The roles of ethylene (ET) or salicylic acid (SA) in plant response to low temperature (LT, 5 °C) have been implicated. However, the combined effect of ET- and SA-signaling on plant growth and metabolism under LT remains to be evaluated. In this study, we comparatively analyzed the response of Arabidopsis ethylene insensitive (ein) 2-1 (an ET insensitive mutant), nonexprressor of pathogenesis relative (npr)1-1 (an SA insensitive mutant) and double mutant ein2-1/npr1-1 plants to LT. The results show that a LT of 5 °C induced plant growth retardation to a less degree in ein2-1, an intermediate degree in npr1-1, but a much larger in ein2-1/npr1-1 compared to the wild-type (WT) plants. The LT susceptibility of the ein2-1/npr1-1 plants was correlated to a lower net photosynthetic rate and proline content, and a higher content of H₂O₂ and malondialdehyde and electrolyte leakage relative to the WT plants. Lower activities of superoxide dismutase, peroxidase, and catalase, as well as a lower glutathione content and a ratio of its reduced form to its oxidized form were also observed in the double mutant plants as compared with the WT plants. However, at normal conditions (23 °C), all the tested physiological and biochemical parameters were comparable between the ein2-1/npr1-1 and WT plants, and plant growth was even better in the double mutant than in the WT plants. On the contrary, most of the above-mentioned parameters were advantageous in the ein2-1 and npr1-1 plants over the WT plants under the LT conditions. These data suggest that a parallel function or physiological redundancy of nonexpressor of pathogenesis relative 1 and ethylene insensitive 2 existed in the Arabidopsis plant response to the LT. On the other hand, an interaction between ET- and SA-signaling occurred during this process.

Screening salt-sensitive mutants is a powerful method to identify genes associated with salt tolerance. We used forward genetic screening with sodium azide-mutated rice (Oryza sativa L. cv. Tainung 67) to identify mutants showing hypersensitivity to salt stress. A new mutant line, named salt hypersensitive 1 (shs1) and exhibiting a severe salt-sensitivity when grown under a high NaCl concentration, was identified; the salt hypersensitivity was caused by duplicate recessive epistasis with mutations likely in two different loci. The shs1 salt sensitive phenotypes included a decreased seed germination rate, reduced shoot height and root length, severe and quick wilting, and overaccumulation of sodium ions in shoots as compared with wild-type plants. In addition, shs1 showed a decreased photosynthetic efficiency and enhanced hydrogen peroxide (H₂O₂) production under the salt stress. An increased superoxide dismutase activity and decreased catalase activity were responsible for the hyperaccumulation of H₂O₂ in shs1. The hypersensitivity of shs1 to the salt stress might be caused by an impaired antioxidant machinery and cellular Na⁺ homeostasis.

Caffeine (1,3,7-trimethylxanthine) is purine alkaloid, and it is secondary metabolite produced naturally in plants. It plays a crucial role in defense and stress tolerance. A hydroponic experiment was carried out to study the morphological, biochemical, and ultrastructural effects of caffeine treatment on seedlings of tobacco (Nicotiana tabacum L. cv. Turkish). The plants were grown in a growth chamber for 14 d in Hoagland's nutrient solution supplemented with 0 (control), 25, 50, 100, 1000, and 5000 μM caffeine. Shoot heights as well as root lengths significantly decreased in the plants treated with 1000 and 5000 μM caffeine. Total protein and sugar content in leaves increased significantly in the 5000 μM caffeine-treated plants. Moreover, electron microscopic analysis shows that in plants exposed to 1000 and 5000 μM caffeine, mesophyll cells possessed enlarged chloroplasts with disrupted thylakoid membranes associated with large starch grains and plastoglobules. Scanning electron micrographs exhibit that the vast majority of stomata in the plants treated with 1000 and 5000 μM were closed. In contrast, the 25 and 50 μM caffeine-treated plants showed an enhanced growth and no signs of injury.

Agrobacterium tumefaciens strain EHA105 carrying a binary vector pCAMBIA2301, which contains a neomycin phosphotransferase gene (nptII) and a β-glucuronidase (GUS) gene (uidA) interrupted with an intron, was used for transformation of Vigna mungo cotyledonary node explants. Various factors such as preculture and wounding of explants, manipulations in inoculation and co-cultivation conditions were found to play a significant role in influencing tissue competence, Agrobacterium virulence and compatibility of both, for achieving the maximum transformation frequencies. The stable transformation with 4.31 % efficiency was achieved using the optimized conditions. The transformed green shoots that were selected and rooted on medium containing kanamycin and tested positive for nptII gene by polymerase chain reaction were established in soil to collect seeds. GUS activity was detected in leaves, roots, pollen grains and T₁ seedlings. Southern analysis of T₀ plants showed the integration of nptII into the plant genome.

