We determined the cold (freezing) tolerance of five Spanish populations of the perennial shrub Bituminaria bituminosa (L.) C.H. Stirton (Fabaceae), as the temperature at which 50 % of leaf electrolytes are released (LT₅₀) using leaves of field-grown plants, obtained in two winters and one spring. The freezing tolerance was greater in winter and reflected the minimum temperatures at the original sites from which the populations were obtained. Tolerance in vitro was related to osmotic adjustment in the leaves; more negative osmotic potential values and more positive pressure potential values (MPa) were associated with greater tolerance. Tolerance and osmotic potential were not related to leaf cation contents but to leaf amino acids, soluble sugar and proline contents.

With improved staining and chromosome preparation techniques, meiosis of pollen mother cells (PMCs) and male gametophyte development in autotetraploid cucumber (Cucumis sativus L.) was studied to understand the correlation between chromosomes behaviour and fertility. Various chromosome configurations, e.g. multivalent, quadrivalents, trivalents, bivalents and univalents were observed in most PMCs at metaphase I. Lagging chromosomes were frequently observed at anaphase in both meiotic divisions. In addition, chromosomes segregations were not synchronous and equal in some PMCs during anaphase II and telophase II. Dyads, triads, tetrads with micronuclei and polyads were observed at tetrad stage, and the frequencies of normal tetrad with four microcytes were only 55.4 %. The frequency of abnormal behaviour in each stage of meiosis was counted, and the average value was 37.2 %. The normal meiotic process could be accomplished to form the microspore tetrads via simultaneous cytokinesis. Most microspores could develop into fertile gametophytes with 2 cells and 3 germ pores through the following stages: single-nucleus early stage, single-nucleus late stage and 2-celled stage. The frequency of abnormalities was low during the process of male gametophyte development. The germination rate of pollen grains was 46.9 %. These results suggested that abnormal meiosis in PMCs was the reason for low pollen fertility in the autotetraploid cucumber.

We tried to determine the mechanisms by which Ca²⁺ mediated NO-induced programmed cell death (PCD) in tobacco protoplasts. Treatment of tobacco protoplasts with the NO donor sodium nitroprusside (SNP) resulted in a rapid [Ca²⁺]_cyt accumulation and decrease in mitochondrial membrane potential (ΔΨ_m) before the appearance of PCD. NO-induced PCD could be largely prevented not only by NO scavenger c-PTIO, but also by EGTA (Ca²⁺ chelator), LaCl₃ (Ca²⁺-channel blocker) or CsA (a specific mitochondrial permeability transition pore inhibitor, which also inhibit Ca²⁺ cycling by mitochondria). All results suggested that NO-induced PCD is mediated through mitochondrial pathway and regulated by Ca²⁺.

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.

Effects of H₂S on seed germination under chromium (Cr) stress were investigated in wheat (Triticum aestivum L.). Under Cr stress, the percentage of germination of wheat seeds decreased, but this decrease could be alleviated by pretreatment with NaHS, an H₂S donor, in a dose-dependent manner. Furthermore, NaHS significantly enhanced the activities of amylase, esterase, superoxide dismutase, catalase, ascorbate peroxidase, and guaiacol peroxidase in Cr-stressed germinating seeds, whereas reduced the Cr-induced increase in lipoxygenase activity and over-production of malondialdehyde (MDA) and H₂O₂, and sustained slightly higher content of endogenous H₂S.

