Effects of Cadmium on Antioxidant Enzyme Activities in Sugar Cane

Sugar cane (Saccharum officinarum L. cv. Copersucar SP80-3280) seedlings were grown in nutrient solution with varying concentrations (0, 2 and 5 mM) of cadmium chloride for 96 h. Leaves were analysed for catalase (CAT), glutathione reductase (GR) and superoxide dismutase (SOD) activities. Although a clear effect of CdCl₂ on plant growth was observed, the activity of SOD was not altered significantly. However, the CAT activity decreased as the concentration of CdCl₂ increased. GR exhibits a significant increase in activity at 2 and 5 mM CdCl₂. CAT and SOD isoenzymes were further characterised by analysis in non-denaturing PAGE. Activity staining for SOD revealed up to seven isoenzymes in untreated control and 2 mM CdCl₂ treated plants, corresponding to Cu/Zn-SOD isoenzymes. At 5 mM CdCl₂, only six Cu/Zn-SOD isoenzymes were observed. No Fe-SOD and Mn-SOD isoenzymes were detected. For CAT, one band of activity was observed.     

Acquired tolerance of tomato (Lycopersicon esculentum cv. Micro‐Tom) plants to cadmium‐induced stress

The effects of varying concentrations of cadmium (Cd) on the development of Lycopersicon esculentum cv. Micro‐Tom (MT) plants were investigated after 40 days (vegetative growth) and 95 days (fruit production), corresponding to 20 days and 75 days of exposure to CdCl₂, respectively. Inhibition of growth was clearly observed in the leaves after 20 days and was greater after 75 days of growth in 1 mM CdCl₂, whereas the fruits exhibited reduced growth following the exposure to a concentration as low as 0.1 mM CdCl₂. Cd was shown to accumulate in the roots after 75 days of growth but was mainly translocated to the upper parts of the plants accumulating to high concentrations in the fruits. Lipid peroxidation was more pronounced in the roots even at 0.05 mM CdCl₂ after 75 days, whereas in leaves, there was a major increase after 20 days of exposure to 1 mM CdCl₂, but the fruit only exhibited a slight significant increase in lipid peroxidation in plants subjected to 1 mM CdCl₂ when compared with the control. Oxidative stress was also investigated by the analysis of four key antioxidant enzymes, which exhibited changes in response to the increasing concentrations of Cd tested. Catalase (EC 1.11.1.6) activity was shown to increase after 75 days of Cd treatment, but the major increases were observed at 0.1 and 0.2 mM CdCl₂, whereas guaiacol peroxidase (EC 1.11.1.7) did not vary significantly from the control in leaves and roots apart from specific changes at 0.5 and 1 mM CdCl₂. The other two enzymes tested, glutathione reductase (EC 1.6.4.2) and superoxide dismutase (SOD, EC 1.15.1.1), did not exhibit any significant changes in activity, apart from a slight decrease in SOD activity at concentrations above 0.2 mM CdCl₂. However, the most striking results were obtained when an extra treatment was used in which a set of plants was subjected to a stepwise increase in CdCl₂ from 0.05 to 1 mM, leading to tolerance of the Cd applied even at the final highest concentration of 1 mM. This apparent adaptation to the toxic effect of Cd was confirmed by biomass values being similar to the control, indicating a tolerance to Cd acquired by the MT plants.     

Effect of salicylic acid on protein composition of Tatar buckwheat Fagopyrum tataricum calluses with different ability for morphogenesis

The effect of salicylic acid on the content of soluble proteins and individual polypeptides in Tatar buckwheat Fagopyrum tataricum calluses differing in ability for morphogenesis was studied. Changes in the protein composition of the calluses cultivated in the dark and in the light indicated the higher sensitivity of the non-morphogenic callus. Different response of callus cultures to salicylic acid and conditions of cultivation (light, darkness) is suggested to be associated with the antioxidant defense system, which is, in particular, characterized by the hydrogen peroxide content in the calluses. Salicylic acid increased the H₂O₂ content in non-morphogenic calluses more strongly than in morphogenic calluses, and the difference was more significant for the calluses cultivated in the light.Translated from Biokhimiya, Vol. 70, No. 3, 2005, pp. 390–396.Original Russian Text Copyright © 2005 by Maksyutova, Galeeva, Rumyantseva, Viktorova.     