The nuclear factor Y (NF-Y) is one of the largest transcription factor families in plants consisting of NF-YA, NF-YB, and NF-YC subunits. It could play important roles in various processes such as flowering time, seed development, and response to drought. In this study, 6 NF-YA, 13 NF-YB, and 8 NF-YC proteins were identified and characterized in Prunus mume. Analyses of a conserved domain indicated that the PmNF-Y subunits shared an elevated degree of homology with the corresponding Arabidopsis NF-Y ones. Phylogenetic analysis showed that each NF-Y subunit family from Prunus mume and Arabidopsis could be divided into 4 or 2 clades based on their full-length proteins. The gene expression patterns of all 27 PmNF-Y genes were examined under abscisic acid (ABA), osmotic, salt, and H₂O₂ treatments using real-time quantitative PCR analyses. PmNF-YA1/2/4/5/6, PmNF-YB3/4/8/10/11/13, and PmNF-YC1/2/4/5/6/8 were found to be up-regulated under the ABA and osmotic treatments. PmNF-YA1/2/3/4/5/6, PmNF-YB1/3/8/10/11/13, and PmNF-YC1/2/5/6/8 were obviously induced by the H₂O₂. In addition, only PmNF-YA2 and PmNF-YB3 expressions were enhanced under the salt stress. These findings could provide an entry point to investigating the roles of PmNF-Y genes during abiotic stress responses.

Nitric oxide (NO) is an important molecule involved in the perception of stress induced by toxic compounds such as arsenic (As). The present study investigated the role of NO applied as sodium nitroprusside (SNP) in cell signalling and the ability of NO to attenuate the toxic effects of As (in the form of sodium arsenate) in water hyacinth (Eichhornia crassipes). Water hyacinth plants were collected and assigned to one of the following treatments: control; 100 μM SNP; 20 μM As; or 20 μM As + 100 μM SNP. The plants remained under these conditions for 0, 4, 12, and 24 h. After each time interval, the plants were collected and As absorption, production of reactive oxygen species (ROS), integrity of membranes, and antioxidant enzyme activities were evaluated. The plants were able to absorb and accumulate large amounts of As, even after only four hours of exposure to the pollutant. The absorption and bioaccumulation factor of As was even greater when plants were exposed to both As and SNP. The accumulation of As triggered increases in ROS production and cell membrane damage. In the presence of SNP, the tolerance index to As increased and damage was mitigated. Therefore, from the present work, it was possible to conclude that exogenous NO influenced the ability of plants to tolerate As; this finding has implications for phytoremediation in areas contaminated by As.

Iron availability affects plant growth depending on soil type. Mangroves are characterized by alkaline soils in which the halophytic wild rice relative Porteresia coarctata thrives. Young plants of P. coarctata grew optimally in the presence of 150 mM NaCl in a hydroponic medium and tolerated iron deficiency and salt up to 21 d without showing any symptoms of stress. A homolog of the rice iron deficiency responsive cis-acting element binding factor (IDEF1) that functions at the base of an iron regulated network was isolated and characterized from P. coarctata. PcIDEF1 had a close paralog in P. coarctata genome and its transcript expression was upregulated by both iron deficient conditions and salt treated conditions for up to three weeks. Sub-cellular localization study suggests nuclear targeting PcIDEF1 protein in guard cells and root tissues of tobacco. In vitro assays for metal binding affinity and binding PcIDEF1 to iron deficiency responsive element 1 (IDE1)-like elements in the 5' flanking region of an iron regulated transporter from P. coarctata suggest that PcIDEF1 could potentially sense iron content in a plant cell and regulate expression of iron responsive genes containing IDE1-like elements in their promoter region. This study provides evidence for a possible cross-talk between iron deficiency and salt responses.

