The effect of different cadmium concentrations (6-120 µM) on Hill reaction activity (HRA) of isolated chloroplasts, contents of chlorophylls (Chls) and carotenoids (Cars), and Cd uptake and accumulation in plant organs of Indian mustard (Brassica juncea L. cv. Vitasso) and mung bean [Vigna radiata (L.) Wilczek] were determined. The Cd stress inhibited photochemical activity of isolated chloroplasts of both species and in both tested developmental stages. On the basis of EC₅₀ values, the mung bean showed a higher sensitivity to Cd treatment than Indian mustard. The higher sensitivity of both species was determined in the earlier than in the older developmental stage. The leaves of Cd-treated plants possessed lower contents of Chls and Cars in both species and the negative effect increased with Cd concentration. A difference between species was also found in Cd uptake and accumulation. In both species, Cd was accumulated more in roots than in shoots, with higher accumulation in Indian mustard than in mung bean.

Esterases (EC 3.1.1.x) represent a diverse group of hydrolases catalyzing the cleavage and formation of carboxyl ester bonds. Their connection with development has made them a suitable marker of development in plants. In the present work, we focused on the fluorimetric determination of the plant esterases in plant cell cultures (tobacco BY-2 cells and early somatic embryos of Norway spruce, clone 2/32) with respect to application the method for the study of programmed cell death and the influence of cadmium(II) ions on the plant cells. The programmed cell death has been triggered by sodium nitroprusside and glucose oxidase. The determination of the esterase activity by the proposed technique in a cell extract determined very small difference in enzyme activity, which was a reliable marker of metabolic changes. In addition, the esterase activity of spruce somatic embryos decreased with the increase in medium Cd concentration.

Plant regeneration via direct somatic embryogenesis from cotyledons, hypocotyls and leaves in seabuckthorn (Hippophae rhamnoides L.) was achieved. The influences of basal media, carbon sources, plant growth regulators (PGRs) with different concentrations and combinations on embryogenesis capacity of explants were studied. The highest frequency of somatic embryos production and germination was obtained on Schenk and Hildebrandt medium (SH) supplemented with 1.0 mg dm^-3 kinetin and 0.2, 0.5 mg dm^-3 indole-3-acetic acid. Granulated sugar was the optimal carbon source. The embryo-derived plantlets with well-developed roots and shoots were transferred successfully to the greenhouse with a maximum survival rate of 55 %. Histological observation revealed that the somatic embryos were similar to those of zygotic embryos.

The photosynthetic performance of Cistus crispus, Centaurea africana and Thymus villosus, three shrubs of the Mediterranean-type maquis vegetation of Central Portugal, were studied between July and December 2003 by means of in vivo chlorophyll a fluorescence measurements. In C. africana total leaf decay occurred between August and October, in T. villosus no clear pattern of variation of the basal fluorescence (F_o) was observed, and in C. crispus an increase of this parameter was observed in late autumn. In all species, the limitation to photosynthesis by photoinhibition and the contribution of non-reversible processes to total photoinhibition were more pronounced in the summer than in the autumn. However, a pronounced decrease of the effective quantum yield of photosystem 2 (Φ_PS2) was found in late autumn, probably due to a decrease of the carboxylation efficiency of the Calvin-Benson cycle, which was also supported by a parallel decrease of the photochemical quenching coefficient (qP).

The C₃-CAM intermediate Clusia minor L. and the C₃ obligate Clusia multiflora H.B.K. plants were exposed for 7 d to a combination of drought stress and high irradiance of about 1200 µmol m^-2 s^-1 for 12 h per day. In both species under these conditions a strong decrease in stomatal conductance was observed at dawn and dusk. Changes in stomatal behaviour of C. minor were accompanied by only a low nocturnal accumulation of malate and citrate. Thus, in C. minor drought stress applied in combination with high irradiance limited CAM expression, and possibly this is the main reason why C. minor prefers semi-shaded sites in the field. The mitochondrial MnSOD, in both well watered and stressed plants of two species showed strong diurnal oscillations with maximum activity at dusk. These oscillations can be explained by the engagement of mitochondria in dissipation of an excess of reducing equivalents. In plants which are able to carry out CAM metabolism tricarboxylic acid cycle is expected to be down regulated in the dark period to prevent breakdown of the entire malate and citrate.