Effects of NaCl on growth, ion accumulation, protein, proline contents and antioxidant enzymes activity in callus cultures of Jatropha curcas

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel crop. The effect of NaCl stress on growth, ion accumulation, contents of protein, proline, and antioxidant enzymes activity in callus cultures of J. curcas was investigated. Exposure of callus to NaCl decreased growth in a concentration dependent manner. NaCl treated callus accumulated Na and declined in K, Ca and Mg contents. Na/K ratio increased steadily as a function of external NaCl treatment. NaCl induced significant differences in quality and quantity of proteins, whereas, proline accumulation remained more or less constant with treatment. NaCl stress enhanced the activity of superoxide dismutase (SOD; E.C. 1.15.1.1) and peroxidase (POX; E.C. 1.11.1.7). Further in the isoenzyme studies, four SOD isoenzymes (SOD 1, 2, 3, and 4) and two POX isoenzymes (POX 1 and 2) were detected with the treatment. NaCl strongly induced activity of SOD 4 isoenzyme in 40, 60, 80 mM and POX 2 isoenzyme in 40 and 80 mM NaCl concentrations. Increase in antioxidant enzymes activity could be a response to cellular damage induced by NaCl. This increase could not stop the deleterious effects of NaCl, but it reduced stress severity and thus allowed cell growth to occur.     

Comparative study on calli from two reed ecotypes under heat stress

The different physiological responses to heat stress in calli from two ecotypes of common reed (Phragmites communis Trin.) plants (dune reed (DR) and swamp reed (SR)) were studied. The relative water content, the relative growth rate, cell viability, membrane permeability (MP), H₂O₂ content, MDA content, proline level, and the activities of enzymes, such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR), and lipoxygenase (LOX) were assayed. Results showed that under heat stress, DR callus could maintain the higher relative growth rate and cell viability than SR callus, while H₂O₂ content, MDA content, and MP in SR callus increased more than in DR callus. The activities of antioxidant enzymes, such as SOD, CAT, POD, APX, and GR in two calli were enhanced by high temperature. However, antioxidant enzymes in DR callus showed the higher thermal stability than those in SR callus. LOX activity increased more in SR callus than in DR callus under heat stress. High temperature markedly elevated proline content in DR callus whereas had no effect on that in SR callus. Taken together, DR callus is more thermotolerant than SR callus, which might be due to the higher activity of antioxidant enzymes and proline level compared with SR callus under heat stress.     

The effect of salt stress on lipid peroxidation and antioxidative enzymes in callus of two <Emphasis Type="Italic">Acanthophyllum</Emphasis> species

The changes in lipid peroxidation and the involvement of the antioxidant system in relation to salt stress tolerance were investigated in the callus of Acanthophyllum glandulosum and Acanthophyllum sordidum. The callus was subjected to NaCl stress (50–200 mM) for 40 d. The callus of A. glandulosum was less sensitive to NaCl stress than that of A. sordidum. Increasing concentrations of NaCl from 50 to 200 mM correlated to increased proline content in A. glandulosum. Total protein content was higher in extracts of A. glandulosum than in extracts of A. sordidum under both control and salinity treatments. Compared with A. sordidum, lipid peroxidation and H₂O₂ content were lower and the activities of superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase, and glutathione reductase were higher in A. glandulosum under salt stress. Activity staining of antioxidant enzymes separated by native polyacrylamide gel electrophoresis (PAGE) revealed that callus of A. sordidum had five Fe-SOD isoforms and one Mn-SOD isoform, all of which were reduced by salinity. In A. glandulosum, two Mn-SOD, three Fe-SOD, and one Cu/Zn-SOD isoforms were detected. Out of these six SOD isoforms, expression of the Mn-SOD and Fe-SOD isoforms was enhanced at 100 mM and higher NaCl concentrations. Two and six GPX isoforms were detected in A. sordidum and A. glandulosum, respectively. Expression of the single CAT isoform in A. sordidum was preferentially reduced by salinity. In A. glandulosum, the two CAT isoforms showed differential down regulation under NaCl stress, with the CAT2 isoform detected only under control condition. These results suggest that A. glandulosum callus is better protected against salinity-induced oxidative damage by maintaining higher activities of antioxidant enzymes than the callus of A. sordidum.     

