The apparent loss of tissue culture competence during leaf differentiation in yams (Dioscorea bulbifera L.)

Explants taken from the leaves of yams (Dioscorea bulbifera L.) at different stages of development were cultured in vitro on a checkerboard using various combinations and/or concentrations of auxin (2,4-d) and cytokinin (6-BAP). An addition of cytokinin to the culture media was not essential for callus induction from explants derived from young leaves in the very early stages of expansion. When the leaves expanded further they required cytokinin and the requirement increased considerably during expansion. Explants taken from fully expanded leaves were no longer able to proliferate, even when extremely high concentrations of cytokinins were applied. Callus grown from highly immature leaves was able to continue proliferating in the absence of cytokinin when subcultured. Callus, that initially required cytokinin in the medium, proliferated in the absence of exogenous cytokinin when subcultured.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylamino purine - 1-NAA 1-naphthalenacetic acid     

Histological analysis of somatic embryogenesis induced in leaf explants ofHelianthus smithii Heiser

Summary According to the hormonal conditions, after one month of culture shoots or somatic embryos could be obtained from leaf explants ofHelianthus smithii. Well-shaped embryos developed on media containing a combination of auxin and cytokinin, while on media containing only cytokinin shoots were observed. The primary leaves of these shoots resembled cotyledons. A detailed histological study of the regeneration process on three media, containing only cytokinin or auxin, or a combination of both, allowed the origin and development of the observed structures to be determined. All three conditions induced somatic embryos, which then developed differently and, within one month, finally gave rise to the two types of structures which were initially observed.Abbrevations BAP 6-benzylaminopurine - MES 2-(N-morpholino)ethane-sulfonic acid - MS medium of Murashige and Skoog - NAA 1 -naphthaleneacetic acid - TDZ thidiazuron     

The physiological significance of the leaf nodules of psychotria

Summary In 6-month growth experiments it was found that leaf-nodulatedPsychotria mucronata seedlings grown in N-poor soil showed a restricted growth and developed severe nitrogen-deficiency symptoms in the leaves. Plants in the same soil supplied with NO₃-N showed healthy growth and dark green leaves. Detached Psychotria leaves bearing leaf nodules exposed to an atmosphere containing N¹⁵-labelled nitrogen gas or acetylene gas gave no evidence of nitrogen fixation, either in the light or in the dark or in both in succession. Therefore nitrogen fixation is probably not associated with the leaf nodules. Chlorophyll retention was observed around the leaf nodules in senescent Psychotria leaves. Psychotria leaf-nodule discs placed on oat leaves cause chlorophyll retention in the oat leaves below the discs. As chlorophyll retention is a common bioassay for cytokinins, these results indicate that a cytokinin-like substance is involved. With the aid of autoradiography and C¹⁴-labelled α-amino-isobutyric acid it was shown that this amino acid accumulates in the leaf nodules. Such directed transport is also a property of cytokinin.     

The effect of calcium bicarbonate on iron absorption and distribution byChrysanthemum morifolium, (Ram.)

Summary Plants grown for two weeks in high-bicarbonate nutrient solution with iron became chlorotic, absorbed less iron, and translocated a lower percentage of absorbed iron than did green plants grown under low bicarbonate with iron. Chlorotic plants, pretreated in low-bicarbonate solutions lacking iron, absorbed more iron and translocated a higher percentage to leaves than the green plants. Plants induced to chlorosis by high bicarbonate absorbed less iron after transfer to low-bicarbonate solution containing iron than did chlorotic plants pretreated with low-carbonate solution lacking iron. Initial localization of iron occurred in the roots. A considerable amount of the iron initially found on the roots was translocated to developing shoots over a nine-week period unless the plants were grown in high bicarbonate solutions. More iron was translocated from roots of plants in minus-iron solutions following initial absorption than when iron was supplied in the nutrient solutions. Journal Series Paper736. University of Georgia, College of Agriculture Experiment Stations, College Station, Athens, Ga. 30601.     