To better understand the mechanisms involved in aluminum toxicity and tolerance in plants, microarray technology was used to evaluate changes in gene expression in Arabidopsis thaliana under Al stress. With the use of Affymetrix Arabidopsis ATH1 Genechip, a comparison of RNA expression profiles was made between control and Al-treated Arabidopsis seedlings. A total of 256 genes were identified as Al-responsive. Ninety-four genes were shown to be up-regulated and 162 were down-regulated; comprising 1.1 % of the 24 000 Arabidopsis genes. Real-time RT-PCR was used to confirm the microarray data. The analysis showed that a large number of transcription factors and several putative signaling components were up-regulated by aluminum. Chloroplast structural and photosynthetic genes were, in general, down-regulated. A number of previously identified Al-responsive genes, e.g. GST, Auxin-regulated, Peroxidase, and Chitinase, were up-regulated by Al-stress, whereas Wali 3 and Wali 4 were down-regulated. We also identified several up-regulated genes involved in vacuolar signaling, sorting and docking. Three genes were also up-regulated by Al-stress, Ras GTP-binding protein, ABC-cassette binding, and the AtELP1 receptor genes, have previously been documented as responsive to drought and/or oxidative stress and may play important roles the detoxification of Al ions by transportation and storage into root vacuoles. Ultrastructural changes in the roots tips cells of Arabidopsis were evaluated using transmission electron microscopy and energy-dispersive X-ray analysis with scanning electron microscopy and results showed Al accumulation in the root tips of Arabidopsis.

In this study we examined the genetic diversity and geographic scale of genotype distribution within the model legume species Medicago truncatula widely distributed in pasture and marginal agricultural lands in Greece and other Mediterranean countries. Thirty one Medicago truncatula and Medicago littorialis accessions were chosen on the basis of their geographical distributions and studied using 9 polymorphic simple sequence repeats (SSR) markers. The number of alleles per locus varied between 3 and 7. A total of 42 alleles were detected with a mean value of 4.66 alleles per locus. Geographic origin was not related with genotypic similarity among accessions. However, there were instances of close genetic relatedness between accessions from neighboring locations in a geographic compartment. In conclusion, the presented data revealed extensive M. truncatula genotype dispersal in Greece pointing to the significance of preserving local genetic resources in their natural environment.

The aim of this work was to investigate the role of the antioxidant enzymes in salt tolerance comparing the salt-sensitive (Pérola) and a salt-tolerant (Pitiúba) cultivar of cowpea [Vigna unguiculata (L.) Walp.]. Salt stress (100 mM NaCl for 8 d) reduced the leaf growth rate more in the sensitive cultivar. The salt-induced decrease in the relative water content, Na⁺ accumulation and increase in leaf electrolyte leakage was similar in both cultivars. Salt stress induced a higher increase in the activities of superoxide dismutase (SOD), ascorbate peroxidase (APX) and phenol peroxidase (POX) in the tolerant cultivar than in sensitive one.

The Triticum aestivum - Aegilops biuncialis (2n=4x=28; U^bU^bM^bM^b) disomic addition lines 2M^b, 3M^b, 7M^b and 3U^b were crossed with the wheat cv. Chinese Spring ph1b mutant genotype in order to induce homoeologous pairing, with the final goal of introgressing Ae. biuncialis chromatin into cultivated wheat. Wheat-Aegilops homoeologous chromosome pairing was studied in metaphase I of meiosis in the F₁ hybrid lines. Using U and M genomic probes, genomic in situ hybridization (GISH) demonstrated the occurrence of wheat-Aegilops homoeologous pairing in the case of chromosomes 2M^b, 3M^b and 3U^b, but not in the case of 7M^b. The wheat-Aegilops pairing frequency decreased in the following order: 2M^b > 3M^b > 3U^b > 7M^b, which may reflect differences in the wheat-Aegilops homoeologous relationships between the examined Aegilops chromosomes. The selection of wheat-Aegilops homoeologous recombinations could be successful in later generations.

In this study, we wanted to inspect whether the evolutionary driven differences in primary sequences could correlate, and thus predict the genetic diversity of related marker loci, which is an important criterion to assess the quality of any DNA marker. We adopted new approach of quantitative symbolic DNA sequence analysis called DNA random walk representation to study multiallelic marker loci from Begonia × tuberhybrida Voss. We described significant correlation of random walk-derived digital invariants to genetic diversity of the marker loci. Specifically, on the 3D-contour plot of multivariate principal component analysis (PCA), we revealed statistical correlation between the first two PCA factors and the number of alleles per marker locus. Based on that correlation, we suggest that DNA walk representation may predict allele-rich loci solely from their primary sequences, which improves current design of new DNA germplasm identificators.