Exogenous Selenium Pretreatment Protects Rapeseed Seedlings from Cadmium-Induced Oxidative Stress by Upregulating Antioxidant Defense and Methylglyoxal Detoxification Systems

The protective effect of selenium (Se) on antioxidant defense and methylglyoxal (MG) detoxification systems was investigated in leaves of rapeseed (Brassica napus cv. BINA sharisha 3) seedlings under cadmium (Cd)-induced oxidative stress. Two sets of 11-day-old seedlings were pretreated with both 50 and 100???M Se (Na₂SeO₄, sodium selenate) for 24?h. Two concentrations of CdCl₂ (0.5 and 1.0?mM) were imposed separately or on the Se-pretreated seedlings, which were grown for another 48?h. Cadmium stress at any levels resulted in the substantial increase in malondialdehyde and H₂O₂ levels. The ascorbate (AsA) content of the seedlings decreased significantly upon exposure to Cd stress. The amount of reduced glutathione (GSH) increased only at 0.5?mM CdCl₂, while glutathione disulfide (GSSG) increased at any level of Cd, with concomitant decrease in GSH/GSSG ratio. The activities of ascorbate peroxidase (APX) and glutathione S-transferase (GST) increased significantly with increased concentration of Cd (both at 0.5 and 1.0?mM CdCl₂), while the activities of glutathione reductase (GR) and glutathione peroxidase (GPX) increased only at moderate stress (0.5?mM CdCl₂) and then decreased at 1.0?mM severe stress (1.0?mM CdCl₂). Monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), catalase (CAT), glyoxalase I (Gly I), and glyoxalase II (Gly II) activities decreased upon exposure to any levels of Cd. Selenium pretreatment had little effect on the nonenzymatic and enzymatic components of seedlings grown under normal conditions; i.e., they slightly increased the GSH content and the activities of APX, GR, GST, and GPX. On the other hand, Se pretreatment of seedlings under Cd-induced stress showed a synergistic effect; it increased the AsA and GSH contents, the GSH/GSSG ratio, and the activities of APX, MDHAR, DHAR, GR, GPX, CAT, Gly I, and Gly II which ultimately reduced the MDA and H₂O₂ levels. However, in most cases, pretreatment with 50???M Se showed better results compared to pretreatment with 100???M Se. The results indicate that the exogenous application of Se at low concentrations increases the tolerance of plants to Cd-induced oxidative damage by enhancing their antioxidant defense and MG detoxification systems.     

NaCl-Dependent growth, ion content and regeneration of calluses initiated from the mangrove plant,Bruguiera sexangula

Calluses initiated from leaves and seedlings of the mangrove,Bruguiera sexangula, were isolated from the original tissues and subcultured. Effects of NaCl on growth and ion content of each callus were measured. The growth rate of calluses derived from leaves (leaf callus) gradually decreased as the NaCl concentration in the medium increased, while that of calluses derived from seedlings (seedling callus) was highest in the medium containing 100 mM NaCl. Concentrations of Na and Cl in both calluses increased with increasing the NaCl concentration in the culture medium. The concentration of K of leaf calluses greatly decreased at 300 mM NaCl, while the K concentration of seedling calluses decreased only slightly and remained relatively high even in the presence of 300 mM NaCl. Transient treatment of leaf calluses with media containing high concentrations of NaCl frequently induced regeneration of adventitious tissues.     

Effect of salicylic acid pretreatment on drought stress responses of zoysiagrass (Zoysia japonica)

The present study was carried out to examine the effects of exogenous salicylic acid (SA) on growth, activities of antioxidant enzymes, and some physiological and biochemical characteristics of zoysiagrass (Zoysia japonica Steud.) plants subjected to drought. Aqueous 0.1, 0.5, or 1.0 mM SA solution was sprayed on the leaves of zoysiagrass for 3 days. Drought was induced by withholding watering for 16 days after SA application. Biomass, chlorophyll content, net photosynthetic rate (P _n), activities of antioxidant enzymes (e.g., superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)), MDA and proline contents were determined. Pretreatments with 0.1 and 0.5 mM SA significantly increased fresh and dry weights and chlorophyll content, while 1 mM SA pretreatment did not show significant change compared to controls. SA pretreatments showed a marked increase in P _n compared with controls from the 7th to 16th day after drought start. Activities of SOD, POD, and CAT were increased by SA pretreatments. MDA and proline contents after 0.1 and 0.5 mM pretreatments were lower than those of controls from the 6th to 12th day of drought, while 1 mM SA pretreatment did not show significant change from the 0th to 9th day of drought. This work suggests that suitable exogenous SA (0.5 mM) helps zoysiagrass to perform better under drought stress by enhancing the net photosynthetic rate and antioxidant enzyme activities while decreasing lipid peroxidation as compared to the controls. SA could be used as a potential growth regulator for improving plant growth under drought stress.     