The pattern of carboxylate exudation in Banksia grandis (Proteaceae) is affected by the form of phosphate added to the soil

Roots of a wide range of plant species exude carboxylates, e.g. citrate, into the rhizosphere, to mobilise sparingly available phosphate. We investigated the carboxylates in root exudates of Banksia grandisWilld. (Proteaceae), which occurs on severely phosphate-impoverished soils in Western Australia. Plants were grown in pots with a nutrient-poor quartz sand, with phosphate, at 25 g P g^–1, added as either K-phosphate, glycerol phosphate, Fe-phosphate or Al-phosphate.Plants grown on Fe-phosphate or Al-phosphate formed `proteoid' or `cluster' roots, and exuded significant amounts of carboxylates. Plants grown on K-phosphate did not form cluster roots; their leaves were chlorotic, and some of these plants died during the experiment. Plants grown on glycerol phosphate did have cluster roots, but their leaves also became chlorotic, albeit later in the experiment.Tri- and dicarboxylates (citrate, 60%; malate, 25%; trans-aconitate, 14%) were the major carboxylates in root exudates when P was supplied as Al-phosphate. The same tri- and dicarboxylates were also exuded when P was supplied as Fe-phosphate (31, 14 and 12%, respectively). In addition, these plants exuded monocarboxylates (lactate, 30%; acetate, 12%). We analysed the effect of the different carboxylates on the mobilisation of phosphate and Fe in two different types of soils. The ecological significance of the difference in exudate spectrum for the mobilisation of nutrients and for the detoxification of aluminium is discussed.Because the leaves of plants grown with K-phosphate or glycerol-phosphate appeared chlorotic, we analysed the concentrations of P, Fe, Zn, Mn and Cu in these leaves. Only the concentration of total P was considerably higher in leaves of plants grown with K- or glycerol-phosphate than that in leaves of plants grown with Fe- or Al-phosphate. Both the concentration of total Fe and that of reduced Fe was the same in chlorotic leaves as that in leaves of plants grown with Fe- or Al-phosphate, which had a healthy appearance. It is concluded that P-induced chlorosis was not due to a lack of total or reduced Fe; it may have been due to precipitation of Fe by phosphate.     

Genotype and plant growth regulator-dependent response of somatic embryogenesis from Gentiana spp. leaf explants

Gentiana kurroo (Royle), Gentiana cruciata (L.), Gentiana tibetica (King. ex Hook. f.), Gentiana lutea (L.), and Gentiana pannonica (Scop.) leaves derived from axenic shoot culture were used as explants. For culture initiation, leaves from the first and second whorls from the apical dome were dissected and cultured on Murashige and Skoog (MS) basal medium supplemented with three different auxins: 2,4-dichlorophenoxyacetic acid, 1-naphthaleneacetic acid (NAA), or 3,6-dichloro-o-anisic acid (dicamba) in concentrations of 0.5, 1.0, or 2.0 mg/l; and five different cytokinins: zeatin, 6-furfurylamonopurine (kinetin), N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (TDZ), N-(2-chloro-4-pyridyl)N′-phenylurea, or 6-benzylaminopurine (BAP). The cytokinin concentrations used were dependent on the type of cytokinin and varied between 0.25 and 3.0 mg/l. After 2 mo. of culture, the morphogenic response of explants was assessed. Frequency of embryogenesis was the highest for G. kurroo (54.7%) and dependent on plant growth hormones (PGRs). This gentian was the only species showing morphogenic capabilities on media supplemented with all applied combinations of PGRs, while none of the 189 induction media permutations stimulated somatic embryogenesis from G. lutea explants. G. tibetica and G. cruciata both produced an average of 6.6 somatic embryos per explant, while G. pannonica and G. kurroo regenerated at 15.7 and 14.2 somatic embryos per explant, respectively. Optimum regeneration was achieved in the presence of NAA combined with BAP or TDZ. This auxin also stimulated abundant rhizogenesis. Somatic embryos were also regenerated from adventitious roots of G. kurroo, G. cruciata, and G. pannonica. Somatic embryos converted into plantlets on half strength MS medium.     

