The use of expressed sequence tag-simple sequence repeat (EST-SSR) markers might reflect the better relationship among species or cultivars than markers previously used. The first set of 30 EST-SSR was developed in hop (Humulus lupulus L.). They represent 25 gene loci with total of 1268 EST sequences. They were used for characterization of 11 hop samples and cross-amplification in Humulus japonicus Sieb. et Zucc. The number of alleles per locus ranged from two to nine. The observed and expected heterozygosities ranged from 0.182 to 0.956 and from 0.233 to 0.775, respectively. We used EST-SSR markers for cluster analysis of hop genotypes. Dendrogram well matched with genealogical and geographical data for hop genotypes.

We evaluated the combined effects of elevated CO₂ and water availability on photosynthesis in barley. Soil and plant water content decreased with water stress, but less under elevated CO₂ concentration (EC) compared with ambient CO₂ concentration (AC). During water stress, stomatal conductance, carboxylation rate, RuBP regeneration, and the rate of triose phosphate utilisation (TPU) were decreased but less when plants grew under EC. Drought treatments caused only a slight effect on maximum photochemical efficiency (variable to maximum fluorescence ratio, F_v/F_m), whereas the actual quantum yield (Φ_PS2), maximum electron transport rate (J_max) and photochemical quenching (qP) were decreased and the non photochemical quenching (NPQ) was enhanced. Under water deficit, the allocation of electrons to CO₂ assimilation was diminished by 49 % at AC and by 26 % at EC while the allocation to O₂ reduction was increased by 15 % at AC and by 12 % at EC.

A two-step protocol for improving the frequency of shoot regeneration from oilseed rape (Brassica napus L.) hypocotyl explants was established. The protocol consists of a pre-culture on callus induction medium (CIM) and a subsequent shoot regeneration on shoot induction medium (SIM). The SIM was Murashige and Skoog medium supplemented with different concentrations of 6-benzylaminopurine (BA; 2-5 mg dm^-3) and naphthaleneacetic acid (NAA; 0.05-0.15 mg dm^-3). Maximum frequency of shoot regeneration (13 %) was on the SIM medium containing 4 mg dm^-3 BA and 0.1 mg dm^-3 NAA, but it increased to 24.45 % when 20 μM silver thiosulphate (STS) was added. Strikingly, an extremely high frequency of shoot regeneration up to 96.67 % was reached by a two-step protocol when hypocotyl explants had been pre-cultured for 7 d on a CIM medium containing 1.5 mg dm^-3 2,4-dichlorophenoxyacetic acid. In addition, the shoot emergence was also 7 d earlier than that observed by use of the one-step protocol. The two-step protocol was also applied for regeneration of transgenic plants with cZR-3, a nematode resistance candidate gene. As a result, 43 plants were generated from 270 shoots and from these 6 plants proved to be transgenic.

Programmed cell death (PCD) was induced by the Yariv reagent in Nicotiana tabacum cv. Bright Yellow-2 cell suspension. The analyses of proteins extracts by 2-D electrophoresis clearly show massive protein degradation which was mainly due to cysteine protease activity. In contrast, some proteins remained unchanged up to 72 h after PCD induction. Peptide mass fingerprints of these proteins, obtained by MALDI-TOF, identified calreticulin, heat shock protein (HSP) 60, HSP70, malate dehydrogenase and mitochondrial ATP synthase β-subunit.

Protein extracts, made to leaves harvested from the stolons of the pasture legume white clover (Trifolium repens L.) at two developmental stages (newly initiated; onset of senescence) were purified further using reverse-phase and ionexchange chromatography. Fractions enriched with the ethylene biosynthetic enzyme 1-aminocyclopropane-1-carboxylate (ACC) oxidase were selected for each stage and the final, partially purified fraction was subjected to twodimensional gel electrophoresis (2DE). Antibodies raised against a recombinant ACC oxidase (ACO) from white clover (antiTR-ACO2) recognised a series of spots of differing pI suggesting that ACO undergoes post-translational modifications. Further, the pattern differed between the ACO proteins partially purified from newly initiated leaves with leaves at the onset of senescence suggesting that the environmental and developmental cues that operate in each tissue influences the type and/or degree of post-translational modifications of the ACO protein. MALDI-TOF mass spectrometry was used to identify protein spots associated with the ACO proteins. Protein with identities to an ACO isoform from Oryza sativa, and a phosphoribulokinase from Arabidopsis thaliana were identified in the 2DE separations from newly initiated leaves, while an isoflavone reductase from Medicago sativa was identified in the 2DE separation of the senescent leaf extract.

