Recovery of cowpea seedling roots from exposure to toxic concentrations of trace metals

Rhizotoxic effects of many trace metals are known, but there is little information on recovery after exposure. Roots of 3-d-old cowpea (Vigna unguiculata (L.) Walp. cv. Caloona) seedlings were grown for 4 or 12 h in solutions of 960 μM Ca and 5 μM B at two concentrations (which reduce growth by 50 or 85%) of nine trace metals that rupture the outer layers of roots. Measured concentrations were 34 or 160 μM Al, 0.6 or 1.6 μM Cu, 2.2 or 8.5 μM ?Ga, 2.3 or 12 μM Gd, 0.8 or 1.9 μM Hg, 1.0 or 26 μM In, 2.4 or 7.3 μM La, 1.8 or 3.8 μM Ru, and 1.3 or 8.6 μM Sc. Roots were rinsed, transferred to solutions free of trace metals, and regrowth monitored for up to 48 h. Recovery from exposure to Hg occurred within 4 h, but regrowth was delayed for ≥?12 h with Al, Ga, or Ru. There was poor regrowth after 4 or 12 h exposure to Cu, Gd, In, La, or Sc. Roots recovered after being grown for 12 to 48 h in 170 μM Al, 5.1 μM? Ga, 2.0 μM Hg, or 1.4 μM Ru, but the extent of recovery was reduced with longer exposure time. Microscopy showed marked differences in symptoms on roots recovering from exposure to the various trace metals. Differences in (i) concentrations that are toxic, (ii) ability of roots to recover, (iii) time for recovery to occur, and (iv) symptoms that develop, suggest that each trace metal has a unique combination of rhizotoxic effects.     

Primary stress response induced by different elements is mediated through auxin signalling in barley root tip

Short-term exposure (15 min) of barley roots to different chemical elements revealed that Cd, Cu, Hg and Pb were the most toxic ones causing a marked root growth inhibition even at µM concentrations. Gd, La, Al, Cr, As, Zn, Ni and Se inhibited root growth to a similar extent only at mM concentrations. Despite the high 20 mM concentration, Co caused only a slight, while Mn, Mg or Ca did not evoke any root growth inhibition. Elements at concentrations inhibiting root growth caused a considerable accumulation of indole-3-acetic acid in the root apex. While Cr, As and Zn inhibited, Cd, Cu, Hg, Pb, Gd, La and Al markedly stimulated the generation of reactive oxygen species in the beginning of differentiation zone. Auxin signalling inhibitor alleviated or prevented root growth inhibition, reactive oxygen species generation and the stimulation of lipoxygenase and glutathione peroxidase activity by various elements, indicating a key role of auxin signalling in the stress response of barley root tip. On the other hand, it did not affect or even had an additive effect on dehydroascorbate reductase and ascorbic acid oxidase activity in combination with different elements. Our results indicate that the primary response of barley roots to the presence of various chemical elements during the short-term treatment is not a specific but rather a general adaptive stress response enabling the plant to survive adverse conditions.     

Soil microbial biomass, nutrient availability and nitrogen mineralization potential among vegetation-types in a low arctic tundra landscape

Copper uptake, localisation and biochemical and physiological traits were studied in hydroponically-grown Erica andevalensis plants at different increasing concentrations of Cu (1 µM, 50 µM, 100 µM, 250 µM, and 500 µM). Increasing Cu concentration in the nutrient medium led to a significative reduction in plant growth rate, an increase in root Cu concentration, leaf photosynthetic pigments and root peroxidase activity. Copper accumulation followed the pattern roots>stems>leaves, a typical behaviour of metal-excluders. Copper treatments led to significant changes in the free amino acid composition in shoots and roots and the concentration of polyamines in shoots. Analysis by scanning electron microscopy coupled with elemental X-ray analysis (SEM–EDX) showed a partial restriction of upward Cu transport by root vascular tissues. In leaf tissues, Cu mostly accumulated in the abaxial epidermis, suggesting a mechanism of compartmentalization to restrict mesophyll accumulation. The toxic effects of excess Cu were avoided to a certain extent by root immobilization, tissue compartmentalization, synthesis of complexing amino acids and induction of enzymes to prevent oxidative damage are among mechanisms adopted by Erica andevalensis to thrive in acidic-metalliferous soils.     