杯山药零余子愈伤组织诱导及植株再生的研究

对怀山药（Ｄｉｏｓｃｏｒｅａ ｏｐｐｏｓｉｔａ）零余子愈伤组织的诱导、分化、再生苗的生根和移栽进行了研究。结果表明：⑴在不同激素组合的培养基上怀山药零余子均能产生愈作组织,而且具有一次成苗的能力。ＢＡ２ｍｇ／Ｌ＋ＮＡＡ２ｍｇ／Ｌ的培养基对诱导愈伤组织最有利,其出愈率达１００％;⑵在愈伤组织的分化中,ＢＡ１ｍｇ／Ｌ＋ＮＡＡ１ｍｇ／Ｌ的激素组合是最佳的,其分化率为６３．６％,且多形成丛生芽;⑶再生植株     

A novel approach for the definition of the inorganic medium components for micropropagation of yellow passionfruit (Passiflora edulis sims. F. Flavicarpa Deg.)

Summary Mineral deficiency symptoms were observed in leaves of yellow passionfruit plantlets grown in MS medium (Murashige and Skoog, 1962) with 1.0 mg l⁻¹ (3.0 μM) gibberellic acid. Initially, leaves showed interveinal chlorosis, followed by bleaching of the leaves and retarded growth. Leaf mineral analysis was done and compared to mineral requirements suggested for passionfruit in the literature. Several modifications were made to the inorganic composition of MS medium, according to mineral deficiencies, mainly of Fe and Ca, and possible toxicity of Cl. The concentration of the elements in the new medium (MSM) was based on the mineral composition of leaves of healthy plants. The chemical equilibrium was checked using the software Geochem (Sposito and Mattigod, 1980) and final adjustments were made to ensure good availability of nutrients. To test the efficiency of the modified medium nodal segments were cultured in both MS and MSM supplemented with 3.0 mg l⁻¹ (13.3 μM) 6-benzyladenine. After three subcultures mineral analysis of the leaves was done. Severe mineral deficiency was observed on the leaves of plantlets cultured in MS, while plantlets cultivated in MSM had green leaves. A comparison of the mineral analysis of plantlets in both media showed a fairly large increase in Ca, Cu, Fe, Mg and S and decrease in levels of B and Cl in plantlets cultivated in MSM. A slight increase or decrease in other elements was also observed. Subculture of the chlorotic plantlets into MSM showed that the visual symptoms of mineral deficiency disappeared in 2–4 wk.     

Morphogenetic responses from in vitro cultured seedling explants of mung bean (Vigna radiata L. Wilczek)

The morphogenetic responses of seedling explants of mung bean (Vigna radiata L. Wilczek cv ML-5) were studied in vitro. Direct induction of shoots/plants was possible from shoot tip, cotyledon and cotyledonary node explants. Dedifferentiation of the explants viz; Shoot tip, cotyledons, cotyledonary node, primordial leaves and roots was obtained on basal medium supplemented with auxin and cytokinin. Shoot regeneration was limited to primary calli while rhizogenesis was of common occurrence in established calli. In addition to differences in hormonal requirements, the various explants showed preferential growth in different basal media.     

Formation in vitro of ripe tomato fruits from thin layer explants of flower pedicels

Thin longitudinal sections cut from pedicels of fifteen cultivars of tomato (Lycopersicon esculentum) were grown in vitro on Murashige-Skoog medium supplemented with various concentrations of different auxins and cytokinins. Isatin (an auxin precursor slowly converted to an active auxin) was the most effective source of auxin for the formation of buds without prior root formation, while zeatin was the most effective cytokinin for growth and development of the buds. Flower buds and ripe fruits developed consistently from explants of the cultivar Pixie Hybrid II treated with 10 M isatin plus 3 M zeatin as the cytokinin. Fruits developed parthenocarpically, grew to a diameter of about 15 mm, ripened promptly, and possessed normal color and flavor.Abbreviations BAP benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - IPA isopentyladenosine - NAA -napthaleneacetic acid     

Photosynthetic carbon fixation in iron-chlorotic and recovered green sugarcane leaves