Differences among three clones of Gentiana punctata L. hairy root shoot regenerants were investigated in relation to their growth patterns, production of secondary metabolites and 2D protein profiles. Prominent differences in growth parameters were stable thus qualifying regenerant clones as true somaclones. Marked differences in protein spots were registered among the regenerant clones but not in comparison with the non-transformed control. Southern blot hybridization of regenerants showed the absence of rolA, B and C genes, initially present in the main hairy root lines. Orf13 and rolD were present and orf8 was missing in all three regenerant clones whereas orf3 was missing only in clone 2. Although lacking the three major rol genes, plants of regenerant clones retained characteristics of the hairy root phenotype.

This study was carried out to investigate the in vitro competition in tissue culture of three flax (Linum usitatissimum L.) cultivars using different distances among hypocotyl explants cultured. Hypocotyl fresh and dry masses, shoot regeneration percentage, shoot number per hypocotyl, regenerated shoot length and total chlorophyll content were examined during shoot regeneration, while plantlet height, number of roots and length of roots were recorded during rooting. With decreasing distance among explants we observed increased shoot regeneration and rooting till a certain point from where stress initiated and significant decreases in all parameters observed. Explants cultured at distance 1.0 cm were found to be at their optimum.

The effect of exogenously applied nitric oxide on the heat tolerance of Chrysanthemum morifolium was investigated by applying the NO donor sodium nitroprusside (SNP). We found that the SNP partially alleviated the heat stress by slowing down the reduction of photosynthetic pigment content and net photosynthetic rate. SNP treatment also lowered the increase in the non-photochemical quenching of fluorescence and malondialdehyde content and maintained higher activities of superoxide dismutase, peroxidase, catalase and ascorbate peroxidase.

Short-time direct and indirect effects of 25 μM Cd on the growth of dicotyledon (Phaseolus coccineus) and monocotyledon (Allium cepa) plants were investigated in the presence of inhibitors of ethylene synthesis, NADPH oxidase, and the octadecanoid pathway. Only 5 min-long action of Cd was enough for inhibition of growth in bean roots, but its recovery time was extended to several days. After 7 h treatment, Cd was significantly accumulated in bean roots, but maximum H₂O₂ accumulation was seen after 1 h. Cd-induced H₂O₂ accumulation decreased especially after addition of ethylene inhibitor silver thiosulphate (STS). Low Cd accumulation and high growth inhibition were observed also in bean leaves and in A. cepa roots. The inhibitors of the octadecanoid pathway greatly weakened the inhibitory effect of Cd in P. coccineus roots, while no significant effect was observed in A. cepa. NADPH oxidase and ethylene blockade reversed (in the case of bean plants and indirectly treated A. cepa plants) or significantly diminished Cd action. Cd-induced growth inhibition of P. coccineus leaves was also alleviated by most inhibitors of the jasmonate pathway and by STS. These results indicate that Cd may have indirect and direct effects on growth processes.

In this study we identified and characterized a major latex lectin - designated as EtLLH - with antimicrobial activity from the succulent African milk tree Euphorbia trigona. The lectin is highly concentrated in the latex of E. trigona and appears to be composed of at least two subunits with a molecular mass of 32 kDa. EtLLH shares significant similarities to known plant lectins - ricin from Ricinus communis and agglutinin from Viscum album coloratum - which specifically bind D-galactose and N-acetyl-D-glucosamine, the major building blocks of many fungal cell walls. Antimicrobial activity assays revealed an impact of EtLLH on three phytopathogenic filamentous ascomycetes. The germination of the conidiospores and the hyphal growth of Aspergillus niger and Fusarium graminearum were severely inhibited by EtLLH already at concentrations below 0.1 mg cm^-3, while the effect on germination of the melanized conidiospores of Botrytis cinerea was less significant.