In situ distribution and speciation of toxic copper, nickel, and zinc in hydrated roots of cowpea

The phytotoxicity of trace metals is of global concern due to contamination of the landscape by human activities. Using synchrotron-based x-ray fluorescence microscopy and x-ray absorption spectroscopy, the distribution and speciation of copper (Cu), nickel (Ni), and zinc (Zn) was examined in situ using hydrated roots of cowpea (Vigna unguiculata) exposed to 1.5 μm Cu, 5 μm Ni, or 40 μm Zn for 1 to 24 h. After 24 h of exposure, most Cu was bound to polygalacturonic acid of the rhizodermis and outer cortex, suggesting that binding of Cu to walls of cells in the rhizodermis possibly contributes to the toxic effects of Cu. When exposed to Zn, cortical concentrations remained comparatively low with much of the Zn accumulating in the meristematic region and moving into the stele; approximately 60% to 85% of the total Zn stored as Zn phytate within 3 h of exposure. While Ni concentrations were high in both the cortex and meristem, concentrations in the stele were comparatively low. To our knowledge, this is the first report of the in situ distribution and speciation of Cu, Ni, and Zn in hydrated (and fresh) plant tissues, providing valuable information on the potential mechanisms by which they are toxic.     

Accumulation of low-molecular-weight organic acids in roots and leaf segments of Zea mays plants treated with cadmium and copper

Maize seedlings grown in a nutrient solution were treated with Cd (50, 100 µM) or Cu (50, 100 µM). Roots and separated leaf sections (from the youngest––basal, through the middle––mature, to the oldest––apical) were analysed. Shoot and root fresh weight, and root net growth rates were reduced significantly after Cu application in comparison to Cd. Copper (50, 100 µM) and cadmium (100 µM) decreased the sum of chlorophyll and total carotenoid pools mostly in mature and old leaf sections. The concentration of Cu and Cd increased in the old and mature leaf sections. Analysis of organic acids showed that both metals differently influenced the low-molecular-weight organic acids (LMWOAs) content in maize leaf sections. In mature sections, the excess Cu increased the succinate and tartrate contents, whereas in the young ones mainly the tartrate level was elevated. Cadmium elevated citrate accumulation in mature and old sections, compared to the control plants. Malate, the main LMWOA in maize leaves, increased only after addition of 100 µM of Cd (mature and old sections) or 50 µM of Cu (old sections). Analysis of LMWOAs in roots showed that the excess of Cd or Cu induced higher accumulation of tartrate and malate and, additionally, copper increased the citrate content.     

Metal ion effects on hydraulic conductivity of bacterial cellulose–pectin composites used as plant cell wall analogs

Low concentrations of some trace metals markedly reduce root elongation rate and cause ruptures to root rhizodermal and outer cortical cells in the elongation zone. The interactions between the trace metals and plant components responsible for these effects are not well understood but may be linked to changes in water uptake, cell turgor and cell wall extensibility. An experiment was conducted to investigate the effects of Al, La, Cu, Gd, Sc and Ru on the saturated hydraulic conductivity of bacterial cellulose (BC)–pectin composites, used as plant cell wall analogs. Hydraulic conductivity was reduced to ≈30% of the initial flow rate by 39 µM Al and 0.6 µM Cu, ≈40% by 4.6 µM La, 3 µM Sc and 4.4 µM Ru and ≈55% by 3.4 µM Gd. Scanning electron microscopy (SEM) revealed changes in the ultrastructure of the composites. The results suggest that trace metal binding decreases the hydraulic conductivity through changes in pectin porosity. The experiment illustrates the importance of metal interactions with pectin, and the implications of such an interaction in plant metal toxicity and in normal cell wall processes.     