Summary Sugarcane var. Co 740 is grown in various parts of Maharashtra (India) and is susceptible to chlorosis due to physiological non-utilization of iron. The physiological disorder is seen over a large area and it results in poor yield. Low sucrose yield can be recovered by foliar sprays of ferrous sulphate. The nonchlorotic and chlorotic leaves were used for the photosynthetic studies. The leaves after ferrous sulphate treatment show an increase in total chlorophyll contents and at the same time show an improved chlorophylla to chlorophyllb ratio which is affected in the chlorotic ones. The recovered green leaves have higher uptake of nitrogen, phosphorus, potassium and iron.¹⁴CO₂ fixation studies for short- and long-term experiments reveal that recovered green leaves can synthesize malate more efficiently and also utilize it for sucrose synthesis more rapidly than in the chlorotic ones. On the contrary more amino acids, reducing sugars and sugar phosphates are synthesized in the chlorotic leaves. There is also an accumulation of citrate, glutamate, and tartrate in the chlorotic leaves. Our results indicate that sucrose synthesis is disturbed in the chlorotic leaves and can be corected byfolia: sprays of ferrous sulphate.     

The morphological and physiological properties of the genetic tumours from a Nicotiana interspecific hybrid

Many plant tumours have been loosely referred to as genetic tumours when no obvious external cause was identifiable. In this study, the morphology of genetic tumours was detected by scanning electron microscopy (SEM) and the influence of exogenous phytohormones on genetic tumour growth was examined. Genetic tumours in Nicotiana hybrid were first observed in the hypocotyl area and later appeared from stems as a cluster complex. In vitro, genetic tumours were produced 5-6 days after explant treatment near each of the cut ends. The morphology of genetic tumours was investigated using SEM. Genetic tumours are mainly composed of epidermal cells surrounded by a well developed extracellular matrix (ECM), that is organized in a network linking surface cells. Globular structures containing several cells were formed from these clusters of epidermal cells. Vigorous tumourous growth from the leaf discs of the Nicotiana hybrid was obtained on medium supplemented with auxin and cytokinin at 0 to 10 muM, but 100 muM of auxin and cytokinin inhibited genetic tumour growth. Leaf discs treated with 10 muM cytokinin or auxin alone produced genetic tumours, but 10 muM cytokinin supplemented with 100 muM auxin stopped growth. Therefore genetic tumours are tissues that are, clearly, disturbed in endogenous hormone balance or have altered sensitivity to hormones and response to exogenous hormones.     

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with ³H and auxin was found to promote conversion of zeatin-type cytokinins to ³H-labelled adenine derivatives. When the very rapid metabolism of exogenous [³H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.Abbreviations Ade adenine - Ados adenosine - BA 6-benzylaminopurine - C control - Con A concanavallin A - CP cellulose phosphate - IPT isopentenyl transferase - NAA 1-naphthylacetic acid - NP normal phase - NPPU N-(3-nitrophenyl)-N-phenylurea - RIA radioimmunoassay - RP reversed phase We wish to thank Dr. J. Zwar for supplying phenylurea derivitives.     

Exogenous auxin and cytokinin dependent activation of CDKs and cell division in leaf protoplast-derived cells of alfalfa

Alfalfa leaf protoplast cultures were used to study the role ofexogenously supplied auxin and cytokinin on the level and activity ofCdc2-related protein kinases and progression through the first celldivision cycle after re-activation of cell division. Among the threealfalfa Cdc2-related kinases studied, the Cdc2MsA/B kinase (PSTAIRE)showed only significant activity during the first four days ofprotoplast culture while the Cdc2MsD (PPTALRE) and Cdc2MsF kinases(PPTTLRE) exhibited only low or undetectable activity, respectively,during this period. Although the Cdc2MsA/B protein could be detectedin leaves and freshly isolated protoplasts in variable amounts, thekinase was never active in these cells. The kinase protein disappearedfrom protoplast-derived cells at the beginning (8h) of culture but itssynthesis re-commenced dependent on the presence of exogenous auxin butnot cytokinin. The cytokinin response of alfalfa protoplast-derivedcells varied significantly in different experiments although cytokininwas always required for completion of the first cell division cycle.Frequently both auxin and cytokinin was required for DNA replication asnot more than 5% of cells could incorporate BrdU into their DNAduring three days and significant Cdc2MsA/B activity could not bedetected in the absence of exogenous cytokinin. In other protoplastpopulations, the Cdc2MsA/B kinase was activated by auxin alone andallowed the protoplast-derived cells to enther the S-phase at a similarrate observed in parallel cultures with both auxin and cytokinin. Evenin these cultures, however, ca. 95% of the protoplast-derivedcells were arrested before mitosis without exogenous cytokinin supplywhich could be correlated with decreasing Cdc2MsA/B activity. Theseobservations suggest, that although cytokinin is required for bothG0-G1/S and G2/M cell cycle transitions, in certain cultures theG1/S requirement is overcome by some unknown factors (e.g.conditions of explants; endogenous cytokinins etc.). Furthermore, ourexperiments indicate, that the roles of cytokinin are related to thepost-translational regulation of the Cdc2MsA/B kinase complex atboth cell cycle transition points in alfalfa leaf protoplast-derivedcells. Finally, as a marker for the transition from the differentiated(G0) stage to the activated (G1) stage, we suggest using the parametersof nuclear morphology (size and ratio ofnucleus/nucleolus).     