Drought stress presents a considerable threat to the global crop production. As a dominant source of vegetarian diet, cereals and grain-legumes remain crucial to meeting the growing dietary demands worldwide. Therefore, breeding cultivars of these staple crops with enhanced drought tolerance stands to be one of the most sustainable solutions to enhance food production in changing climate. Given the context, a more focused survey of environment-defined germplasm sets is imperative to comprehend such adaptive traits. In parallel, uncovering the genetic architecture and the molecular networks that collectively contribute towards drought tolerance is urgently required through rationally combining large-scale genomics, proteomics, and metabolomics data. Also, attention needs to be directed to reasonably quantify the epistatic as well as environmental influences, thereby warranting deployment of analyses like metaquantitative trait loci (QTL) that encompass multiple environments and diverse genetic backgrounds. Further, innovative techniques like genomic selection (GS) and genome wide association study (GWAS) would help to capture the quantitative variation underlying drought tolerance. Equally importantly, integration of physiological traits-based techniques with ever-evolving 'omics' technologies and the new-generation phenotyping platforms will be of immense importance in advancing our existing knowledge about the genetically-complex and poorly-understood phenomena, such as plant drought response, and a deeper understanding would likely to provide a great impetus to the progress of crop breeding for drought tolerance.

Assam tea (Camellia assamica) is perennial crop susceptible to moisture stress. We used its tender roots to construct a cDNA library for the identification, functional annotation, and analysis of transcripts. A total of 811 full-length expressed sequence tags were generated. After processing and assembly, 207 unigenes comprising 58 contigs and 149 singletons were registered. Finally, 35.75 % of the unigenes could be assigned to functional categories based on the Arabidopsis proteome. There was 43 % of a coding GC content and 1 272 coding DNA sequences found in the unigenes. Codon usage analysis shows leucine as the highest (9.92 %) and tryptophan (2.0 %) as the lowest coded amino acids. Further, a comparative study with drought-induced genes of young roots (reported earlier) reveals that 4.83 % of genes required for normal growth of roots were also induced by a drought stress. Expressions of 10 unigenes under different abiotic stresses, such as drought, cold, and salinity, were further confirmed by RT-qPCR. The sequence tags generated in this study will be valuable resources for functional genomics study of tea and other woody crop plants in future.

Involvement of DNA polymerase (pol) enzymes in meiotic DNA repair has not been clearly understood in plants. DNA polymerase λ is involved in short patch DNA synthesis and base excision repair in both plants and animals. The presence and activity of the pol λ enzyme was studied in a protein isolated from spikelets during flower development stages of rice (Oryza sativa L.) and maize (Zea mays L.). Western blot analysis showed a 2- to 2.5-fold higher accumulation of pol λ in spikelets than in shoots. Assays of pol and in-gel activity showed the dideoxynucleotide triphosphate sensitive pol λ enzyme in spikelets of both the plants. An enhanced presence of the enzyme and its high activity suggests an active role of pol λ in meiotic recombination during microspore development.

Little is known about the contributions of DNA methylation/demethylation to plant aging and senescence. We used Arabidopsis thaliana to study how increasing age of an annual plant species influences DNA methylation. Based on methylation-sensitive DNA fragmentation assay, it could be concluded that aging A. thaliana was accompanied by DNA demethylation. Bisulfite sequencing reveals that cytosine methylation within the Actin2 3' untranslated region and internal transcribed spacer with 5.8S rRNA (ITS1-5.8SrRNA-ITS2) DNA regions decreased with A. thaliana growth and aging. We show that transcription of methyltransferase genes, chromomethyltransferase AtCMT3 and methyltransferse AtMETI, significantly decreased during development and aging of the A. thaliana plants, whereas expression of demethylase genes - repressor of silencing AtROS1, demeter AtDME, and demeter-like AtDML2 and AtDML3 - increased at least at some stages of plant development. The data obtained in the present study suggest that plant DNA regions may undergo demethylation during plant aging via reduction of DNA methylation processes and activation of active DNA demethylation.

The effects of NaCl on the H₂O₂ content and the activities of catalase (CAT) and superoxide dismutase (SOD) were studied in diverse group of plants, such as a unicellular alga, Chlorella sp., an aquatic macrophyte, Najas graminea, and a mangrove plant, Suaeda maritima, all showing high tolerance to NaCl. Significant accumulation of H₂O₂ was observed in all the tested plants upon their exposure to 255 mM NaCl. The activity of both CAT and SOD increased significantly in response to the NaCl treatment. Growing the plants in presence of 255 mM NaCl also resulted in the synthesis of new isoforms of both CAT and SOD.

