Glutathione in adaptation of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> to cadmium stress

The role of glutathione (GSH) in the adaptation of wild type Arabidopsis thaliana plants to Cd stress was investigated. The nutrient solution (control or containing 50 or 100 μM Cd) was supplemented with buthionine sulfoximine (BSO; 50, 100, 500 μM, to decrease the GSH content in plants) or GSH (50, 100, 500 μM, to increase its content in plants) in order to find how GSH content could regulate Cd stress responses. BSO application did not influence plant biomass, while exogenous GSH (especially 500 μM) reduced root biomass. BSO (500μM) in combination with Cd (100 μM) increased Cd toxicity on root growth (by over 50 %), most probably due to reduced GSH content and phytochelatin (PC) accumulation (by over 96 %). On the other hand, combination of exogenous GSH (500 μM) with Cd (100 μM) was also more toxic to plants than Cd alone despite a significant increase in GSH and PC accumulation (up to 2.7 fold in the roots). This fact could indicate that the natural content of endogenous GSH in wild type A. thaliana plants is sufficient for Cd-tolerance. A decrease in this GSH content led to decreased Cd-tolerance of the plants but an increase in GSH content did not enhance Cd-tolerance, and it showed even toxic effect on the plants.     

Glutathione reductase fromSaccharomyces cerevisiae undergoes redox interconversionin situ andin vivo

Redox interconversion of glutathione reductase was studiedin situ withS. cerevisiae. The enzyme was more sensitive to redox inactivation in 24 hour-starved cells than in freshly-grown ones. While 5 μM NADPH or 100 μM NADH caused 50% inactivation in normal cells in 30 min, 0.75 μM NADPH or 50 μM NADH promoted a similar effect in starved cells. GSSG reactivated the enzyme previously inactivated by NADPH, ascertaining that the enzyme was subjected to redox interconversion. Low EDTA concentrations fully protected the enzyme from NADPH inactivation, thus confirming the participation of metals in such a process. Extensive inactivation was obtained in permeabilized cells incubated with glucose-6-phosphate or 6-phosphogluconate, in agreement with the very high specific activities of the corresponding dehydrogenases. Some inactivation was also observed with malate, L-lactate, gluconate or isocitrate in the presence of low NADP⁺ concentrations. The inactivation of yeast glutathione reductase has also been studiedin vivo. The activity decreased to 75% after 2 hours of growth with glucono-δ-lactone as carbon source, while NADPH rose to 144% and NADP⁺ fell to 86% of their initial values. Greater changes were observed in the presence of 1.5 μM rotenone: enzymatic activity descended to 23% of the control value, while the NADH/NAD⁺ and NADPH/NADP⁺ ratios rose to 171% and 262% of their initial values, respectively. Such results indicate that the lowered redox potential of the pyridine nucleotide pool existing when glucono-δ-lactone is oxidized promotesin vivo inactivation of glutathione reductase.     

Modification of chromium (VI) phytotoxicity by exogenous gibberellic acid application in <Emphasis Type="Italic">Pisum sativum</Emphasis> (L.) seedlings

Effects of exogenous gibberellic acid (GA; 10 and 100 μM) application on growth, protein and nitrogen contents, ammonium (NH₄ ⁺) content, enzymes of nitrogen assimilation and antioxidant system in pea seedlings were investigated under chromium (VI) phytotoxicity (Cr VI; 50, 100 and 250 μM). Exposure of pea seedlings to Cr and 100 μM GA resulted in decreased seed germination, fresh and dry weight and length of root and shoot, and protein and nitrogen contents compared to control. Compared to control, Cr and 100 μM GA led to the significant alteration in nitrogen assimilation in pea. These treatments decreased root and shoot nitrate reductase (NR), glutamine synthetase (GS) and glutamine 2-oxoglutarate aminotransferase (GOGAT) activities (except 50 μM Cr alone for GOGAT) while glutamate dehydrogenase (GDH) activity and NH₄ ⁺ content increased. Compared to control, the root and shoot activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) increased (except APX activity at 250 μM Cr + 100 μM GA) while catalase (CAT), glutathione reductase (GR) and dehydroascorbate reductase (DHAR) activities were decreased (except GR at 100 μM GA alone) following exposure of Cr and 100 μM GA. Total ascorbate and total glutathione in root and shoot decreased by the treatments of Cr and 100 μM GA while their levels were increased by the application of 10 μM GA compared to Cr treatments alone. It has been reported that application of 10 μM GA together with Cr alleviated inhibited levels of growth, nitrogen assimilation and antioxidant system compared to Cr treatments alone. This study showed that application of 10 μM GA counteracts some of the adverse effects of Cr phytotoxicity with the increased levels of antioxidants and sustained activities of enzymes of nitrogen assimilation; however, 100 μM GA showed apparently reverse effect under Cr phytotoxicity.     

