectively, from 1,000 anthers inoculated. This technique was then used to produce fertile DH plants of potential agronomic interest from a collection of ten F₁ crosses involving cultivars of high breeding value. From these crosses 849 green plants were obtained and seed was harvested from 702 plants indicating that 83% of green plants were fertile and therefore were spontaneously DHs. No aneuploid plant was obtained. The 702 plants yielded enough seeds to be field tested. One of the DH lines obtained by microspore embryogenesis, named ‘Lanuza’, has been sent to the Spanish Plant Variety Office for Registration by the Batlle Seed Company. This protocol can be used instead of the labor-intensive inter-generic crossing with maize as an economically feasible method to obtain DHs for most crosses involving the durum wheat cultivars grown in Spain.     

Transfer of water from roots into dry soil and the effect on wheat water relations and growth

This investigation was performed to study the effect on plant water relations and growth when some of roots grow into dry soil. Common spring water (Triticum aestivum) plants were grown from seed in soil in 1.2 m long PVC (polyvinyl chloride) tubes. Some of the tubes had a PVC partition along their center so that plants developed a split root system (SPR). Part of the roots grew in fully irrigated soil on one side of the partition while the rest of the roots grew into a very dry (-4.1 MPa) soil on the other side of the partition. Split root plants were compared with plants grown from emergence on stored soil moisture (STOR) and with plants that were fully irrigated as needed (IRR). The experiment was duplicated over two temperature regimes (10°/20°C and 15°/25°C, night/day temperatures) in growth chambers. Data were collected on root dry matter distribution, soil moisture status, midday leaf water potential (LWP), leaf relative water content (RWC) and parameters of plant growth and yield.Some roots were found in the dry side of SPR already at 21 DAE (days after emergence) at a soil depth of 15 to 25 cm. Soil water potential around these roots was -0.7 to -1.0 MPa at midday, as compared with the initial value of -4.1 MPa. Therefore, water apparently flowed from the plant into the dry soil, probably during the night. Despite having most of their roots (around 2/3 of the total) in wet soil, SPR plants developed severe plant water stress, even in comparison with STOR plants. Already at 21 DAE, SPR plants had a LWP of -1.5 to -2.0 MPa, while IRR and STOR had a LWP of -0.5 MPa or higher. As a consequence of their greater plant water stress, SPR as compared with IRR plants were lower in tiller number, ear number, shoot dry matter, root dry matter, total biomass, plant height and grain yield and had more epicuticular wax on their leaves.It was concluded that the exposure of a relatively small part of a plant root system to a dry soil may result in a plant-to-soil water potential gradient which may cause severe plant water stress, leading to reduced plant growth and yield.     

The effect of inoculation with Azospirillum brasilense on growth and nitrogen utilization by wheat plants

Azospirillium brasilense is a rhizosphere bacteria that has been reported to improve yield when inoculated on wheat plants. However, the mechanisms through which this effect is induced is still unclear. In the present work, we have studied the effects of inoculating a highly efficient A. brasilense strain on wheat plant grown in 5 kg pots with soil in a greenhouse, under three N regimes (0, 3 or 16 mM NO₃ ^–, 50 ml/pot once or twice-a -week), and in disinfected or non-disinfected soil. At the booting stage, the inoculated roots in both soils showed a similar colonization by Azospirillum sp. that was not affected by N addition. The plants grown in the disinfected soil showed a higher biomass, N content and N concentration than those in the non-disinfected soil, and in both soils the inoculation stimulated plant growth, N accumulation, and N and NO₃ ^– concentration in the tissues.At maturity, the inoculated plants showed a higher biomass, grain yield and N content than the uninoculated ones in both soils, and a higher grain protein concentration than the uninoculated. It is concluded that in the present experiments, A. brasilenseincreased plant growth by stimulating nitrogen uptake by the roots.     

Copper uptake and phytotoxicity as assessed in situ for durum wheat (Triticum turgidum durum L.) cultivated in Cu-contaminated, former vineyard soils

This work assessed in situ, copper (Cu) uptake and phytotoxicity for durum wheat (Triticum turgidum durum L.) cropped in a range of Cu-contaminated, former vineyard soils (pH 4.2–7.8 and total Cu concentration 32–1,030 mg Cu kg⁻¹) and identified the underlying soil chemical properties and related root-induced chemical changes in the rhizosphere. Copper concentrations in plants were significantly and positively correlated to soil Cu concentration (total and EDTA). In addition, Cu concentration in roots which was positively correlated to soil pH tended to be larger in calcareous soils than in non-calcareous soils. Symptoms of Cu phytotoxicity (interveinal chlorosis) were observed in some calcareous soils. Iron (Fe)–Cu antagonism was found in calcareous soils. Rhizosphere alkalisation in the most acidic soils was related to decreased CaCl₂-extractable Cu. Conversely, water-extractable Cu increased in the rhizosphere of both non-calcareous and calcareous soils. This work suggests that plant Cu uptake and risks of Cu phytotoxicity in situ might be greater in calcareous soils due to interaction with Fe nutrition. Larger water extractability of Cu in the rhizosphere might relate to greater Cu uptake in plants exhibiting Cu phytotoxic symptoms.