This study concerns the efficacy of partial agar substitution by galactomannans as support in plant regeneration media for Nicotiana tabacum. The production of multiple shoots from leaf-derived callus and their rooting were evaluated. The galactomannans applied were obtained from Cassia fastuosa (cassia) and Cyamopsis tetragonolobus (guar gum - a commercial galactomannan) seeds. The results obtained on media solidified with mixtures of agar/galactomannan (3 g dm^-3 each) gels were compared with those on media gelled with a standard concentration of agar (6 g dm^-3). The in vitro performance allowed to conclude that the use of galactomannans raised the number of shoots and improved their quality. Furthermore, the length of roots and the size of leaves were significantly higher in the media solidified with agar/guar galactomannan mixtures.

Nicotiana tabacum 1 (NT-1) cells were transformed with four different expression cassettes of hepatitis B surface antigen (HBsAg). The transformed nature of the cells was confirmed by polymerase chain reaction (PCR). The expression levels were assayed by enzyme linked immunosorbent assay (ELISA). The expressivities varied among the different cassettes and the maximum expression of 16.6 ng g^-1(f.m.) of cells was noted in pEFEHER transformed cells. Salicylic acid (100 µM) treatment resulted in 1.8 fold increase of expression in pEFEHBS transformed cells. The effect of different concentrations of kanamycin and geneticin was studied on the growth of transformed cells and HBsAg expression. The cell growth was optimum at lower concentrations of the antibiotics, and the maximum expression was noted at 200 mg dm^-3 of kanamycin.

Free polyamine contents and expressions of five genes encoding for polyamine biosynthetic enzymes were investigated at four different stages during peach (Prunus persica L. Batsch cv. Akatsuki) flower development. Fresh mass of peach flowers increased, accompanied by reduction in contents of total polyamines and putrescine/spermidine ratio due to decrease in putrescine content. Spermidine, the largest fraction, and spermine, the least part, underwent minor change. Expressions of the five key genes involved in polyamine biosynthesis during flower development did not parallel the changes in free polyamines.

This study was the first attempt to extract RNA from black willow (Salix nigra Marshall) that contains numerous secondary products and to examine the photosynthetic gene expression of black willow under a wide range of soil moisture regimes. Black willow cuttings were grown under control, continuous flooding, periodic flooding and periodic drought for 42 d. A modified lithium chloride precipitation method was used for RNA extraction. Results of real-time polymerase chain reaction showed reduced gene expression of oxygen evolving complex, large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenasse and ferredoxin on day 7 as well as the latter two on day 14 in response to flooding. Therefore, decreased expression of these three genes may have contributed to the observed reduced photosynthetic capacity in response to flooding.

The responses of water relations, stomatal conductance (g_s) and growth parameters of tomato (Lycopersicon esculentum Mill. cv. Royesta) plants to nitrogen fertilisation and drought were studied. The plants were subjected to a long-term, moderate and progressive water stress by adding 80 % of the water evapotranspirated by the plant the preceding day. Well-watered plants received 100 % of the water evapotranspirated. Two weeks before starting the drought period, the plants were fertilised with Hoagland's solution with 14, 60 and 110 mM NO₃ ^- (N14, N60 and N110, respectively). Plants of the N110 treatment had the highest leaf area. However, g_s was higher for N60 plants and lower for N110 plants. At the end of the drought period, N60 plants showed the lowest values of water potential (Ψ_w) and osmotic potential (Ψ_s), and the highest values of pressure potential (Ψ_p). N60 plants showed the highest Ψ_s at maximum Ψ_p and the highest bulk modulus of elasticity.

To understand the physiological mechanism that confers Cd sensitivity, root morphology and Cd uptake kinetics of the Cd-sensitive mutant and wild type rice were investigated. The root length, root surface area, and root number of mutant rice decreased more significantly with increasing Cd concentration in growth media compared with the wild type rice. The uptake kinetics for ¹⁰⁹Cd²⁺ in roots of both the mutant and wild type rice were characterized by a rapid linear phase during the first 6 h and a slower linear phase during the subsequent period. Concentration-dependent Cd²⁺ influx in both species could be characterized by the Michaelis-Menten equation, with similar apparent K_m values for mutant and wild type rice (2.54 and 2.37 µM, respectively). However, the V_max for Cd²⁺ influx in mutant root cells was nearly 2-fold higher than that for wild type rice, indicating that enhanced absorption into the root is one of the mechanisms involved in Cd sensitivity in mutant rice.