Zinc alleviates mercury-induced oxidative stress in <Emphasis Type="Italic">Pfaffia glomerata</Emphasis> (Spreng.) Pedersen

The possible role of zinc (Zn) to reverse the oxidative stress caused by mercury (Hg) was investigated in Pfaffia glomerata plantlets. Thirty-day-old acclimatized plantlets of P. glomerata were exposed to four treatments: control, 50 μM Zn, 50 μM Hg and 50 μM Zn + 50 μM Hg for 9 days. In Zn + Hg treatment, shoot and root Hg concentrations were 59 and 24% smaller than that plants exposed to 50 μM Hg added alone. An increase in the Zn concentration in the shoot of plants exposed to Zn + Hg occurred, although in the roots Zn concentration was not altered, when compared to the control. Fresh and dry weights, as well as the activity of δ-aminolevulinic acid dehydratase (δ-ALA-D) in Hg-treated plants were significantly reduced. Percentage survival, fresh and dry weights and δ-ALA-D activity of plants treated by 50 μM Zn + 50 μM Hg were greater than of that treated by Hg alone. Moreover, Zn treatment reduced the lipid peroxidation caused by Hg, being this effect related to increased root superoxide dismutase activity, and shoot catalase and ascorbate peroxidase activities. In conclusion, the presence of Zn in the substrate caused a significant reduction in the oxidative stress induced by Hg.     

Differential responses of pea seedlings to indole acetic acid under manganese toxicity

Present study showed the responses of pea seedlings to exogenous indole acetic acid (IAA; 10 and 100 μM) application under manganese (Mn; 50, 100 and 250 μM) toxicity. Manganese and 100 μM IAA alone as well as in combination decreased growth of pea seedlings compared to control. Moreover, some parameters of oxidative stress—hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) were also increased by single and combined treatments of Mn and 100 μM IAA compared to control. In contrast, addition of 10 μM IAA together with Mn, alleviated Mn toxicity symptoms and promoted growth led to the decrease in H₂O₂ and MDA levels compared to Mn treatments alone. Under single and combined treatments of Mn and 100 μM IAA, catalase activity decreased while superoxide dismutase and ascorbate peroxidase activities increased and glutathione reductase and dehydroascorbate reductase exhibited differential responses. However, addition of 10 μM IAA together with Mn, increased activities of studied enzymatic antioxidants. Root and shoot reduced ascorbate (AA) and reduced glutathione (GSH) and, their reduced/oxidized ratios decreased while dehydroascorbate (DHA) and oxidized glutathione (GSSG) contents increased compared to control following single and combined treatments of Mn and 100 μM IAA. However, supply of 10 μM IAA together with Mn, increased AA and GSH, and their reduced/oxidized ratios in root and shoot compared to Mn treatments alone. This study thus suggests that 10 μM of IAA was able to increase Mn tolerance in pea seedlings under Mn toxicity while opposite was noticed for 100 μM IAA.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of protocorm-like bodies in <Emphasis Type="Italic">Cattleya</Emphasis>