Kinetics and mechanisms of cowpea root adaptation to changes in solution calcium

Background and aims

Close regulation of cellular Ca in roots is required in the face of marked changes in soil solution Ca over time and space. This study’s aims were to quantify and gain insights into the ways in which roots respond to changes in solution Ca.

Methods

Root elongation rate (RER) of cowpea (Vigna unguiculata (L.) Walp.) seedlings was determined at 0.05 to 15 mM Ca for up to 24 h both without and with added K, Mg, or Na. Root tip concentrations of Ca, K, Mg, and Na were determined and binding of cations by root tips estimated by subsequent Cu sorption.

Results

Transfer from higher to lower Ca solutions (and with added K at high Ca) resulted in RER?≥?2 mm h^?1 within minutes. This was attributed to greater cell wall relaxation through lower Ca binding aided by a decrease to pH?≤?5.1 in solution. Transfer to higher Ca solutions, which remained at ~pH 5.6, led to an equally rapid decrease in RER to ~0.5 mm h^?1, an effect ascribed to greater cell wall binding of Ca. Thereafter, a gradual increase in RER to ~1.8 mm h^?1 occurred over 24 h, an effect likely due to reduced cell wall Ca binding as shown by decreasing Cu sorption at a rate of 0.027 mmol Cu kg^?1 FM h^?1 over 24 h.

Conclusion

The kinetics of changes in RER and cations in root tips suggest that roots respond to changes in solution Ca through effects on cell wall relaxation of the rhizodermis and outer cortex in the elongation zone.     

Rhizotoxicity of aluminate and polycationic aluminium at high pH

Although monomeric Al species are often toxic in acidic soils, the effects of the aluminate ion (Al(OH) ₄ ⁻ ) on roots grown in alkaline media are still unclear. Dilute, alkaline (pH 9.5) nutrient solutions were used to investigate the effects of Al(OH) ₄ ⁻ on root growth of mungbean (Vigna radiata L.). Root growth was reduced by 13% after 3 d growth in solutions with an Al(OH) ₄ ⁻ activity of 16 μM and no detectable polycationic Al (Al₁₃). This decrease in root growth was associated with the formation of lesions on the root tips (due to the rupturing of the epidermal and outer cortical cells) and a slight limitation to root hair growth (particularly on the lateral roots). When roots displaying these symptoms were transferred to fresh Al(OH) ₄ ⁻ solutions for a further 12 h, no root tip lesions were observed and root hair growth on the lateral roots improved. The symptoms were similar to those induced by Al₁₃ at concentrations as low as 0.50 μM Al which are below the detection limit of the ferron method. Thus, Al(OH) ₄ ⁻ is considered to be non-toxic, with the observed reduction in root growth in solutions containing Al(OH) ₄ ⁻ due to the gradual formation of toxic Al₁₃ in the bulk nutrient solution resulting from the acidification of the alkaline nutrient solution by the plant roots.     

Interaction of boron and aluminum on the physiological characteristics of rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) seedlings

It has been reported that aluminum (Al) toxicity is a major limiting factor for plant growth and production on acidic soils. Boron (B) is indispensable micronutrient for normal growth of higher plants, and its addition could alleviate Al toxicity. The rape seedlings were grown under three B (0.25, 25 and 500 μM) and two Al concentrations [0 (?Al) and 100 μM (+Al) as AlCl₃·6H₂O]. The results indicated that Al stress severely hampered root elongation and root activity at 0.25 μM B while the normal (25 μM) and excess (500 μM) B improved the biomass of rape seedlings under Al exposure. Additionally, normal and excess B treatment reduced accumulation of Al in the roots and leaves under Al toxicity, which was also confirmed by hematoxylin with light staining. This indicates that both normal and excess B could alleviate Al toxicity. Furthermore, it also decreased the contents of malondialdehyde and soluble protein under Al toxicity. Likewise, superoxide dismutase activity (SOD) improved by 97.82 and 131.96% in the roots, and 168 and 119.88% in the leaves at 25 and 500 µM B, respectively, while the peroxidase and catalase activities dropped as a result of Al stress. The study results demonstrated that appropriate B application is necessary to avoid the harmful consequences of Al toxicity in rape seedlings.     