Inorganic nutrient manipulation for highly improved <Emphasis Type="Italic">in vitro</Emphasis> plant regeneration in finger millet—<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.

Summary Growth and morphogenesis of plant tissues under in vitro conditions are largely influenced by the composition of the culture media. In this study, effects of different inorganic nutrients (ZnSO₄ and CuSO₄) on callus induction and plant regeneration of Eleusine coracana in vitro were examined. Primary callus induction without ZnSO₄ resulted in improved shoot formation upon transfer of calluses to normal regeneration medium. CuSO₄ increased to 5x the normal concentration in the media for primary seed callus induction and plant regeneration resulted in a 4-fold increase in number of regnnerated shoots. For long-term callus cultures, 2x KNO₃ or 4x Fe-EDTA could replace the requirement for α-naphthaleneacetic acid in the regeneration medium, while 60 μM ZnSO₄ or 0.5 μM CuSO₄ was optimal for plant regeneration from callus cultures.     

High-frequency vibration improve callus growth via antioxidant enzymes induction in <Emphasis Type="Italic">Hyoscyamus kurdicus</Emphasis>

Effect of high-frequency vibration on growth rate, membrane stability and activities of some antioxidant enzymes were studied in callus tissues of Hyoscyamus kurdicus. Calli initiated from leaf (LE), shoot (SE) and root (RE) explants, and sinusoidal vibrations at 0, 50, 100 and 150 Hz for 30 min were applied on the H. kurdicus calli. Results showed that sinusoidal vibration at 50 and 100 Hz promoted the growth rate as compared to control, and the optimum growth was found at 50 Hz. Sinusoidal vibration increased significantly protein and proline contents and activity of superoxide dismutase (SOD), ascorbate peroxidase (APX) and peroxidase (POX) enzymes, and decreased total carbohydrate, H₂O₂ level and CAT activity as compared to control. Lipid peroxidation also decreased under sinusoidal vibration in all the calli, and the maximum percentage of decrease was observed at 50 Hz. Native polyacrylamide gel electrophoresis indicated different isoform profiles in vibration treated and untreated plants concerning antioxidant enzymes. The responses of different types of calluses were different, and RE callus showed the highest growth, membrane stability and antioxidant enzymes activity as compared to LE and SE calli. These results suggest sinusoidal vibration at optimum frequency could improve callus growth by induction of antioxidative enzymes activity and membrane stability in calli of H. kurdicus.     

Polyamines reduce salt-induced oxidative damage by increasing the activities of antioxidant enzymes and decreasing lipid peroxidation in Virginia pine

Polyamines play an important role in the plant response to adverse environmental conditions including salt and osmotic stresses. In this investigation, the responses of polyamines to salt-induced oxidative stress were studied in callus cultures and plantlets in Virginia pine (Pinus virginiana Mill.). Our results demonstrated that polyamines reduce salt-induced oxidative damage by increasing the activities of antioxidant enzymes and decreasing lipid peroxidation. Among different polyamines used in this study, putrescine (Put) is more effective in increasing the activities of ascorbate peroxidase (APOX), glutathione reductase (GR), and superoxide dismutase (SOD), reducing the activities of acid phosphatase and V-type H+-ATPase, and decreasing lipid peroxidation in Virginia pine, compared to both spermidine (Spd) and spermine (Spm). When 2.1 mM Put, Spd, and Spm were separately added to the medium, higher diamine oxidase (DAO) and polyamine oxidase (PAO) activities were observed in callus cultures and plantlets, compared to the concentrations of 0.7 and 1.4 mM. The activities of these two enzymes produce hydrogen peroxide (H₂O₂), which may act in structural defense as a signal molecule and decreasing the protection of polyamines against salt-induced oxidative damage in Virginia pine.     