A protocol for in vitro induction of crape myrtle tetraploids using nodes from in vitro-grown shoots (2n = 48) was established. Nodal buds were excised from in vitro-grown shoots, maintained on proliferation medium containing Murashige and Skoog medium supplemented with 4.44 μM 6-benzyladenine , 0.54 μM α-naphthaleneacetic acid, and treated with a range of concentrations of colchicine under three different conditions. Nodal bud explants treated in liquid proliferation medium supplemented with either 15 or 20 mM colchicine for 24 h turned necrotic and died; whereas, those cultured on solid proliferation medium supplemented with either 125 or 250 μM colchicine for 30 days survived, but no tetraploid plants were obtained. However, when explants were cultured in liquid proliferation medium containing 250, 500 or 750 μM colchicine for 10 days, tetraploid plants (2n = 96) were obtained. Incubation of explants in medium containing 750 μM colchicine promoted the highest frequency of survival (40%) of explants and of recovered tetraploids (60%). Morphological and anatomical characteristics of leaves, including leaf index, stomata size and number, stomata index (length/width), and number of chloroplasts in guard cells correlated with ploidy of crape myrtle plants. The number of chloroplasts in guard cells of stomata was a stable and reliable marker in discriminating plants of different ploidy levels. Chromosome counts and flow cytometry confirmed these findings.     

Modulation of copper toxicity-induced oxidative damage by nitric oxide supply in the adventitious roots of <Emphasis Type="Italic">Panax ginseng</Emphasis>

Nitric oxide (NO) is a highly reactive, membrane-permeable free radical, which has recently emerged as an important signalling molecule and antioxidant. Here we investigated the protective effect of NO against the toxicity caused by excess CuSO₄ (50 μM) in the adventitious roots of mountain ginseng. It was found that NO donor, sodium nitroprusside (SNP), was effective in reducing Cu-induced toxicity in the mountain ginseng adventitious roots. Protective effect of SNP, as indicated by extent of lipid peroxidation, was reversed by incorporation of 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (CPTIO), a NO scavenger, in the medium suggesting that the protective effect of SNP is attributable to NO released, which was revealed from in situ confocal laser scanning microscopic localization of NO in the adventitious roots of mountain ginseng. Results obtained in the present study suggest that reduction of excess Cu-induced toxicity by SNP is most likely mediated through the modulation in the activities of antioxidant enzymes involved in H₂O₂ detoxification (catalase, peroxidase, ascorbate peroxidase) and in the maintenance of cellular redox couples (glutathione reductase), and contents of molecular antioxidants (particularly non-protein thiol, ascorbate and its redox status). Exogenous NO supply also improved the activity of superoxide dismutase, an enzyme responsible for O₂ ^·− dismutation, and NADPH oxidase, an enzyme responsible for O₂ ^·− generation, in excess Cu supplied adventitious roots of mountain ginseng.     

Salicylic Acid-induced Nitric Oxide and ROS Generation Stimulate Ginsenoside Accumulation in Panax ginseng Roots

We evaluated the involvement of nitric oxide (NO) in salicylic acid (SA)-induced accumulation of ginsenoside in adventitious roots of Panax ginseng and its mediation by reactive oxygen species (ROS). Related effects of SA on components of the antioxidant system were also sought. Adventitious roots of P. ginseng were grown in suspension culture for 3 weeks in MS medium and treated over 5 days with SA (100 μM) alone, SA in combination with the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), or PTIO alone. Nitric oxide, the superoxide anion (O₂^·−), H₂O₂, nitrite, nonprotein thiol, and ascorbate were monitored together with ginsenoside, NADPH oxidase activity, and several antioxidant enzymes. Salicylic acid did not inhibit root growth but induced accumulation of ginsenoside, lipid peroxidation, and generation of NO and O₂^·−. It also enhanced activities of NADPH oxidase, superoxide dismutase, catalase, and peroxidase, including ascorbate peroxidase. These effects were suppressed by PTIO. Salicylic acid also decreased glutathione reductase activity. Inclusion of PTIO with SA decreased the activity of glutathione reductase further. Treatment with SA plus PTIO also decreased nonprotein thiol and ascorbate contents but caused nitrite to overaccumulate. Salicylic acid applied to adventitious roots in culture induced accumulation of ginsenoside in an NO-dependent manner that was mediated by the associated increases in O₂^·−, which gave other antioxidant responses that were dependent on NO.     