Limited information is available on the role of brassinosteroids (BRs) in response of plants to nutrient deficiency. To understand the functions of BRs in response to iron deficiency, we investigated the effect of 24-epibrassinolide (EBR) on activities of ferric-chelate reductase (FCR), H⁺-ATPase, Ca²⁺-ATPase, nitrate reductase (NR), antioxidant enzymes, Fe and other minerals content and distribution, chlorophylls, soluble protein, free proline, reactive oxygen species, and malondialdehyde in peanut (Arachis hypogea L.) plants subjected to Fe deficiency (10^-5 M Fe(III)-EDTA) with foliar application of EBR (0, 10^-8, 5.0×10^-8, 10^-7, 5.0×10^-7, and10^-6 M). Results show that EBR increased Fe translocation from roots to shoots and increased Fe content in cell organelles. Activities of antioxidant enzymes increased and so the ability of resistance to oxidative stress was enhanced. As result of enhancement of H⁺-ATPase and Ca²⁺-ATPase activities, the inhibition of Fe, Ca, Mg, and Zn uptake and distribution was ameliorated. Chlorophyll, soluble protein, and free proline content also increased and consequently, chlorosis induced by Fe deficiency was alleviated. The results demonstrate that EBR had a positive role in regulating peanut growth and development under Fe deficiency and an optimal concentration appeared to be 10^-7 M.

Phenylalanine ammonia lyase (PAL) is a specific branch point enzyme of primary and secondary metabolism. The Citrus reticulata Blanco PAL gene was cloned and designated as CrPAL1. The cDNA sequence of CrPAL1 was 2 166 bp, encoding 721 amino acid residues. Sequence alignment indicates that CrPAL1 shared a high identity with PAL genes found in other plants. Both the dominant and catalytic active sites of CrPAL1 were similar to PAL proteins observed in Petroselinum crispum. Phylogenetic tree analysis indicates that CrPAL1 was more closely related to PALs in Citrus clementina × C. reticulata and Poncirus trifoliata than to those from other plants. Subcellular localization reveals that CrPAL1-green fluorescent protein fusion protein was specifically localized in the plasma membrane. Activity of PAL as well as CrPAL1 expression increased under Fe deficiency. A similar result was noted for total phenolic content. The root exudates of C. reticulata strongly promoted reutilization of apoplastic Fe in roots. Furthermore, Fe was more desorbed from the cell wall under Fe deficiency than in sufficient Fe supply.

Turnip (Brassica rapa L.) plants were grown in Perlite with low (< 2.5 μM) or adequate (25 μM) boron supply under well-watered and drought conditions for 12 weeks. Dry mass of leaves and roots was reduced under drought by about 61 and 56 % in plants supplied adequately with B, while up to 84 and 74 % under B starvation. Drought reduced B content by about 70 and 82 % for B-sufficient and B-deficient plants, respectively. According to the chlorophyll fluorescence parameters, the photosynthesis processes conserved their normal activities under low B supply in well-watered plants, while a serious damage to photosystem 2 occurred under drought stress. Stomatal limitation was the most important cause for a 17 % lower net photosynthetic rate (P_N) of drought stressed B-sufficient plants. In B-deficient plants, however, both stomatal and non-stomatal limitations were involved in 53 % reduction of P_N. Low B supply reduced strongly leaf water potential.

Physiological changes associated with senescence of flowers and abscission of floral parts in Oncidesa (formerly Oncidium) cv. Gower Ramsey are caused by a plant hormone ethylene which is produced by pollinia cap dislodgment during postharvest handling and transportation. The ethylene receptor gene OgERS1 of Oncidesa has been previously cloned and characterized. To analyze promoter activity of OgERS1, transgenic Arabidopsis thaliana plants were generated to express the ß-glucuronidase (GUS) reporter gene under the control of 5'-upstream sequence of OgERS1 from Oncidesa. The expression pattern of the OgERS1 promoter at the cellular level was investigated by analysis of GUS activity. This promoter can activate gene expression in both actively dividing young tissues and abscission-related aging tissues. Expression of GUS was detected in the shoot meristem uniquely in 10 to 30 d-old-plants and was found in flower buds, axillary buds, flower stems, and abscission layers during later development. In 2- to 3-week-old transgenic Arabidopsis, exogenous ethylene, glucose, lactose, and maltose enhanced promoter activity implying that crosstalk between sugar and an ethylene receptor may exist. However, indole-3-acetic acid, benzylaminopurine, abscisic acid, heat, wounding, salinity, drought, and flooding slightly suppressed promoter activity. These results demonstrate that the promoter of OgERS1 was developmentally and environmentally regulated, and imply a potential for application of this bi-functional promoter to increase branching or enhanced dwarfing.