Effect of nitric oxide and putrescine on antioxidative responses under NaCl stress in chickpea plants

Chickpea plants were subjected to salt stress for 48 h with 100 mM NaCl, after 50 days of growth. Other batches of plants were simultaneously treated with 0.2 mM sodium nitroprusside (NO donor) or 0.5 mM putrescine (polyamine) to examine their antioxidant effects. Sodium chloride stress adversely affected the relative water content (RWC), electrolyte leakage and lipid peroxidation in leaves. Sodium nitroprusside and putrescine could completely ameliorate the toxic effects of salt stress on electrolyte leakage and lipid peroxidation and partially on RWC. No significant decline in chlorophyll content under salt stress as well as with other treatments was observed. Sodium chloride stress activated the antioxidant defense system by increasing the activities of peroxidase (POX), catalase (CAT) superoxide dismutase (SOD) and ascorbate peroxidase (APX). However no significant effect was observed on glutathione reductase (GR) and dehydro ascorbate reductase (DHAR) activities. Both putrescine and NO had a positive effect on antioxidant enzymes under salt stress. Putrescine was more effective in scavenging superoxide radical as it increased the SOD activity under salt stress whereas nitric oxide was effective in hydrolyzing H₂O₂ by increasing the activities of CAT, POX and APX under salt stress.     

Endogenous hormone concentrations and embryogenic callus development in wheat

Immature zygotic embryos of two wheat (Triticum aestivum L.) genotypes, known for their different ability to generate embryogenic callus, were used as initial explants to establish callus cultures. Embryogenic and non-embryogenic calluses were obtained from the competent genotype (`Combi'), while only non-embryogenic callus was produced by the incompetent one (`Devon'). The morphogenetic competence of each callus type was evaluated by transferring some segments to regeneration conditions. The endogenous hormone concentrations (free indole-3-acetic acid [IAA], abscisic acid [ABA], gibberellins ₁, ₃ and ₂₀ [GAs], zeatin/zeatin riboside [Z/ZR] and N ⁶[²-isopentenyl] adenine/ N ⁶[²-isopentenyl] adenosine; [iP/iPA]) of the initial explants were determined by means of radio-immunoassay and showed that the only difference was the higher concentration of ABA found in the embryos of the most competent genotype; whose embryos showed a reduced rate of precocious germination. When analysing the endogenous hormone concentrations in the various callus types generated in each genotype, it was found that only differences in the free IAA concentrations were associated with variations in the morphogenic properties of the calluses. Higher concentrations of endogenous free IAA were typical of embryogenic callus cultures. It was also observed that a loss in the embryogenic competence of the calluses, due to a prolonged time of culture, occurred concomitantly with a reduction in free IAA concentrations, practically to the concentrations found in the non-embryogenic calluses.     

Pathways of ROS homeostasis regulation in Mesembryanthemum crystallinum L. calli exhibiting differences in rhizogenesis

A comparison of the hydrogen peroxide (H₂O₂) content, proline and betacyanin concentration and activities of some antioxidant enzymes (catalase, superoxide dismutase, guaiacol and ascorbate peroxidases) was made in Mesembryanthemum crystallinum L. calli differing in rhizogenic potential. Callus was induced from hypocotyls of 10-day-old seedlings on a medium containing 1?mg?l^?1 2,4-dichlorophenoxyacetic acid and 0.2?mg?l^?1 kinetin, which was either supplemented with 40?mM NaCl (CIM-NaCl medium) or did not contain any salt (CIM medium). The callus obtained on CIM-NaCl was rhizogenic, whereas the callus induced on the medium without salt was non-rhizogenic throughout the culture. The rhizogenic callus differed from the non-rhizogenic callus in lower betacyanin and H₂O₂ content, but the rhizogenic callus displayed a higher proline level. The activity of H₂O₂ scavenging enzymes, such as catalase (CAT), ascorbate peroxidase (APX) and guaiacol peroxidase (POD), was markedly higher in the rhizogenic callus than in the non-rhizogenic callus, but the total activity of superoxide dismutase (SOD) was higher in the non-rhizogenic callus than in the rhizogenic callus. Aminotriazole (CAT inhibitor) and diethyldithiocarbamate (SOD inhibitor) were added solely to the CIM and CIM-NaCl media to manipulate the concentration of reactive oxygen species (ROS) in the cultured tissues. Both CAT and SOD inhibitors brought about an increase in H₂O₂ content in calli cultured on CIM-NaCl and the loss of rhizogenic potential. Conversely, the addition of inhibitors to the medium without salt led to a decrease in H₂O₂ content. This corresponded with a significant decrease in the endogenous concentration of betacyanins, but did not change the lack of rhizogenic ability.     