Regulated alterations in redox and energetic status are the key mediators of salinity tolerance in the halophyte <Emphasis Type="Italic">Sesuvium portulacastrum</Emphasis> (L.) L

The present work addresses the importance of antioxidant, redox and energetic parameters in regulating salt-tolerance in Sesuvium portulacastrum. Experiments were conducted on 45 days old plants subjected to 250 and 1,000 mM NaCl stress for 2–8 days. Plants showed no significant change in growth parameters (shoot length, dry weight, and water content) at 250 mM NaCl as compared to control. However, growth of plants was significantly affected at 1,000 mM NaCl. The differential growth behaviour could be attributed to a greater decline in the energetic parameters (in terms of ratios of NADP/NADPH and ATP/ADP) at 1,000 mM NaCl than at 250 mM NaCl. The osmotic stress imposed to plants at 250 mM NaCl was presumably balanced by the accumulation of sodium ions (Na⁺), an energetically favorable process, and did not require an increased synthesis of proline. In contrast, to counter osmotic stress at 1,000 mM NaCl, plants accumulated Na⁺ as well as proline and were, therefore, energetically stressed. Further, the response of enzymatic and molecular antioxidants at 1,000 mM was either close to or even lower than that at 250 mM, which resulted in oxidative damage at 1,000 mM, particularly on longer durations. In conclusion, it is suggested that altered redox and energetic status of the plants could play a key role in mediating the tolerance of Sesuvium under salinity stress.     

Arsenic-induced root growth inhibition in mung bean (<Emphasis Type="Italic">Phaseolus aureus</Emphasis> Roxb.) is due to oxidative stress resulting from enhanced lipid peroxidation

Arsenic (As) toxicity and its biochemical effects have been mostly evaluated in ferns and a few higher plants. In this study, we investigated the effect of As (10.0 and 50.0 μM) on seedling growth, root anatomy, lipid peroxidation (malondialdehyde and conjugated dienes), electrolyte leakage, H₂O₂ content, root oxidizability and the activities of antioxidant enzymes in mung bean (Phaseolus aureus Roxb.). Arsenic significantly enhanced lipid peroxidation (by 52% at 50.0 μM As), electrolyte leakage and oxidizability in roots. However, there was no significant change in H₂O₂ content. Arsenic toxicity was associated with an increase in the activities of superoxide dismutase (SOD), guaiacol peroxidase (GPX) and glutathione reductase (GR). In response to 50.0 μM As, the activities of SOD and GR increased by over 60% and 90%, respectively. At 10.0 μM As, the activity of ascorbate peroxidase (APX) increased by 83%, whereas at 50.0 μM it declined significantly. The catalase (CAT) activity, on the other hand, decreased in response to As exposure, and it corresponded to the observed decrease in H₂O₂ content. We conclude that As causes a reduction in root elongation by inducing an oxidative stress that is related to enhanced lipid peroxidation, but not to H₂O₂ accumulation.     

Changes in the Growth, Chemical Composition, and Antioxidant Activity in the Aquatic Plant Wolffia arrhiza (L.) Wimm. (Lemnaceae) Exposed to Jasmonic Acid

The present study was undertaken to test the influence of exogenously applied jasmonic acid (JA) at concentrations of 0.01–100 μM upon the growth and metabolism of the aquatic plant Wolffia arrhiza (Lemnaceae). JA acted in a concentration-dependent manner. JA at 0.1 μM stimulated plant growth and accumulation of cellular components (proteins, monosaccharides, chlorophylls, phaeophytins, and carotenoids). Treatment with JA at 0.1 μM enhanced W. arrhiza viability by the induction of biomass production and increased the level of photosynthetic pigments, monosaccharides, and soluble proteins. Moreover, JA at 0.1 μM activated the enzymatic (catalase, ascorbate peroxidase, NADH peroxidase) and nonenzymatic antioxidant (ascorbate, glutathione) system in W. arrhiza and, therefore, suppressed lipid peroxidation. In contrast, decreases in fresh weight, major photosynthetic pigments, monosaccharides, and soluble protein content were observed in W. arrhiza exposed to 100 μM JA. JA applied at 100 μM also stimulated the formation of lipid peroxides which are responsible for membrane damage. In the presence of 100 μM JA, antioxidant enzyme (catalase, ascorbate peroxidase, NADH peroxidase) activity and ascorbate as well as glutathione content were inhibited. The data support the hypothesis that JA plays an important role in W. arrhiza growth and metabolism, regulating oxidative status by direct influence on the enzymatic as well as nonenzymatic antioxidant machinery.     

Enhanced recovery of doubled haploid lines from parthenogenetic plants of melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.)