Suspension cultures of Habanero pepper (Capsicum chinense Jacq.) were exposed to salicylic acid or methyl jasmonate to change secondary metabolism. Both treatments led to the accumulation of capsaicinoids and their late biosynthetic intermediate, vanillin. Both elicitors had a positive effect on the activities of phenylalanine ammonia lyase and coumarate O-methyltransferase, but none of them represented the main limiting step for capsaicinoid accumulation since vanillin contents were two orders of magnitude higher than those of capsaicinoids.

Morphological and molecular characteristics were studied in six wild species of Passiflora. There were statistically significant differences among these six species for all characteristics studied. Intra-specific variability was observed for number of flowers, number of fruits, number of seeds, fruit length, fruit width and leaf area. Cluster analysis using morphological data showed three groups: 1) P. palmeri var. sublanceolata, P. morifolia and P. foetida var. foetida, 2) P. coriacea and P. micropetala, and 3) P. suberosa. The dendrogram constructed using randomly amplified polymorphic DNA (RAPD) data showed six different groups for each species. The genetic distances among the 24 accessions ranged from 0.05 (between P. morifolia accessions P1 and P3) to 0.95 (P. coriacea accession 31 and P. palmeri var. sublanceolata accession 49). The species showed high morphological and molecular inter- and intraspecific variability.

In order to investigate the function of chloroplast ascorbate peroxidase under temperature stress, the thylakoid-bound ascorbate peroxidase gene from tomato leaf (TtAPX) was introduced into tobacco. Transformants were selected for their ability to grow on medium containing kanamycin. RNA gel blot analysis confirmed that TtAPX in tomato was induced by chilling or heat stress. Over-expression of TtAPX in tobacco improved seed germination under temperature stress. Two transgenic tobacco lines showed higher ascorbate peroxidase activity, accumulated less hydrogen peroxide and malondialdehyde than wild type plants under stress condition. The photochemical efficiency of photosystem 2 in the transgenic lines was distinctly higher than that of wild type plants under chilling and heat stresses. Results indicated that the over-expression of TtAPX enhanced tolerance to temperature stress in transgenic tobacco plants.

Embryogenic cell suspension cultures of Santalum album were transformed with Agrobacterium tumefaciens harboring pD35SHER plant expression vector having hepatitis B small surface antigen (HBsAg) with a C-terminal ER retention signal. The transformed colonies were selected on culture medium supplemented with kanamycin and subsequently the transgenic nature of these colonies was confirmed by PCR analysis. The expression of HBsAg was confirmed by RT-PCR analysis and Western blot analysis and the expression was quantified using monoclonal antibody-based ELISA. Cell suspension cultures were initiated from the colony with expression of 11.09 μg(HBsAg) g^-1(f.m.). To further increase the expression of HBsAg, transgenic S. album suspensions were cultured on media with various medium additives and cells growing in medium with 30 mM trehalose showed the expression of 19.95 μg(HBsAg) g^-1(f.m.).

Zephyr lily (Zephyranthes grandiflora), an important ornamental plant has been micropropagated in vitro after controlling microbial contamination by a pretreatment with 0.2 % Bavistin and 0.1 % Pantomycin for 4 h before final sterilization with 0.1 % mercuric chloride. In 67 % of the sterile cultures, 11 shoots on average were regenerated directly from basal half of bulb scales in Murashige and Skoog (MS) medium containing 3 % sucrose and 2 mg dm^-3 benzylaminopurine (BAP). Shoots emerged in bunches on a basal achlorophyllous bulbous part. Combination of 2 mg dm^-3 BAP with 1 mg dm^-3 gibberellic acid (GA₃) enhanced shoot growth. Stout roots (maximum of 5-6 per shoot) were developed in presence of 1 mg dm^-3 indole-3-butyric acid (IBA). Micro-bulbs showed potential of regeneration and could be used as secondary explants. The morphologically identical plants derived by in vitro propagation were genetically identical as shown by PCR based ISSR marker analysis of genomic DNA.