Effects of salt and proline on Medicago sativa callus

In this study, two cultivars of Medicago sativa (cv. Yazdi and cv. Hamedani) were used for callus production. Calluses were transferred to MS medium containing 0, 30, 60, 90, and 120 mM NaCl and 0, 5, 10 mM proline. After 4–5 weeks dry weight and intracellular free proline of the calluses were measured. The growth of callus in both cultivars decreased with increasing salt concentration. Addition of exogenous proline to the culture medium increased the dry weight and free proline content of callus. The difference between control and treated calluses with 10 mM exogenous proline in the medium was significant. The data obtained from experiments indicated that the responses of two Medicago cultivars was genotype dependent.     

Efficient plant regeneration from cell cultures of ornamental statice, Limonium sinuatum mill.

Summary A plant regeneration system from cell suspension cultures was established in an important ornamental crop, Limonium sinuatum Mill. cv. ‘Early Rose’. Friable callus was initially induced from leaf segments of in vitro-cultured seedlings on 0.25% gellan gum-solidified half-strength Murashige and Skoog [1/2MS] medium containing 1.0 mg l⁻¹ (4.14 μM) picloram. These calluses were maintained as cell suspension cultures, which showed high proliferation ability with about 80 times increase in fresh weight during the 2-wk interval of subculture. Shoot regeneration from these cell cultures was achieved by cytokinins, especially zeatin, which was the most effective in producing normal shoots with reduced hyperhydration when used in combination with 0.5% gellan gum. Shoot regeneration ability was different among the cell lines originated from each different seedling. Shoot formation was observed at different frequencies on four of five cell lines whereas one cell line showed no shoot differentiation. Regenerated shoots detached from callus readily rooted 1 mo. after the transfer onto 0.5% gellan gum-solidified 1/2MS medium lacking plant growth regulators. The plantets were successfully transferred to the greenhouse after acclimatization. No ploidy changes were observed in the callus induced or in the regenerated plantlets. The regenerated plantlets that were transferred to the greenhouse after acclimatization grew normally and did not any morphological signs of somaclonal variation.     

Plant regeneration of the arsenic hyperaccumulator <Emphasis Type="Italic">Pteris vittata</Emphasis> L<Emphasis Type="Italic">.</Emphasis> from spores and identification of its tolerance and accumulation of arsenic and copper

An in vitro plant regeneration system was established from the spores of Pteris vittata and identification of its tolerance, and accumulation of gametophytes and callous, to arsenic (As) and copper (Cu) was investigated. The highest frequency (100%) of callus formation was achieved from gametophyte explants treated with 0.5 mg l^?1 6-benzylaminopurine (6-BA) + 0.5 mg l^?1 gibberellin acid (GA). Furthermore, sporophytes were differentiated from the callus tissue derived from gametophyte explants on MS medium supplemented with 0.5 mg l^?1 6-BA, 0.5–1.0 mg l^?1 GA and additional 300 mg l^?1 lactalbumin hydrolysate (LH) for 4 weeks. The optimum combination of ½ MS + 1.0 mg l^?1 GA + 0.5 mg l^?1 6-BA + 300 mg l^?1 LH promoted sporophyte formation on 75 ± 10% of the callus. Every callus derived from gametophyte explants could achieve 3–4 sporophytes. The in vitro growth of gametophyte and callus was accelerated in the medium containing Na₃AsO₄ lower than 0.5 mM, but this growth was inhibited with 2 mM Na₃AsO₄. And with the increase of Na₃AsO₄ in the culture medium from 0 to 2 mM, the As accumulation in gametophytes and callus increased and achieved a level of 763.3 and 315.4 mg kg^?1, respectively. Gametophytes and calluses transplanted to culture medium, supplemented with different concentrations of CuSO₄, are similar to those in Na₃AsO₄, and the Cu accumulation in gametophytes could achieve 7,940 mg kg^?1 when gametophytes were subcultured in medium containing 3 mM CuSO₄. These results suggested that the high efficiency propagation system could be a useful and rapid means to identify other heavy metal tolerance and accumulation. Further, the regeneration ability of callus made it possible for genetic transformation of this fern.     