Jatropha curcas L. (physic nut) is native to Central America and now naturalized widely in many tropical and subtropical areas. Microsatellite markers were isolated and characterized. Eleven out of 55 markers showed polymorphisms, and the allelic variation was investigated using 26 accessions of J. curcas collected from several provinces in Thailand. Each marker showed 2 to 5 alleles and the average polymorphic information content (PIC) was 0.49. Thirty four markers (62 %) were also successfully amplified in J. integerrima, J. gossypifolia and J. podagrica.

Mature pollen grain represents a highly desiccated structure with an extremely tough cell wall. Thanks to it, it resists common proteomic protocols. Instead, a robust homogenization has to be performed since proteins are needed to burst out of the cell to be included in the extracted proteome fraction. Here, a novel way of pollen homogenization employing Roche MagNA Lyser Instrument is presented, sparing time and laborious work. However, plant proteomics does not rely solely on perfect homogenization; also the choice of the extraction protocol is of key importance. The composition of the extraction buffer has a decisive influence on which proteome fraction will be extracted. Therefore the second part of our study is dedicated to the comparison of different extraction protocols with respect to subsequent proteomic analyses.

Three cultivars of tomato Pusa Ruby, Arka Vikas and Pusa Early Dwarf were subjected to osmotic stresses induced by mannitol, polyethylene glycol and NaCl in vitro. Polypeptide patterns were analyzed during each stress treatment to differentiate between tolerant and sensitive cultivars. The stresses induced more stress proteins in cv. Pusa Ruby compared to other two cultivars indicating it to be relatively osmotic stress tolerant.

In this study, we have analyzed the expression of the low oxygen inducible sucrose synthase isozyme SH1 (SUS-SH1) in the phloem of maize (Zea mays L.) infected with maize bushy stunt phytoplasma. Immunolocalization and Western blot analysis revealed several fold induction of SUS-SH1 in companion cells of phytoplasma inhabited phloem of leaf sheaths and stems. The results imply higher rates of sucrose metabolism and intensified hypoxia in the phloem.

Cucumis sativus L. seedlings were pretreated 1 h with 10 μM paraquat (PQ) and then were subjected to normal (25/18 °C) or elevated (42/38 °C) temperature to investigate whether PQ can protect plants against heat stress. Heat stress inhibited fresh and dry masses of the second leaf, root dry mass and shoot fresh mass. In leaves, the stress disintegrated membranes of 84.97 % chloroplasts and elevated contents of malondialdehyde, superoxide radical and hydrogen peroxide. In contrast, PQ pretreatment altered antioxidant activities in leaves, even after PQ was rinsed off before seedlings were exposed to different temperatures. Under heat stress, PQ pretreatment improved plant growth, decreased percentage of abnormal chloroplasts (53.03 %) and reduced contents of malonaldehyde, superoxide radical and hydrogen peroxide due to increased activities of antioxidant enzymes such as superoxide dismutase, catalase, guaiacol peroxidase, glutathione peroxidase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase.

Tomato plants were treated for two weeks with different concentrations of Cr(III) or Cr(VI) compounds to compare their toxic effects. The concentration of total Cr in plant tissues increased linearly with its concentration in the growth medium and Cr accumulated largely in the roots, regardless of the form in which it was supplied to the plant. All measured plant growth parameters were negatively affected by Cr, but Cr(VI) showed much more pronounced toxic effects. Leaf net photosynthetic rate (P_N) was decreased by both Cr forms, and the decrease was also greater for Cr(VI). Cr(III) caused no significant effect on leaf stomatal conductance, whereas Cr(VI) reduced it. Cr(VI) also markedly reduced the variable to maximum chlorophyll a fluorescence ratio, measured in dark-adapted leaves.

The removal of Mg²⁺ is an important step in the chlorophyll degradation pathway and extracts from senescent and presenescent Arabidopsis thaliana leaves were analyzed for Mg-dechelatase activity, using chlorophyllin, an artificial derivative of the natural substrate, chlorophyllide. The optimum temperature and pH for this reaction were determined to be at approximately 50 °C and 7.2, respectively. Mg-dechelatase activity was enhanced by addition of EDTA and inhibited by MgCl₂, HgCl₂ and reduced glutathione, indicating phenomenons such as retroinhibition by reaction products and dependence on the redox state of the mixture. Size exclusion chromatography was performed on Arabidopsis leaf extracts, and Mg-dechelatase activity was found in the fraction corresponding to molecular mass of about 42 kDa, which indicates that the Mg-dechelating compound in Arabidopsis is considerably larger than in other systems. During dark-induced senescence, the activity increased over time until reaching a maximum at day 4, and then decreased. The addition of plant growth regulators indicated that the accumulation of Mg-dechelatase was activated by ethylene and delayed by 6-benzylaminopurine.