Tissue-specific distribution of basic β-1,3-glucanase (Glu2), basic class II chitinase (Ch2), basic class IV chitinase (Ch4), and acidic class III chitinase (SE2) were examined both in leaves and roots of sugar beet treated with salicylic acid (SA), benzothiadiazole (BTH) and glycine betaine. Protein localization was monitored by immunohistological analysis using specific antibodies. BTH, SA as well as glycine betaine induced both Glu2 and chitinase isozymes in leaves and roots of treated plants. The enzymes were accumulated in extracellular space and cell walls. They were mostly deposited in parenchyma cells of leaves and cortex parenchyma and endodermis of roots. In leaf tissues, BTH and SA induced proteins more effectively than glycine betaine but the effect of glycine betaine in roots was as efficient as BTH and SA. Glycine betaine induced the formation of extracellular globuli containing Ch4. Induced proteins were spatially distributed over the whole plant regardless the site of the inducer application.

Seasonal changes in foliage nitrogen (N) and carbon (C) concentrations and δ¹⁵N and δ¹³C ratios were monitored during a year in Erica arborea, Myrtus communis and Juniperus communis co-occurring at a natural CO₂ spring (elevated [CO₂], about 700 μmol mol^-1) and at a nearby control site (ambient [CO₂], 360 μmol mol^-1) in a Mediterranean environment. Leaf N concentration was lower in elevated [CO₂] than in ambient [CO₂] for M. communis, higher for J. communis, and dependent on the season for E. arborea. Leaf C concentration was negatively affected by atmospheric CO₂ enrichment, regardless of the species. C/N ratio varied concomitantly to N. Leaves in elevated [CO₂] showed lower δ¹³C, and therefore likely lower water use efficiencies than leaves at the control site, regardless of the species, suggesting substantial photosynthetic acclimation under long-term CO₂-enriched atmosphere. Leaves of E. arborea showed lower values of δ¹⁵N under elevated [CO₂], but this was not the case of M. communis and J. communis foliage. The use of the resources and leaf chemical composition are affected by elevated [CO₂], but such an effect varies during the year, and is species-dependent. The seasonal dependency and species specificity suggest that plants are able to exploit different available water and N resources within Mediterranean sites.

An efficient technique for isolation of resistant gene analogs (RGAs) in pepper from silver stained denaturing polyacrylamide gel was developed using a modified amplified fragment length polymorphism (AFLP) strategy. Pepper DNA was digested, ligated and pre-amplified as in a normal AFLP method. The selective amplification was made by using combinations with oligonucleotide primers based on conserved motifs in and around nucleotide binding site (NBS) of known NBS-leucine-rich repeats resistance proteins from known resistant genes. The amplified products were separated by using denaturing polyacrylamide gels and silver staining instead of radioactive labelling. We isolated specific polymorphic AFLP bands directly from the gels with one round of polymerase chain reaction amplification, in order to confirm, after sequencing, that these bands have homologies with products of resistance genes described so far. Two bands (R2: 250 bp and R6: 150 bp) are particularly highlighted because they could be considered as RGAs related to resistance to Phytophthora capsici in pepper, because their sequences have a very high homology with other resistant gene analogs that have already been described. Besides, they were only detected in the resistant parent and in the bulked resistant segregants but not in the susceptible parent or susceptible F₂ segregants. We can conclude that the technique used is clean, quick and efficient for the isolation of RGAs in pepper.

The responses of tobacco plants over-expressing trans-zeatin O-glucosyltransferase gene under constitutive or senescence-inducible promoter (35S:ZOG1 and SAG12:ZOG1) and of wild type (WT) plants to water stress and subsequent rehydration were compared. In plants sufficiently supplied with water, both transgenics have higher net photosynthetic rate (P_N) in upper and middle leaves and higher stomatal conductance (g_s) in middle leaves than WT. Water use efficiency (WUE = P_N/E) was higher in both transgenics than in WT. During prolonged water stress, both P_N and E declined to a similar extent in both transgenics and WT plants. However, 7 d after rehydration P_N in SAG:ZOG (upper and middle leaves) and 35S:ZOG (upper leaves) was higher than that in WT plants. Increased content of endogenous CKs in 35S:ZOG plants did not prevent their response to ABA application and the results obtained did not support concept of CK antagonism of ABA-induced stomatal closure. The chlorophyll (Chl) a+b content was mostly higher in both transgenics than in WT. During water stress and subsequent rehydration it remained unchanged in upper leaves, decreased slightly in middle leaves only of WT, while rapidly in lower leaves. Total degradation of Chl, carotenoids and xanthophyll cycle pigments (XCP) was found under severe water stress in lower leaves. Carotenoid and XCP contents in middle and upper leaves mostly increased during development of water stress and decreased after rehydration. While β-carotene content was mostly higher in WT, neoxanthin content was higher in transgenics especially in 35S:ZOG under severe stress and after rehydration. The higher content of XCP and degree of their deepoxidation were usually found in upper and middle leaves than in lower leaves with exception of SAG:ZOG plants during mild water stress.