A comparative study on the aluminium- and copper-induced organic acid exudation from wheat roots

It is well established that aluminium (Al) and some heavy metals can elicit organic acid exudation from a range of species. In the present research we found that copper (Cu) can also induce organic acid exudation from the roots of wheat, rye, triticale, maize and soybean. Using intact wheat plants, we made a comparative study of Al- and Cu- induced organic acid exudation. In 5-day-old wheat seedlings, severe Cu stress (40 µ M CuCl₂) mainly induced the exudation of malate and citrate, and Al-tolerant genotypes could release significantly greater amounts of malate than Al-sensitive genotypes. The time course of the exudation of malate and citrate from the roots of 5-day-old seedlings of wheat (cv. Atlas) in 200 µ M AlCl₃ was similar to that in 40 µ M CuCl₂. In older wheat plants (15-day-old), moderate Cu stress (12 µ M CuCl₂) induced the exudation of large amounts of citrate and addition of Al or La sharply reduced Cu-induced citrate exudation, while Cu or La did not affect Al-induced malate efflux. When half of the root system of Atlas wheat was immersed in Al- or Cu-containing solution and the remaining half in Al- or Cu-free solution, organic acids were only exuded into the solution containing Al or Cu. This suggests that no long distance signal transport is involved in organic acid exudation induced by Al or Cu, and that direct contact of Al or Cu with plant roots is a prerequisite for the induction of organic acid exudation. The anion-channel inhibitor niflumic acid (NIF) significantly stimulated the exudation of both citrate and malate from 5-day-old wheat seedlings under severe Al or Cu stress. Our results suggest that Cu-induced organic acid efflux may be a common response, which may play a role in alleviating Cu toxicity in plants.     

Root endophytic Chaetomium cupreum promotes plant growth and detoxifies aluminum in Miscanthus sinensis Andersson growing at the acidic mine site

Miscanthus sinensis Andersson grows naturally at the Hitachi mine. The root‐zone soil was acidic and contained high concentrations of Cu, Pb, Zn and exchangeable Al. Adventitious roots accumulated high concentrations of Al and Fe, but not other heavy metals. The purpose of this study was to elucidate the mechanism of tolerance of Al in M. sinensis, focusing on its chemical interaction with root endophytes. We isolated Chaetomium cupreum, which produced siderophores, from adventitious roots of M. sinensis via CAS assay. In inoculation tests, C. cupreum promoted M. sinensis seedling growth and increased Al and Fe uptake in the roots, although C. cupreum did not stimulate M. sinensis to produce Al detoxicants, such as citric and malic acids. Observation of the pattern of Al localization in the roots clarified that C. cupreum reduced Al toxicity in M. sinensis via compartmentalizing Al into fungal mycelia surrounding the roots and creating a less toxic Al‐localization pattern, allocating Al to the epidermis, endodermis and stele of roots. In conclusion, our results indicated that C. cupreum increases Al tolerance in M. sinensis growing at the acidic mine site.     

Spatial coordination of aluminium uptake, production of reactive oxygen species, callose production and wall rigidification in maize roots

Aluminium (Al) toxicity associated with acid soils represents one of the biggest limitations to crop production worldwide. Although Al specifically inhibits the elongation of root cells, the exact mechanism by which this growth reduction occurs remains controversial. The aim of this study was to investigate the spatial and temporal dynamics of Al migration into roots of maize (Zea mays L.) and the production of the stress response compound callose. Using the Al-specific fluorescent probe morin, we demonstrate the gradual penetration of AI into roots. Al readily accumulates in the root's epidermal and outer cortical cell layers but does not readily penetrate into the inner cortex. After prolonged exposure times (12-24 h), Al had entered all areas of the root apex. The spatial and temporal accumulation of Al within the root is similarly matched by the production of the cell wall polymer callose, which is also highly localized to the epidermis and outer cortical region. Exposure to Al induced the rapid production of reactive oxygen species and induced a significant rigidification of the cell wall. Our results suggest that Al-induced root inhibition in maize occurs by rigidification of the epidermal layers.     