An experiment was conducted with two contrasting pigeon pea (Cajanus cajan L.) genotypes, ICPL 84023 (tolerant) and ICP 7035 (susceptible), to study the physiological and molecular basis of waterlogging tolerance in relation to oxidative stress and antioxidant enzyme activities. Waterlogging resulted in visible yellowing and premature senescence of leaves, and greater decline in relative water content, chlorophyll content, and membrane stability index in ICP 7035 than in ICPL 84023. Superoxide radical and hydrogen peroxide contents increased at day 4 and 6 of waterlogging probably due to activation of NADPH-oxidase. O₂ ^.- production was inhibited, by diphenylene iodonium chloride, a specific inhibitor of NADPH oxidase and expression of NADPH oxidase-mRNA was increased under waterlogging condition in ICPL 84023. ICP 7035 showed higher contents of ROS in control condition and after recovery, however, during waterlogging the O₂ ^.- production was higher in ICPL 84023. Activities of antioxidant enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase and catalase increased under waterlogging more in ICPL 84023 than in ICP 7035. Cu/Zn-SOD and APX-mRNA expression in 24-h waterlogged plants showed enhanced expression in ICPL 84023 compared to ICP 7035. The cloning and sequencing of APX gene of tolerant and susceptible genotypes yielded cDNAs of 622 and 623 bp, having 95 % homology with each other and 92 % with the corresponding sequences of Vigna unguiculate APX-gene.

In this communication, we report the binding of abscisic acid responsive elements (ABREs) of rice Osem, namely motif A and motif B, with a cognate trans-acting factor present in the nuclear extract of tobacco leaf. The binding is specific as both the complexes were disrupted with an excess of homologous non-radioactive DNA like motif A or motif B themselves or with cis-elements of rice Rab16A, motif I (ABRE) and motif IIa (non-ACGT ABRE-like sequences). Four tandem repeats of ABRE from wheat Em (4X ABRE) or two tandem repeats of Em ABRE, plus two copies of coupling element (CE1) from barley HVA22 (2X ABRC), also showed specific complexes, that were competed out by an excess of homologous competitors like motif I, motif IIa, motif A, motif B, 4X ABRE and 2X ABRC, but not by the unrelated 4X DRE sequence. Elution of the protein from all the complexes showed a single 26 kDa polypeptide band. Introgression of two of the above synthetic promoters 4X ABRE and 2X ABRC, each fused with minimal promoter of cauliflower mosaic virus 35S (CaMV 35S), could induce the expression of the reporter gene β-glucuronidase (gus) in transgenic tobacco in response to high NaCl concentration, dehydration or abscisic acid, but not at the constitutive level, proving that they can be used as efficient stress-inducible promoters. Our work shows both in vivo and in vitro activity of the promoters from monocot genes in the model dicot plant tobacco.

Thalli of Xanthoparmelia somloensis with natural content of polyols (control) and polyol-free thalli (acetone-rinsed) were used to study ribitol effects at low temperatures. Thalli segments were cultivated in ribitol concentration of 32 or 50 mM for 168 h at temperatures +5, 0, and -5 °C. The chlorophyll fluorescence parameters (potential yield of photochemical reactions in PS 2 (variable to maximum fluorescence ratio, F_v/F_m), effective quantum yield of photochemical reactions in PS 2 (Φ_PS2), and non-photochemical quenching (NPQ) were monitored in 24-h intervals using an imaging system. The effect of 32 mM ribitol on F_v/F_m and Φ_PS2 was apparent only at -5 °C, however, the effect was seen throughout the whole exposure. Surprisingly, 50 mM ribitol concentration treatment led to a decrease in F_v/F_m and Φ_PS2 and to an increase in NPQ values at -5 °C, while no change was observed at 0 °C and +5 °C. Acetone-rinsing caused decrease of F_v/F_m, Φ_PS2 and NPQ.