The effect of interspecific competition and element additions (N and P) on four grassland species (Poa pratensis, Lolium perenne, Festuca valida, Taraxacum officinale) grown under field conditions was studied. Two grasses (L. perenne, F. valida) grown in monoculture (absence of competition) showed lower carbon isotope discrimination (Δ¹³C) and enriched δ¹⁵N values. Nitrogen addition (as urea) had inconsistent effects on species Δ¹³C while caused enrichment of δ¹⁵N of P. pratensis and F. valida but strong depletion of δ¹⁵N of T. officinale. Phosphorous had no significant effect on Δ¹³C but depleted δ ¹⁵N of all species.

Plants of two cytotypes (2n=2x=20, and 2n=3x=30) of pinto peanut (Arachis pintoi Krapov. & W.C. Gregory) were regenerated through somatic embryogenesis. Embryogenic calli were induced from shoot tips or immature leaves dissected from in vitro growing plants. In the case of the diploid peanut the best somatic embryogenesis was achieved when shoot tips were cultured on Murashige and Skoog (MS) medium supplemented with 10 mg dm^-3 Picloram (PIC) and 0.1 mg dm^-3 6-benzylaminopurine (BAP) or when explants from immature leaves were cultured on MS + 10 mg dm^-3 PIC + 0.01 mg dm^-3 BAP. In the case of triploid peanut the highest number of somatic embryos was obtained when shoot tips were cultured on MS + 10 mg dm^-3 PIC + 0.01 mg dm^-3 BAP or when immature leaves were cultured on MS + 20 mg dm^-3 PIC + 0.01 mg dm^-3 BAP. Somatic embryos were converted into plants by culture on MS + 0.01 mg dm^-3 naphthaleneacetic acid + 0.01 mg dm^-3 BAP. Plants were successfully transferred to pots in greenhouse.

Growth parameters and cadmium accumulation were investigated in alfalfa seedlings treated with 10 μM salicylic acid (SA) at the beginning of seed imbibition. Shoot and root growths were accelerated by SA treatment and suppressed by Cd both in presence and absence of SA. Cd accumulation was stimulated by SA in alfalfa seedlings in dependence of the treatment duration. K, Mg, Ca and Fe contents in roots are decreased in the presence of Cd alone, while SA induces a decrease of Mg, Ca and Fe. Shoot K, Mg and Ca concentrations are increased by Cd only in the absence of SA, while SA induces also an increase of these concentrations, but only in the absence of Cd. High negative correlation of Cd concentration with K and Ca concentrations in root indicates a competition for the same carrier not regulated by SA. Positive correlation between Cd and Mg concentrations in shoots, which is decreased by SA pre-treatment, together with the increase of positive correlation between Cd and Fe concentrations in shoots under the influence of SA, indicates a possible mechanism of SA action through maintenance of ionic homeostasis.