Recovery of doubled haploid (DH) progeny from haploid melon plants for use in breeding programs requires efficient chromosome doubling procedures. We describe improved procedures for recovery of fruits and viable seeds from parthenogenetic melon plants. Plant regeneration from nodal explants treated with 500 mg/L colchicine for 12 h was increased from 40 to 88% by transferring the treated explants to medium supplemented with a combination of growth regulators [5 μM IAA; 5 μM BA; 1 μM ABA; 30 μM AgNO₃). Prolonged exposure (2–7 days) to colchicine inhibited regeneration from nodal explants but had less effect on shoot tip explants. Many colchicine-treated plantlets flowered in vitro, allowing early assessment of their male fertility. Production of stained pollen in plants from nodal explants was highest after 0.5–2 days of colchicine treatment and on plants from shoot tips after 1–2 days. In vitro pollen counts correlated well with counts from greenhouse grown plants and with fruit set. The fruit set rate for colchicine-treated plants with a high pollen number was 47%. Appropriate colchicine treatment and culture of nodal explants as well as tip explants can substantially increase the number of fertile plants and DH lines recovered from parthenogenetic melons.     

Shoot organogenesis in elite clones of <Emphasis Type="Italic">Eucalyptus tereticornis</Emphasis>

An efficient shoot organogenesis system has been developed from mature plants of selected elite clones of Eucalyptus tereticornis Sm. Cultures were established using nodal explants taken from freshly coppice shoots cultured on Murashige and Skoog medium containing 58 mM sucrose, 0.7% (w/v) agar (MS medium) and supplemented with 2.5 μM benzyladenine (BA) and 0.5 μM α-naphthaleneacetic acid (NAA). Shoot organogenesis was achieved from leaf segments taken from elongated microshoots on MS medium supplemented with 5.0 μM BA and 1.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The addition of cefotaxime to the medium promoted shoot differentiation, whereas carbenicillin and cephalexin inhibited shoot differentiation. Maximum shoot bud organogenesis (44.6%) occurred in explants cultured on MS medium supplemented with 5.0 μM BA, 1.0 μM 2,4-D and 500 mg/l cefotaxime. Leaf maturity influenced shoot regeneration, with maximum shoot organogeneisis (40.5%) occurring when the source of explants was the fifth leaf (14–16 days old) from the top of microshoot. Shoot organogenic potential also varied amongst the different clones of E. tereticornis. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analyses indicated clonal uniformity of the newly formed shoots/plants, and these were also found to be true-to-type.     

Reduction of ascorbate free radical by the plasma membrane of synaptic terminals from rat brain

Synaptic plasma membranes (SPMV) decrease the steady state ascorbate free radical (AFR) concentration of 1 mM ascorbate in phosphate/EDTA buffer (pH 7), due to AFR recycling by redox coupling between ascorbate and the ubiquinone content of these membranes. In the presence of NADH, but not NADPH, SPMV catalyse a rapid recycling of AFR which further lower the AFR concentration below 0.05 μM. These results correlate with the nearly 10-fold higher NADH oxidase over NADPH oxidase activity of SPMV. SPMV has NADH-dependent coenzyme Q reductase activity. In the presence of ascorbate the stimulation of the NADH oxidase activity of SPMV by coenzyme Q₁ and cytochrome c can be accounted for by the increase of the AFR concentration generated by the redox pairs ascorbate/coenzyme Q₁ and ascorbate/cytochrome c. The NADH:AFR reductase activity makes a major contribution to the NADH oxidase activity of SPMV and decreases the steady-state AFR concentration well below the micromolar concentration range.     

Effect of salicylic acid and methyl jasmonate on antioxidant systems of <Emphasis Type="Italic">Haematococcus pluvialis</Emphasis>