Biochemical and molecular markers have been used on eleven species of Cucurbitaceae collected from lower Gangetic plains. Six enzyme systems were selected. Among 40 primers examined, 14 random amplified polymorphic DNA (RAPD) and 10 inter-simple sequence repeat (ISSR) primers were selected for the analysis. Generated RAPD (100) and ISSR (100) fragments showed high variations among the species. Jaccard similarity coefficients were used for the evaluation of pairwise genetic divergence; cluster analysis of the similarity matrices was performed to estimate interspecific diversity. Further, principal coordinate analysis was performed to evaluate the resolving power of the three marker systems to differenciate among the species.

Unfavourable environment brings many kinds of stresses to plants. To survive such stresses, efficient resistance is required for the plants. Multifunctional genes enable the cross-talk among the various abiotic stress resistance systems. This paper reviews the action mechanisms of multifunctional genes. These genes can be classified into three groups: genes encoding diverse proteins through mRNA splicing (e.g. AOX in rice); genes like BADH, P5CS and HAV that control drought, salinity, osmotic and heat stress resistance; and a gene family, for example AQP, controlling transport of many compounds including water and nutrients. These genes participate in signal sensing and transduction, transcriptional regulation and functional gene activation during stress resistance induction. Furthermore, it should be noted that, under abiotic stresses, the regulation cascades are mutually interdependent and there also exists a close correlation between those cascades and normal plant growth and development.

Content of polyamines and activities of antioxidative enzymes in response to stripe rust disease caused by Puccinia striiformis have been studied in two wheat (Triticum aestivum L.) cultivars PBW 343 (resistant) and HD 2329 (susceptible). Various infection stages ranging from traces to 100 % were collected. Infection leads to stimulation of peroxidase (POD), superoxide dismutase (SOD), catalase, diamine oxidase and polyamine oxidase activities along with increase in putrescine, spermidine and spermine content while ascorbate, tocopherol and chlorophyll content decreased in HD 2329 and no visible symptoms appeared in PBW 343. Histochemical localization pattern of POD and SOD activities revealed correlation with lignin deposition in cell walls.

The effect of various hormonal combinations on regeneration of shoots and roots from meristem-derived callus of Crocus sativus L. and activities of antioxidant enzymes have been studied. The most efficient regeneration occurred with 1.0 mg dm^-3 1-naphthaleneacetic acid (NAA) + 1.0 mg dm^-3 thidiazuron and 1.0 mg dm^-3 NAA + 2.0 mg dm^-3 kinetin. For sprouting, regenerated shoot were subcultured on Murashige and Skoog medium containing 1.0 mg dm^-3 NAA + 1.0 mg dm^-3 benzylaminopurine (BAP). Protein content and superoxide dismutase activity decreased in regenerated shoots and roots and increased in sprouting shoots, while catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) activities increased during organogenesis and decreased in sprouting shoots. High CAT and PPO activities were detected in regenerated roots, whereas high POX activity was observed in regenerated shoot.

An important aspect of heat-shock response of lupin (Lupinus albus cv. Rio Maior) is the formation of cytoplasmic granular aggregates, called heat-shock granules (HSGs). In this study, two-dimensional electrophoresis (2-DE) was used to detect the component proteins of HSG complexes formed in vivo. Evaluation of 2-DE revealed differential expression of several proteins under heat shock conditions when compared with control. Among them, small heat-shock proteins (sHSPs) of 15 to 30 kDa were found to be the major representative proteins along with other proteins of relative molecular mass ranging from 36 to 45 kDa and above.