Methylglyoxal (MG) is a highly reactive α-oxoaldehyde, demonstrating anticancer effect on plant neoplastic tumours. In in vivo studies it was observed that MG destroyed crown gall tumours in Nicotiana tabacum produced by Agrobacterium tumefaciens, without any adverse effect on the host. The efficacy of MG in comparison to other anticancer drugs viz. cisplatin and ellagic acid in the treatment of crown gall was investigated. A slight degeneration of galls was noted in plants treated with cisplatin and ellagic acid but the plants died subsequently. With MG however, crown galls were completely cured and the plants completed their usual life cycle by flowering and producing seeds. MG inhibited the respiration of crown gall calluses suggesting that energy depletion resulted in tumour destruction.

To determine the effects of leaf colour on gas exchange and chlorophyll fluorescence, two genotypes of Begonia semperflorens with green leaves or red leaves were compared. The red leaves showed a high accumulation of anthocyanins and high absorbance at 282 and 537 nm while the green leaves exhibited a higher net photosynthetic rate and lower thermal dissipation of light energy. It seems likely that anthocyanins in the vacuoles restricted the absorption of green light to the chloroplasts, leading to a decrease in the efficiency of excitation capture by open PS 2 centres, photochemical quenching and CO₂ assimilation.

The growth and ion content of salt sensitive Lycopersicon esculentum Mill. cv. M82 and salt tolerant L. pennellii Correll accession LA716 were examined under both control and stress conditions (150 mM NaCl). L. esculentum grew more vigorously than L. pennellii under optimal conditions, however, L. pennellii was able to maintain its growth better than cultivated tomato when the plants were exposed to salinity. Sodium content of both L. esculentum and L. pennellii increased as a result of NaCl stress. In addition, both species showed reduced potassium and calcium content due to salinity. The physiological traits were also measured in a population of 52 L. pennellii introgression lines grown under both normal and stress conditions. A total of 311 quantitative trait loci (QTL) were identified for the studied traits: plant height, stem diameter, leaf number, leaf and root fresh and dry mass, and sodium, potassium and calcium contents. Some of the loci (124) were identified under both control and stress conditions while 86 QTL were identified only under non-stress conditions and 101 loci were identified only under NaCl stress.

To broaden our knowledge of flower development, two floral homeotic genes, LbAG and LbSEP3, were isolated from the flower of Lycium barbarum L. The open reading frame length of LbAG and LbSEP3 were 1090 and 992 bp encoding 249 and 242 amino acids, respectively. Sequence alignment and phylogenetic analysis indicated that LbAG belonged to C-type MADS-box gene and that LbSEP3 was E-type MADS-box gene. Compared with other floral homeotic proteins, LbAG held the conserved AG motif I, II and LbSEP3 conserved SEP3 motif I, II. Expression profile showed that LbAG transcripts were abundant in inner two whorls and fruit but not in root, leaf, sepal, and petal, and that LbSEP3 constitutively expressed in root, leaf, fruit, and all the four floral whorls.

Sour pummelo (Citrus grandis) seedlings were irrigated with nutrient solution containing four boron concentrations (i.e., 2.5, 10, 25 and 50 μM H₃BO₃) and two aluminum concentrations [i.e., 0 (-Al) and 1.2 mM AlCl₃ . 6 H₂O (+Al)]. It was found that B did not affect, but Al increased, the Al content in the roots. The Al and citrate contents in the -Al leaves either did not change or slightly increased with increasing B concentration. On the other hand, the Al and citrate contents in the +Al leaves rapidly decreased as B concentration increased from 2.5 to 50 μM, then decreased at the highest B concentration. The Al and citrate contents were higher in the +Al than in the -Al leaves, except for at 25 μM B when they were similar. The leaf malate content did not change in response to B or Al, except for an increase in the +Al leaves and a decrease in the -Al leaves at 2.5 μM B. Similarly, the root malate and citrate contents did not change in response to B with or without Al, except for a decrease in the malate and citrate contents in the +Al roots at 50 μM B and an increase in the citrate content in the -Al roots at 50 μM B. The activities of acid-metabolizing enzymes were less affected by B-Al interactions in the roots than in the leaves.