The influence of phytohormones, salicylic acid (SA) and methyl jasmonate (MJ) on the antioxidant systems in Haematococcus pluvialis was investigated. Both SA and MJ at 500 μM concentration reduced the growth of alga with salicylic acid, having more pronounced effect. Carotenoid and chlorophyll contents were decreased by SA and increased by MJ. Salicylic acid (100 μM) increased astaxanthin content to 6.8-fold under low light (30 μmol m⁻² s⁻¹), while MJ (10 μM) showed marginal increase in astaxanthin. Salicylic acid (500 μM) increased superoxide dismutase activity to 4.5- and 3.3-fold and ascorbate peroxidase (APX) activity to 15.5- and 7.1-fold under low and high light, respectively. Methyl jasmonate increased catalase activity (1.4-fold) under high light and APX activity (5.4-fold) under low light. Different mechanism of oxidative stress induced antioxidant production may be the plausible reason for this varied response for salicylic acid and methyl jasmonate. Higher concentrations of SA and MJ inhibited astaxanthin accumulation by different mechanisms either by scavenging the free radicals or by increasing primary carotenoids production. At lower concentrations, these phytohormones could be used for elicitation of secondary carotenoid production.     

Azadirachta indica and Ocimum sanctum leaf extracts alleviate arsenic toxicity by reducing arsenic uptake and improving antioxidant system in rice seedlings

In the present study the potentials of aqueous extracts of the two plants, neem (Azadirachta indica) and Tulsi (Ocimum sanctum) were examined in alleviating arsenic toxicity in rice (Oryza sativa L.) plants grown in hydroponics. Seedlings of rice grown for 8 days in nutrient solution containing 50 μM sodium arsenite showed decline in growth, reduced biomass, altered membrane permeability and increased production of superoxide anion (O2·−), H2O2 and hydroxyl radicals (·OH). Increased lipid peroxidation marked by elevated TBARS (thiobarbituric acid reactive substances) level, increased protein carbonylation, alterated levels of ascorbate, glutathione and increased activities of enzymes SOD (superoxide dismutase), CAT (catalase), APX (ascorbate peroxidase) and GPX (glutathione peroxidase) were noted in the seedlings on As treatment. Exogenously added leaf aqueous extracts of Azadirachta indica (0.75 mg mL−1, w/v) and Ocimum sanctum (0.87 mg mL−1, w/v) in the growth medium considerably alleviated As toxicity effects in the seedlings, marked by reduced As uptake, restoration of membrane integrity, reduced production of ROS, lowering oxidative damage and restoring the levels of ascorbate, glutathione and activity levels of antioxidative enzymes. Arsenic uptake in the seedlings declined by 72.5% in roots and 72.8% in shoots, when A. indica extract was present in the As treatment medium whereas with O. sanctum extract, the uptake declined by 67.2% in roots and 70.01% in shoots. Results suggest that both A. indica and O. sanctum aqueous extracts have potentials to alleviate arsenic toxicity in rice plants and that A. indica can serve as better As toxicity alleviator compared to O. sanctum.     

Somatic embryogenesis and plant regeneration from root sections of <Emphasis Type="Italic">Allium schoenoprasum</Emphasis> L.

A protocol has been developed for somatic embryogenesis and subsequent plant regeneration in Allium schoenoprasum L. Calli were induced from root sections isolated from axenic seedlings and cultivated on media containing either Murashige and Skoog’s (MS) or Dunstan and Short’s mineral solution supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) in combination with 6-benzylaminopurine (BA), 6-furfurylaminopurine (Kin) or thidiazuron (TDZ) at 1, 5 or 10 μM. The highest frequencies of callus induction were achieved on media with 5 μM 2,4-D in combination with 5 μM TDZ or 10 μM BA (78.9% and 78.4%, respectively). Calli were then transferred to 1 μM 2,4-D, where compact yellow callus turned to segmented yellowish callus with transparent globular somatic embryos at the surface. Calli that were previously grown on media with 5 μM 2,4-D in combination with 10 μM BA or 10 μM TDZ showed the highest frequencies of embryogenic callus formation (45% and 42%) as well as mean number of somatic embryos per regenerating callus. The choice of mineral solution formulation did not significantly affect callus induction or embryogenic callus formation. The embryos could complete development into whole plants on plant growth regulator (PGR)-free medium, but inclusion of Kin (0.5, 2.5 and 5 μM) in this phase improved somatic embryo development and multiplication. Subsequently transferred to 1/2 MS PGR-free medium, all embryos rooted and the survival rate of the plants in a greenhouse was 96%.     

Thidiazuron-induced high-frequency direct shoot organogenesis of <Emphasis Type="Italic">Cannabis sativa</Emphasis> L.