Bacillus cereus C1L was demonstrated to induce systemic resistance in Lilium formosanum against leaf blight caused by Botrytis elliptica. Suppression subtractive hybridization library of L. formosanum triggered by B. cereus C1L were screened and 3 differentially expressed genes were identified. Based on sequence analysis, these genes encoding putative glycine-rich protein, metallothionein-like protein, and PsbR protein of photosystem 2, were designated LfGRP1, LfMT1, and LfPsbR, respectively. The results of Northern blot analysis showed that expressions of LfGRP1, LfMT1 and LfPsbR increased in response to B. elliptica infection. On the other hand, expression of LfMT1 increased but expressions of LfGRP1 and LfPsbR decreased when the rhizosphere of L. formosanum was drenched with suspension of B. cereus C1L with or without subsequent challenge with B. elliptica on lily leaves. Similar expression profiles of homologues of LfGRP1, LfMT1, and LfPsbR (named LsGRP1, LsMT1, and LsPsbR, respectively) were presented in Lilium oriental hybrid Star Gazer. In addition, application of the photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, on the leaves reduced disease severity and expressions of LsGRP1 and LsPsbR just as that in response to B. cereus C1L treatment.

5-Methyltryptophan (5MT), a tryptophan analog, resistant M4 rice mutants with high free amino acid contents were obtained through in vitro mutagenesis. To evaluate the 5MT resistance mechanism, a cDNA library was constructed by using the leaves and roots of the 5MT resistant mutant plants. Expressed sequenced tags (ESTs) of 1 019 randomly selected clones were analyzed and then assembled 588 unigens. A total of 389 unigenes had significant homologies with known protein sequences at the NCBI database and the remaining 199 unigenes were designated unidentified genes. These unigens were grouped into 13 categories according to their putative functions. Of the 233 randomly selected clones, 25 were identified as differentially expressed genes between 5MT resistant and 5MT sensitive wild type plants. For further study of the differential expression of the genes, expression patterns of 12 genes related to various biological functions were evaluated in response to 5MT treatment in both the resistant plants and sensitive plants. All of the tested 12 genes exhibited higher expression levels in mutant plants than wild type plants under the 5MT inhibition. These expression patterns of the 12 genes suggested that the genes related to 5MT resistance in the rice mutants have a variety of functions, and yield remarkably diverse expression patterns upon 5MT treatment. Many genes that were identified tend to be related to defense and stress responses, suggesting "cross-talking" between biotic/abiotic stresses including the 5MT treatment. Therefore, 5MT resistant mutants might be of value for identifying genes related to plant defenses and stresses.

Endogenous contents of indolyl-3-acetic acid (IAA) and abscisic acid (ABA) were quantified in excised roots of Catasetum fimbriatum (Orchidaceae) cultured in vitro on solidified Vacin and Went medium with 1, 2, 4, 6, 8 and 10 % sucrose, as well as 2 % sucrose plus mannitol. Maximum root growth was observed in media with 4 % sucrose and 2 % sucrose plus 2.2 % mannitol, suggesting that a moderate water or osmotic stress promotes orchid root growth. Contents of both ABA and IAA increased in parallel to increasing sucrose concentration and a correlation between root elongation and the ABA/IAA ratio was observed. Incubating isolated C. fimbriatum roots with radiolabeled tryptophan, we showed an accumulation of IAA and its conjugates.

AP2 (APETALA2)/EREBPs (ethylene responsive element binding proteins) are the primary members of a family of transcription factors and OsAP211 was isolated from Oryza sativa L using the yeast one-hybrid system. It can specifically bind to the promoter containing three tandem repeats of DRE core sequence: TACCGACAT and activate the transcription of the downstream lacZ gene in the yeast one-hybrid system. OsAP211 contained a single open reading frame of 225 amino acids and encoded a protein containing a conserved AP2/EREBP domain featuring the DREB family. The semi-quantitative RT-PCR (s-Q RT-PCR) analysis indicated OsAP211 was strongly induced by low temperatures but not by NaCl and drought. It accumulates primarily in shoot tips during the tillering stage and young spikes during the booting stage. OsAP211 might function as a DRE-binding transcription factor in stress signal transduction pathways in rice.