Changes in pigments contents, leaf area, leaf dry mass per unit area (LMA), photosynthetic rate and chlorophyll a fluorescence were investigated in developing leaves of Fagopyrum dibotrys Hara. mutant. Anthocyanins transiently accumulate below the upper epidermis during leaf ontogeny of this mutant. Red leaves possessed lower Chl content, LMA, photosynthetic rate and apparent carboxylation efficiency than green leaves. However, content of anthocyanins declined and above mentioned parameters increased during further leaf development. In both red and green leaves, chronic photoinhibition did not take place according to variable to maximum chlorophyll fluorescence ratio (F_v/F_m). Red leaves had higher non-photochemical quenching (NPQ) and higher PS 2 efficiency.

Long-distance electrical signals generated in locally stimulated plants are linked with systemic physiological responses. The propagation of electrical signal through a plant can be measured by multiple electrodes attached to different sites of a plant body. As this signal has to be measured with the sensitivity of tens of microvolts, it can be easily disturbed by power-line hums or external electromagnetic fields. These disturbances can mimic the action potentials generated by a plant. In this work, we present a brief summary of various experimental approaches to the measurement of surface electrical potential (SEP) on a plant and a description of our multi-channel device for the SEP measurement. The main advantages of our measuring system are galvanic separation of the measuring unit, resulting in the elimination of power-line disturbances, and simple and stable contact of Ag/AgCl-peletted electrodes with the plant surface, facilitated by an ordinary gel used in human electrocardiography. These improvements enabled us to detect unperturbed variation (slow) and action (fast) potentials on a plant, as demonstrated by the four-electrode measurement of the electrical signal propagation in a locally wounded tomato plant.

To determine genetic diversity among 47 elite sweet sorghum (Sorghum bicolor ssp. bicolor L.) genotypes, 46 simple sequence repeat (SSR) markers evenly distributed on all 10 chromosomes were selected. All SSR markers used were polymorphic among the genotypes studied. A total of 228 alleles were identified with an average of 4.96 alleles per marker. Furthermore, the genotypes studied showed medium genetic diversity. Clustering analysis grouped the 47 genotypes into 5 distinct clusters.

Low expression of osmotically responsive genes 2 (LOS2) encodes an enolase (2-phospho-D-glycerate hydrolase, EC 4.2.1.11) that converts 2-phospho-D-glycerate (PGA) to phosphoenolpyruvate (PEP) in the glycolytic pathway in Arabidopsis. Meanwhile, it is a transcriptional activator of cold-responsive gene, negatively controlling the expression of STZ/ZAT10, a zinc finger transcriptional repressor of cold-responsive gene from Arabidopsis. A novel LOS2 gene, designated PtrLOS2 (GenBank accession number GQ144341), was isolated from trifoliate orange [Poncirus trifoliata (L.) Raf.]. The PtrLOS2 cDNA is 1 662 bp in length with a 1 338 bp open reading frame (ORF), encoding a deduced 445 amino acid residue protein with a predicted molecular mass of 47.79 kDa and an isoelectric point of 5.54. The deduced protein of the PtrLOS2 gene shares high identity (over 86 %) with other plant species enolase, which suggests that the PtrLOS2 probably encodes an enolase. Sequence alignment showed that PtrLOS2 protein has a conserved DNA-binding and a repression domain. Moreover, a conserved start site of alternative translation for the c-myc promoter binding protein (MBP-1) was also found in PtrLOS2 protein. PtrLOS2 was constitutively expressed in leaves, stems and roots. PtrLOS2 expression in roots and stems was much higher than that in leaves under normal conditions, however, the expression of PtrLOS2 was up-regulated in leaves, but down-regulated in roots after cold treatments. The PtrLOS2 expression in stems was firstly up-regulated and then down-regulated after cold treatments. Meanwhile, after ABA treatment, the expression of PtrLOS2 was up-regulated in leaves but in stems and roots firstly down-regulated followed with up-regulation.