In vitro proliferation of saffron (Crocus sativus L.) stigma

Excised young intact stigmas plus ovaries of Crocus sativus L. were cultured on Linsmaier-Skoog media supplemented with either a cytokinin or an auxin alone or in combinations. Benzyladenine and kinetin at concentrations of 0.1, 1, and 5 mgl^-1 supported growth, and crocin was biosynthesized in the stigmas in vitro. Auxins had little effect. Young excised single stigmas or half ovaries were also cultured so as to form stigma-like structures in order to explore a possible new approach to industrial production of the spice, saffron. On Linsmaier-Skoog and Nitsch media supplemented with kinetin at concentration of 1 or 5 mgl^-1 and alpha-naphthalene acetic acid or indole-butyric acid at concentration of 0.1 or 10 mgl^-1 in combinations, stigma-like structures appeared directly and indirectly (through meristematic tissue), grew and matured. The maximum number of structures were 75 per half ovary. Three kinds of yellow pigments including crocin were tentatively identified by TLC in the stigma-like structures as was the case for the in vivo grown natural stigma, although the contents were lower.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA indole-acetic acid - IBA indole-butyric acid - NAA alpha-naphthalene-acetic acid - TLC thin layer chromatography     

Interspecific hybridization of red clover (Trifolium pratense L.) with T. sarosiense Hazsl. Using in vitro embryo rescue

Summary Interspecific hybrid clover plants from the cross Trifolium sarosiense Hazsl. X T. pratense L. were obtained in the present investigation. Immature hybrid embryos were excised aseptically from the pistillate parent, T. sarosiense (2 n = 48), and cultured in vitro prior to in situ abortion. Agar-solidified nutrient media modified from that developed previously for tissue and cell cultures of red clover (2 n = 14) were used for embryo rescue.The heart shaped embryos obtained were cultured for 8 to 14 days on a medium containing a high level of sucrose, a moderate level of auxin, and low cytokinin activity. Viable embryos were then transferred to a standard medium with low auxin and moderate cytokinin levels for the direct germination of shoots. Some embryos produced only callus. Plants were regenerated from callus using an alternate culture scheme. Hybrid shoot numbers were increased on a low auxin, high cytokinin medium and subsequently rooted before transfer to soil in the greenhouse.About 10% of the hybrid embryos were rescued using the optimal culture sequence. Five full-sib families of the F₁ hybrid were successfully grown to maturity. Root-tip cells of hybrid plants possessed the expected somatic chromosome number of 31. The genetically determined leaf-mark trait carried by the staminate parent and the rhizomatous root habit of the pistillate parent were expressed in hybrid plants.The investigations reported in this paper (No. 81-3-151) were performed in connection with projects of the Kentucky Agricultural Experiment Station and the paper is published with the approval of the Director     

Micropropagation of Sesbania aculeata (Pers.) by adventitious organogenesis

Multiple shoots differentiated from hypocotyl explants of Sesbania aculeata (Pers.) syn S. cannabina (Retz.) Pers., a leguminous woody shrub, when cultured on Murashige and Skoog's basal medium supplemented with auxin (IBA, NAA) or auxin and cytokinin (IBA + BAP, NAA + BAP). Shoot budding occurred directly from the explant as well as from callus. Differentiation of shoot and root occurred in one step in the same concentration of auxin or auxin and cytokinin. Elongation of shoots occurred in the shoot induction medium.     