Induction of high-frequency shoot regeneration using nodal segments containing axillary buds from a 1-yr-old mother plants of Cannabis sativa was achieved on Murashige and Skoog (MS) medium containing 0.05–5.0 μM thidiazuron. The quality and quantity of regenerants were better with thidiazuron (0.5 μM thidiazuron) than with benzyladenine or kinetin. Adding 7.0 μM of gibberellic acid into a medium containing 0.5 μM thidiazuron slightly increased shoot growth. Elongated shoots when transferred to half-strength MS medium supplemented with 500 mg l⁻¹ activated charcoal and 2.5 μM indole-3-butyric acid resulted in 95% rooting. The rooted plants were successfully acclimatized in soil. Following acclimatization, growth performance of 4-mo-old in vitro propagated plants was compared with ex vitro vegetatively grown plants of the same age. The photosynthesis and transpiration characteristics were studied under different light levels (0, 500, 1,000, 1,500, or 2,000 μmol m⁻² s⁻¹). An increase in photosynthesis was observed with increase in the light intensity up to 1,500 μmol m⁻² s⁻¹ and then decreased subsequently at higher light levels in both types of plants. However, the increase was more pronounced at lower light intensities below 500 μmol m⁻² s⁻¹. Stomatal conductance and transpiration increased with light intensity up to highest level (2000 μmol m⁻² s⁻¹) tested. Intercellular CO₂ concentration (C _i) and the ratio of intercellular CO₂ concentration to ambient CO₂ (C _i/C _a) decreased with the increase in light intensity in both in vitro as well as ex vitro raised plants. The results show that in vitro propagated and hardened plants were functionally comparable to ex vitro plants of same age in terms of gas and water vapor exchange characteristics, within the limits of this study.     

Chloroplast glutathione reductase: Purification and properties

Glutathione reductase was partially purified from isolated pea chloroplasts ( Pisum sativum L. cv. Progress #9). A 1600-fold purification was obtained and the purified enzyme had a specific activity of 26 μmol NADPH oxidized (mg protein)⁻¹ min⁻¹. The enzyme had a native molecular weight of approximately 156 kdalton and consisted of two each of two subunits of about 41 and 42 kdalton. The K_m for oxidized glutathione was 11 μ M and the K_m for NADPH was 1.7 μ M . Enzyme activity was affected by the ionic strength of the assay medium, and maximum activity was observed at an ionic strength of between 60 and 100 m M . The enzyme was inactivated by sulfhydryl modifying reagents and the presence of either oxidized glutathione or NADPH affected the extent of inactivation. Chloroplast glutathione reductase probably serves in the removal of photosynthetically derived H₂O₂ by reducing dehydroascorbate for ascorbate-linked reduction of H₂O₂. Intermediates of this reaction sequence, dehydroascorbate, ascorbate, reduced glutathione, and NADPH had no effect on enzymic activity.     

The use of aeroponics to investigate antioxidant activity in the roots of <Emphasis Type="Italic">Xerophyta viscosa</Emphasis>

In order to ultimately understand the whole plant mechanism of attaining desiccation tolerance, we undertook to investigate the root tissues of the resurrection plant Xerophyta viscosa, as previous work has only been conducted on the leaf tissues of resurrection plants. An aeroponic plant growth system was designed and optimised to observe the root’s response to desiccation without the restrictions of a soil medium, allowing easy access to roots. Successful culture of both X.viscosa and the control, Zea mays, was achieved and dehydration stress was implemented through reduction of nutrient solution spraying of the roots. After drying to the air dry state (achieved after 7 days for roots and 10 days for shoots), rehydration was achieved by resumption of root spraying. X.viscosa plants survived desiccation and recovered but Z. mays did not. The activity of the antioxidant enzymes superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase and quantities of ascorbate and glutathione were determined during root desiccation. There was an initial decline in activity in all enzymes upon drying to 80% RWC, but activity thereafter remained constant, at rates indicative of potential metabolic activity, to the air-dry state. This data suggests that these enzymes are not denatured by desiccation of the root tissue. Ascorbate and glutathione content remained constant at concentrations of 70 and 100 μM, respectively during drying. Thus root tissues appear to retain antioxidant potential during drying, for use in recovery upon rehydration, as has been reported for leaf tissues of this and other resurrection plants.