Arabidopsis thaliana plants (wild type accessions Col and N1438) were grown in nutrient solution for 34 d with or without 50 mM NaCl. Salt stress inhibited plant growth rate more in Col than in N1438 and a decrease in K⁺, Ca²⁺ and nitrogen contents was observed in both accessions. NaCl diminished accumulation of malate, fumarate and citrate only in Col accession. To measure the effect of NaCl on transcript level of superoxide dismutase (SOD) isoforms and copper chaperone for SOD genes, a semi-quantitative polymerase chain reaction (RT-PCR) method was developed using cDNA normalized against the EF1a gene in parallel with quantitative real time RT-PCR (Q-PCR) technique. Both methods gave the same results. The abundance of transcripts of the three genes coding for Cu/Zn-SOD responded similarly to NaCl in both accessions: CSD1 gene was overexpressed, and CSD2 and CSD3 genes were repressed. However, the genes coding for Fe-SOD (FSD1), Mn-SOD (MSD1) and Cu-chaperone for SOD (CCS) responded to NaCl differently in Col and N1438: the former gene was overexpressed in Col and repressed in N1438, and the opposite behaviour was observed for the latter two genes.

We investigated whether changes in abscisic acid (ABA) content in leaves of Mikania micrantha infected by the holoparasite Cuscuta campestris at five growth stages, influenced the host stomatal conductance (g_s), transpiration rate (E) and net photosynthetic rate (P_N). C. campestris infection caused a negative effect on g_s, E and P_N of the host plants. ABA content in host leaves infected by C. campestris was significantly lower at 6 d after parasitization (DAP) and significantly higher at 13 and 33 DAP, relative to uninfected controls. In the parasite, ABA content was lowest at 13 DAP and then sharply increased to the maximum at 26 DAP. Moreover, the ABA content in the parasite was always lower than in the infected host leaves. The results suggest that an increase in host ABA concentration contributes to reduced host g_s, E and P_N in the holoparasitic C. campestris - M. micrantha association.

LAF1 (Long after far-red radiation 1) is a R2R3 Myb transcription factor and a signal transducer of far-red radiation. To investigate the role of LAF1 in leaf and root development, the leaf growth and vein patterning in laf1 mutants under short day conditions were examined. The length of rosette leaves was reduced and the width of the midvein was increased in laf1 mutants compared to their wild-type (WT) counterparts. In addition, cell size and cell number were both decreased in the laf1 mutant in comparison to the WT plant. A comparative analysis of gene expression showed that the transcript levels of PIN and IAA genes, encoding auxin carrier and response proteins, were decreased in laf1 mutants. LAF1 expression was also shown to be induced by 1-naphthaleneacetic acid. These results suggest that both auxin transport and auxin responses are impaired in laf1 mutants, and that LAF1 is involved in the regulation of leaf and root development mediated by auxin signaling under short day conditions.

The objective of this study was to examine the role of NADPH oxidase on superoxide radical production under waterlogging in mung bean (Vigna radiata) cvs. T 44 (tolerant) and Pusa Baisakhi (PB) (susceptible), and wild species Vigna luteola. Two days of waterlogging caused decline in superoxide radical (O₂ ^.-) contents in all the genotypes, however, further waterlogging up to 8 d caused significant increase in O₂ ^.- contents. In control and revived plants O₂ ^.- contents were higher in PB, while under waterlogging stress T 44 and V. luteola showed greater increases in the O₂ ^.- contents. During waterlogging the increase in O₂ ^.- content was found to be due to the diphenylene iodonium chloridesensitive NADPH oxidase (NOX). This was further confirmed by the waterlogging induced increase in NOX activity, which was higher in tolerant genotypes T 44 and V. luteola compared with PB. Gene expression studies showed enhanced expression of NOX in the roots of waterlogged V. luteola and T 44, while little expression was observed in control or treated plants of PB. PCR band products were cloned and sequenced, and partial cDNAs of NOX was obtained. Results suggest that increase in O₂ ^.- content during waterlogging could be due to the induction of membrane linked NOX.