Callus induction and thallus regeneration from callus of phycocolloid yielding seaweeds from the Indian coast

The tissue culture of phycocolloid yielding seaweeds included preparation of axenic explants, callus induction, subculture of excised callus and regeneration of plantlets from pigmented callus in the laboratory. Treatment of algal material with 0.1–0.5% detergent for 10 min and 1–2% betadine for 1–5 min and 3–5% antibiotic treatment for 48–72 h successively enabled viable axenic explants to be obtained as high as 60% for Gracilaria corticata, Sargassum tenerrimum and Turbinaria conoides and 10% for Hypnea musciformis. Callus induction was more conspicuous in T. conoides than in the other three species investigated. Of the irradiances investigated, 30 μmol photons m⁻² s⁻¹ produced calluses in as many as 40% explants in G. corticata and T. conoides and 10% in H. musciformis and S. tenerrimum. The explants cultured at 5 and 70 μmol photons m⁻² s⁻¹ did not produce any callus in all the species studied except for H. musciformis in which 10% explants developed callus at 5 μmol photons m⁻² s⁻¹. Most of the species investigated showed uniseriate filamentous Type of growths and buds from cut ends and from all over the surface of explants. Nevertheless, T. conoides had three Types of callus developments, namely (1) uniseriate filamentous Type of outgrowths from the centre of the cut end of explant, (2) bubbly Type of callus and (3) club-shaped callus clumps. The subculture of T. conoides callus embedded in 0.4% agar produced two Types of filamentous growth, namely filiform (with elongated cells) and moniliform filaments (with round cells) in the 2 months period after inoculation. Further, friable callus with loose cells was also found associated with excised callus. The moniliform filaments showed prolific growth of micro-colonies resembling to somatic embryo-like growth which, in liquid cultures, differentiated and developed into propagules with deformed shoots and distinct rhizoids. The shoots of these propagules remained stunted with abnormal leaf stalks without forming triangular shaped leaves as the parental plant and rhizoids had prolific growth in the laboratory cultures. The excised callus of G. corticata continued to grow when transferred to liquid cultures and showed differentiation of new shoots within 10 days. The shoots grew to a maximum length of 5–6 cm in the 2 months period in aerated cultures in the laboratory. Dedicated to the memory of Late Dr. Rangarajan.     

Biochemical responses of the ethylene-insensitive Never ripe tomato mutant subjected to cadmium and sodium stresses

In order to further address the known interaction between ethylene and components of the oxidative system, we have used the ethylene-insensitive Never ripe (Nr) tomato (Solanum lycopersicum L.) mutant, which blocks ethylene responses. The mutant was compared to the control Micro-Tom (MT) cultivar subjected to two stressful situations: 100 mM NaCl and 0.5 mM CdCl₂. Leaf chlorophyll, lipid peroxidation and antioxidant enzyme activities in roots, leaves and fruits, and Na and Cd accumulation in tissues were determined. Although we verified a similar growth pattern and Na and Cd accumulation for MT and Nr, the mutant exhibited reduced leaf chlorophyll degradation following stress. In roots and leaves, the patterns of catalase (CAT), glutathione reductase (GR), ascorbate peroxidase (APX), guaiacol peroxidase (GPOX), superoxide dismutase (SOD) enzyme activity as well as malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) production under the stressful conditions tested were very similar between MT and Nr mutant. However, Nr fruits showed increased H₂O₂ production, reduced and enhanced APX activity in NaCl and CdCl₂, respectively, and enhanced GPOX in NaCl. Moreover, through non-denaturing PAGE, a similar reduction of SOD I band intensity in both, control MT and Nr mutant, treated with NaCl was observed. In leaves and fruits, a similar SOD activity pattern was observed for all periods, genotypes and treatments. Overall the results indicate that the ethylene signaling associated with NR receptor can modulate the biochemical pathways of oxidative stress in a tissue dependent manner, and that this signaling may be different following Na and Cd exposure.