Exposure of pea plants to high irradiance (HI) for 60 min caused a reversible photoinhibition as shown by changes in variable to maximum fluorescence ratio (F_v/F_m). A significant decline in F_m was observed in leaves from both pea cultivars subjected to HI, the decrease being higher in JI281 than in JI399 plants. The values recovered during the post-stress period in both cultivars. In both cultivars, minimal fluorescence (F₀) increased under HI, but in cultivar JI399 F₀ recovered to initial value during the post-stress period. The expression of antioxidant enzyme genes was higher in JI399 than in JI281, both in control and stressed plants. In JI281, after 60 min of HI, an induction of the transcripts of CAT, chlMDHAR, cytAPX and cytCu,Zn-SOD was observed, whereas there was a slight increase in PHGPX, stAPX and chlCu,Zn-SOD mRNAs. After 24 h of the recovery period, the induction of some transcripts was not maintained (CAT, cytAPX and cytCu,Zn-SOD), whereas the induction of others was maintained (PHGPX and chlCu,ZnSOD) or even increased (cytGR, stAPX and chlMDHAR). In JI399, CAT and cytAPX were increased strongly after 60 min of HI, and slight increases were observed in cytGR, chlGR and chlMDHAR. In the post-stress period the expression of stAPX, cytGR and chlMDHAR was even slightly higher than after 60 min of HI, however, expression of CAT, cytAPX, cytCu,ZnSOD, chlCu,ZnSOD and chlGR decreased.

We analyzed low molecular mass phenolics, lignin content and both soluble and cell wall bound peroxidase activity in the needles of three Picea omorika (Pancic) Purkyne lines grown in the generative seed orchard. The highest values of the total phenol content as well as of catechine, caffeic acid, coniferyl alcohol, isoferulic acid and lignin concentration were detected in B5 line ("semidichotomy" line). The soluble guaiacol peroxidase activity was the highest in A3 line (line "borealis"). The highest activity of cell wall bound peroxidases was measured in B5 line, and it was in correlation with lignin content.

Ageing of plant organs is accompanied by an increased production of free radicals what results in membrane lipid peroxidation. Non-polar aldehydes originating from this process interact with the cellular material to form the fluorescent end-products, lipofuscin-like pigments (LFP). Their formation was studied both qualitatively and quantitatively in ageing of bean cotyledons. The concentration of lipofuscin-like pigments increased 9-fold in 14-d-old (senescent) cotyledons in relation to 8-d-old (young) cotyledons. HPLC fractionation patterns indicate changes in their composition during ageing. The LFP increase in old cotyledons was accompanied by elevated levels of non-polar aldehydes that increased during ageing to 167 %. The composition of aldehydes was studied by mass spectrometry. The most abundant fraction in both young and old cotyledon was represented by C12 aldehydes, which comprised both saturated and unsaturated species. We have observed differences in abundances of individual aldehydes between the young and the old cotyledons that might explain the differences in the composition of lipofuscin-like pigments. These results support the involvement of free radicals in plant ageing; however, it is suggested that plant aldehydic products of lipid peroxidation differ from those found in animals.

Two genotypes (cv. Smaragd and line DP1059) of Pisum sativum with different susceptibility to Fusarium oxysporum and F. solani and influence of pathogenesis on enzyme activities were studied. The increase of activity of studied enzymes was mostly observed in both roots and shoots during pathogenesis. Only activity of acid phosphatase decreased in the root and increased in shoots. The correlation between enzyme activity change and susceptibility of pea cultivars to F. oxysporum or F. solani was observed.

In this study, a relationship between lipid peroxidation, the antioxidant defense system and salt stress in salt-sensitive cultivated tomato (Lycopersicon esculentum) and its salt-tolerant wild relative (L. pennellii) was established. Superoxide dismutase (SOD) activities were significantly higher in the leaves of L. pennellii than those of L. esculentum after 12 and 84 d. POX activity showed a gradual increase in both cultivars under 70 mM NaCl. POX activity in L. pennellii significantly increased after 6 and 84 d whereas showed no remarkable change in leaves of L. esculentum under 140 mM NaCl. A higher salinity tolerance of L. pennellii was also correlated with a lower lipid peroxidation, which might be due to a higher content of antioxidant enzymes studied.

Direct somatic embryogenesis from ray floret explants of five chrysanthemum cultivars has been obtained within 12 - 15 d on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA). Scanning electron microscopic observation also confirmed the direct origin of somatic embryos from explants. Somatic embryos developed asynchronously on the adaxial surface of explants. Among the five cultivars tested, Birbal Sahani was best responding (40 % explants responded on 4 mg dm^-3 2,4-D and 2 mg dm^-3 BA supplemented medium). Precocious germination of somatic embryos was noticed on the same medium. The best sucrose concentration in the medium was found to be 60 g dm^-3 where 70 % explants responded while 55 % embryogenic response was obtained on medium supplemented with 400 mg dm^-3 inositol. Plants developed from somatic embryos were transferred to soil and produced true-to-type flowers.