Differential response of wheat roots to Cu, Ni and Cd treatment: oxidative stress and defense reactions

Wheat seedlings cv. Zyta were treated with Cu, Ni and Cd at the concentrations causing approximately 50 % root growth inhibition, i.e. 12.5, 50 and 60 μM, respectively. Tissue metal accumulation, membrane permeability, lipid peroxidation, protein oxidation, concentration of thiol compounds as well as protease, glutathione S-transferase (GST) and peroxidase (POD) activities were studied in roots after 7 days of metal exposure. The metals showed different concentrations in root tissues with Cu and Cd being accumulated to the smallest and to the greatest extent, respectively. Membrane permeability was significantly enhanced by Cu and Ni but not by Cd treatment. All metals induced similar increase in protein oxidation, while significant enhancement of lipid peroxidation was observed only in the case of Cu treatment. The detected thiol compounds: cysteine (Cys), homocysteine (Hcy), γ-glutamylcysteine (γ-GluCys) and glutathione (GSH) were differently influenced by the metal treatment. Ni appeared to be the most effective inductor of GSH accumulation while both Cu and Ni similarly increased Cys content in the roots. Accumulation of γ-GluCys was found in response to Cu and Cd applications. Concentration of Hcy was enhanced by Cd treatment but exposure to Ni decreased its content below the level of detection. The activity of GST was considerably elevated by Cd and Ni treatments, while POD activity was increased only in response to Cu application. Our study showed that wheat roots differently responded to treatment with metals used at the concentrations having similar impact on growth.     

A de novo synthesis citrate transporter, Vigna umbellata multidrug and toxic compound extrusion, implicates in Al-activated citrate efflux in rice bean (Vigna umbellata) root apex

Al-activated organic acid anion efflux from roots is an important Al resistance mechanism in plants. We have conducted homologous cloning and isolated Vigna umbellata multidrug and toxic compound extrusion (VuMATE), a gene encoding a de novo citrate transporter from rice bean. Al treatment up-regulated VuMATE expression in the root apex, but neither in the mature root region nor in the leaf. The degree of up-regulation of VuMATE was both partially Al concentration and time dependent, consistent with the delay in the onset of the Al-induced citrate efflux in rice bean roots. While La(3+) moderately induced VuMATE expression, Cd(2+) and Cu(2+) did not induce the expression. Electrophysiological analysis of Xenopus oocytes expressing VuMATE indicated this transporter can mediate significant anion efflux across the plasma membrane. [(14) C]citrate efflux experiments in oocytes demonstrated that VuMATE is a H(+) -dependent citrate transporter. In addition, expression of VuMATE in transgenic tomato resulted in increased Al resistance, which correlated with an enhanced citrate efflux. Taken together, these findings suggest that VuMATE is a functional homolog of the known citrate transporters in sorghum, barley, maize and Arabidopsis. The similarities and differences of all the known citrate transporters associated with Al stress in the MATE family are also discussed.     

Functional characterization of plasma membrane-localized organic acid transporter (CsALMT1) involved in aluminum tolerance in <Emphasis Type="Italic">Camelina sativa</Emphasis> L.