Limestone gravel as growth medium in hydroponics

Summary Plants were grown in gravel beds of basalt or limestone. With the standard nutrient solution yields for the plants grown in the limestone gravel were very much reduced and the plants were chlorotic. This chlorosis and reduced yields remained even if the gravel was acid or water washed beforehand, or if it was pretreated in high phosphate concentrations.High yields, comparable to those obtained in basalt beds were obtained if phosphate and iron were added in small amounts (0.1mM P, 1 mg/1 Fe) at each irrigation. This technique to avoid lime induced chlorosis can be easily and economically accomplished in hydroponics. It proved successful here with lettuce, cucumber, tomato and eggplants.     

Bicarbonate,the most important factor inducing iron chlorosis in vine grapes on calcareous soil

Summary In pot experiments grape vine was grown on a calcareous and on a non calcareous soil with a low and with a high water saturation. During the growing period soil solution samples were collected and analyzed for their pH and for HCO ₃ ⁻ , phosphate, Fe, and Ca. High water saturation resulted in a pH increase and in an increase of the HCO ₃ ⁻ concentration in both soils. The level in pH and HCO ₃ ⁻ , however, was much higher in the calcareous soil than in the non calcareous soil. The Fe concentration varied much throughout the experimental period, but there was no major differences between soils and water saturation treatments. The Ca concentration of the soil solution increased with time in the calcareous soil; for the non calcareous soil rather the reverse was true. The phosphate level in the soil solution of the non calcareous soil was about 10 times higher than in the calcareous soil. After 3 weeks growth all plants of the calcareous soil with the high water saturation showed first symptoms of Fe deficiency. These became more intense from day to day. Plants of the other treatments did not show any chlorotic symptoms. In the treatment with the chlorotic plants the HCO ₃ ⁻ concentration of the soil solution was the highest, the phosphate concentration the lowest from all treatments. It is therefore concluded that HCO ₃ ⁻ and not phosphate is the primary cause for lime induced Fe chlorosis. Despite the low phosphate concentration in the soil solution, the P concentration in the chlorotic leaves was more than twice as high as the P concentration in green leaves grown on the same soil. It is thus assumed that the high P content frequently found in chlorotic leaves is the result and not the cause for Fe chlorosis.     

The effect of soil moisture on the tolerance of Lupinus pilosus genotypes to a calcareous soil

Commercial narrow-leafed lupins (Lupinus angustifolius L.) grown on calcareous soils commonly display chlorotic symptoms resembling Fe deficiency. The severity of chlorosis increases with concurrent increases in soil moisture content. Our research has indicated that the rough-seeded lupin species, Lupinus pilosus Murr., has a range of adaptation to calcareous soils, from tolerant to intolerant. A pot experiment was conducted comparing a tolerant, a moderately tolerant and a moderately intolerant genotype of L. pilosus. Plants were grown for 35 days in a calcareous soil (50% CaCO₃) at three moisture contents (80%, 100% and 120% of field capacity); the growth was compared with that on a fertile black cracking clay control soil at 70% of field capacity. Visual chlorosis score, chlorophyll meter readings, number of leaves and shoot dry weights were recorded at 14, 21, 28 and 35 days after sowing. Concentrations of chlorophyll, active Fe and nutrients in the youngest fully expanded leaves were also measured. Results showed that increased soil moisture increased the severity of chlorotic symptoms (increased chlorosis score) in all genotypes. The tolerant genotype showed significantly less symptoms than other genotypes at all moisture contents. All genotypes were able to recover from chlorosis symptoms at 80% moisture in the calcareous soil. Chlorosis score negatively correlated with chlorophyll meter readings, chlorophyll concentration and foliar active and total Fe, and Mn concentrations. Visual chlorosis score appeared to be a cost effective, accurate and efficient method enabling classification of the tolerance of genotypes. The chlorotic symptoms were likely to be due to HCO₃ ^- induced nutrient deficiencies or a direct effect of HCO₃ ^- on chlorophyll synthesis. This study indicates that the most probable mechanism of tolerance is related to an ability to prevent uptake of HCO₃ ^- or efficiently sequester it once inside the root which prevents increases in internal pH and transport to the shoots.