Aluminum (Al) toxicity is a major limiting factor that inhibits root elongation and decreases crop production in acidic soils. The symptoms of inhibited root growth include a reduced uptake of nutrients because the roots become stubby and brittle. The release of organic anions from roots can protect a plant from Al toxicity. The mechanism relies on the efflux of organic anions, such as malate or citrate, which protect roots by chelating the Al³⁺. In this study, homologs of TaALMT1, a Camelina gene that encodes an aluminum-activated malate transporter, were investigated. The expression of this gene was induced by Al in the root, but not in the shoots. Using green fluorescent protein (GFP) fusion constructs and Western-blot analysis, we observed that CsALMT1 was localized in the plasma membrane. Also, to determine the degree to which Al tolerance was affected by malate secretion in Camelina root, we generated CsALMT1 overexpressing plants. CsALMT1 overexpressing transgenic plants showed a higher root elongation rate than the wild-type plant. Damaged cell staining analysis by hematoxylin under 25 µM Al treatment for 2, 4, and 6 h showed a pattern of less damage in CsALMT1 transgenic plants than in wild-type plant, especially in the root elongation zone. Furthermore, the rate of increase of secretion of organic acid in overexpressed plants after Al treatment was higher than that in the wild-type plant. In addition, in the Al-specific dye morin staining on root protoplast under 50 µM Al treatment, less Al accumulation was observed in the CsALMT1 transgenic plants than in the wild-type plant. The Al contents in the roots of the transgenic plants were at a lower level than those in the wild-type plant. These results show that the overexpression of CsALMT1 improves Al tolerance by increasing the release of malate from the root to the soil and, thereby, detoxifies the Al³⁺.     

Effects of culture medium and auxins on growth of adventitious root cultures of Cuphea aequipetala Cav. and their ability to produce antioxidant compounds

Cuphea aequipetala Cav. (Lythraceae), a species highly valued for its medicinal properties, is threatened in the wild. To provide an alternative source of material for production of bioactive compounds, we established adventitious root cultures of C. aequipetala and determined their phenolic compounds contents and antioxidant activity. Cultures were initiated from root tips of in vitro C. aequipetala plantlets and were grown in B5 or SH culture medium containing either indole butyric acid (IBA) or α-naphthalene acetic acid at 0, 5 or 10 µM. The maximum root biomass (1.6 g/L dry mass (DM) per L medium) was recorded after 14 days of growth in B5 + 5 µM IBA. Roots in B5 medium remained green, whereas they tended to oxidize in SH medium. The highest contents of total phenolic compounds (9.1 ± 0.1 µg gallic acid equivalents/g DM) and flavonoids (37.5 ± 0.7 µg quercetin equivalents/g DM) were in roots grown in B5 + 5 µM IBA after 14 days of growth. Root cultures accumulated mainly flavan-3-ols, whereas roots or leaves from whole plants accumulated mainly flavonols. We analyzed the antioxidant properties of root extracts using in vitro assays. Roots grown in B5 medium showed stronger free-radical scavenging activity than that of roots grown in SH medium. Our results show that adventitious root cultures of C. aequipetala are a promising system for research on antioxidant compounds biosynthesis and for scaled-up production of useful biological materials.     

Morphological changes in the apex of pea roots during and after recovery from aluminium treatment

We investigated how the pea (Pisum sativum cv. Harunoka) root, upon return to an Al-free condition, recovers from injury caused by exposure to Al. The growing region of the root during and after treatment with Al was examined by marking the root at intervals with India ink. Al-induced cell death was detected by staining with Evans blue. Root growth in 40 μM Al solution relative to that in Al-free solution (RRG) was approximately 45% from 6 h to12 h after the start of the treatment. However, values of RRG from 12 h to 24 h in Al-free solution for recovery or in the same Al solution were about 75% and 35%, respectively, indicating recovery from Al-induced growth inhibition. Images of the root characterized by zonal staining with Evans blue were observed in the sub-apical region (more than 1 mm from the tip) in Al-stressed roots. However, the interval of the stained zone was widened in the root after recovery from Al-induced growth inhibition, though it was narrower and more densely stained with time in the Al-stressed roots. During the recovery, the root apex may resume elongation in a specified region without Al-induced death or injury in cells detected by Evans blue.     

Selenium improves the antioxidant ability against aluminium-induced oxidative stress in ryegrass roots

We investigated the role of selenium (Se) against aluminium (Al) stress in ryegrass by evaluating the growth responses and the antioxidant properties of plants cultured hydroponically with Al (0 or 0.2 mM) and selenite (0–10 µM Se). Al addition significantly reduced the yield and length of shoots and roots, and most Al was accumulated in the roots. Al also enhanced lipid peroxidation and activated the peroxidase (POD), ascorbate peroxidase (APX) and superoxide dismutase (SOD) enzymes in the roots. Se application up to 2 µM improved root growth and steadily decreased thiobarbituric acid reactive substances (TBARS) accumulation in plants treated with 0 and 0.2 mM Al. However, above 2 µM, Se induced stress in plants grown with or without Al. Significant changes in antioxidant enzymes activities were also found as a result of the added Se. At low Se addition levels POD was activated, whereas APX activity decreased irrespective of added Al. Furthermore, Se supplied up to 2 µM greatly decreased root SOD activity in Al-stressed plants. Our study provides evidence that Se alleviated the Al-induced oxidative stress in ryegrass roots through the enhancement of the spontaneous dismutation of superoxide radicals and the subsequent activation of POD enzyme.     

Effects of copper on the growth,photosynthesis and nutrient concentrations of Phaseolus plants

Bean plants (Phaseolus vulgaris L. var. Zargana Kavala) were grown under conditions of increasing Cu concentrations in the growth medium (0.5-160.5 µM). Generally, the Cu concentrations between 0.5-1.5 µM were deficient, 1.5-10.5 µM were optimal, and 10.5-160.5 µM were toxic to plant growth. The Cu toxicity was associated with marked increases in plant tissue Cu concentrations. Under the Cu-deficient and optimal growth conditions, Cu was located primarily in the leaves. Under Cu toxicity, it was primarily sequestered in the roots. With increasing Cu in the growth medium, there was a positive correlation between Cu concentrations in the roots, stems and leaves, Ca in the roots, and K and Mg in the leaves. In contrast, Ca concentrations in the leaves and stems showed a negative correlation. The chlorophyll (Chl) concentration increased with increasing leaf Cu concentration, however, the Chl a/b ratio decreased. Since with an increasing leaf Cu concentration the leaf area decreased more markedly than the leaf dry mass, the net photosynthetic rate (PN) per leaf area increased and per dry mass decreased. The increase in PN per leaf area was almost entirely accounted for by the increase in Chl concentration. The initial Chl fluorescence (F0) increased with increasing leaf Cu concentration. The ratio of variable to maximum fluorescence (Fv/Fm) under Cu toxicity decreased. The half-time for the rise from F0 to Fm (t1/2) remained relatively unchanged with increasing leaf Cu concentration. Therefore the Cu-stress caused a small decrease in the efficiency of photosystem 2 photochemistry, but its primary effect was on growth.     

Efficient plant regeneration from shoot apex explants of maize (Zea mays) and analysis of genetic fidelity of regenerated plants by ISSR markers

An efficacious regeneration system was developed from shoot apex explants of Zea mays using a two-step culture procedure. Seventeen Indian genotypes were assessed for their regeneration potential. The maximum response of shoot induction was obtained from explants cultured on Murashige and Skoog medium supplemented with 4.5 µM thidiazuron and 26.7 µM glycine. Maximum mean number of shoots (17.2) was observed in genotype COH (m)-5 while NPK was the least responsive (6.7). Shoot clumps transferred from shoot induction medium to multiplication (second) medium amended with 1.1 µM thidiazuron and 0.88 µM N ₆ -benzylaminopurine showed increased number of shoots in COH (m)-5 (36.1 shoots); NPK was the least responsive with an average of 9.5 shoots. The best response in root induction, with a larger number of roots (10.5) and longer roots (6.6 cm), was observed in Murashige and Skoog medium supplemented with 7.3 µM indole-3-butyric acid and 7.9 µM phloroglucinol. Analysis of variance indicated that plant regeneration response varied greatly among the genotypes. In vitro raised plants were successfully transferred to the field after hardening, with a 99 % survival rate. Inter simple sequence repeats analysis revealed that the similarity matrix pair-wise value was 1, the Mantel test value was p 1.0; Analysis of molecular variance genetic variances were 93 % within the population and 7 % between populations; Principal component jolliffe cut off was 0.15, Principal component and Principle coordinate analysis % variance was 13.19. These values were congruent for both the mother and the in vitro-raised plants, confirming genetic integrity.