MicroRNAs (miRNAs) are small non-coding RNAs that play crucial regulatory roles in diverse developmental processes via cleavage or translational inhibition of their target mRNAs. Although a growing number of miRNAs and their targets have been predicted and discovered via experimentation in many plants, little is known about conserved miRNAs and their target genes in Pinus densata. In the present study, the conserved miRNAs, miR171 and miR482, from Pinus densata were characterized. Analysis of miR171 and miR482 reveal that these miRNAs were highly conserved in other plant species. In addition, the precursors of miR171 and miR482 were validated by real time-PCR and sequencing. Using real-time quantitative PCR, miR171 and miR482 as well as their corresponding targets were found to be differentially expressed in needles, stems, and roots of Pinus densata. Furthermore two target genes, one GRAS family transcription factor protein gene and one nucleotide-binding site leucine-rich repeat (NBS-LRR) resistance protein gene, were experimentally verified to be the targets of pde-miR171 and pde-miR482, respectively, using RNA ligase-mediated 5'-rapid amplification of cDNA ends (RLM-RACE).

Moth orchids (Phalaenopsis) are among the top-traded blooming potted plants in the world. To explore mitochondrial DNA (mtDNA) markers for species identification, we located simple sequence repeats in the mtDNA of Phalaenopsis aphrodite subsp. formosana and then pre-screened them for polymorphic markers by their comparison with corresponding mtDNA regions of P. equestris. The combination of 13 selected markers located in intergenic spacers could unambiguously distinguish 15 endemic moth orchids. Five most variable markers with polymorphic information content (PIC) ≥ 0.7 could be combined to classify 18 of 19 endemic moth orchids including parental strains most commonly used in breeding programs. The sequences of four selected mtDNA regions were highly variable, and one region (MT2) could be used to completely distinguish 19 endemic moth orchids. Though mitochondrial introns were highly conserved among moth orchids, evolutionary hotspots, such as variable simple sequence repeats and minisatellite repeats, were identified as useful markers. Furthermore, a marker technology was applied to reveal the maternal inheritance mode of mtDNA in the moth orchids. Moreover, phylogenetic analysis indicates that the mtDNA was nonmonophyletic below the Phalaenopsis genus. In summary, we have revealed a set of mtDNA markers that could be used for identification and phylogenetic study of Phalaenopsis orchids.

Secretion of organic acids (OAs) by roots has been suggested to be an important mechanism of Al resistance in many species. In Stylosanthes, the participation of OAs in the mechanism of Al resistance is poorly understood. We aimed to study the production and secretion of OAs by two Brazilian Stylosanthes species with different Al resistance. Stylosanthes capitata and S. guianensis were treated with Al at different concentrations in 0.5 mM CaCl₂ (pH 4.0), and then root elongation, Al and OA content, OA secretion into the external solution, and the activity of citrate synthase (CS) were measured. Al-induced secretion of citric acid was also evaluated in the presence of protein synthesis and anion channel inhibitors. S. guianensis accumulated lower amounts of Al in its roots and displayed less inhibition of root elongation compared to S. capitata. Citric and malic acids were the most abundant OAs in the roots, and their content decreased with the Al treatment, except for citric acid in S. guianensis. Citric acid was the only OA secreted into the nutrient solution by the Al-treated plants of both species, but more by S. guianensis. Citrase synthase activity decreased in S. capitata but increased in S. guianensis with the Al treatment, and it may have a crucial role in the maintenance of citric acid content in the roots of S. guianensis. The use of anion channel and protein synthesis inhibitors reveal that anion channels were likely involved in the secretion of citric acid, and channel protein transcription was up-regulated by exposure to Al in Stylosanthes.

We tested the capability of plants to utilize a mixture of amino acids and peptides as the exclusive source of nitrogen. The aim of this study was to find out how such a nutrition affected plantlet growth, photosynthetic performance, and N assimilation metabolism in tobacco (Nicotiana tabacum L., cv. Petit Havana SR1) grown in vitro. Plantlets grown in a casein hydrolysate-supplemented (CA) medium were compared to plantlets grown in a complete Murashige-Skoog (MS) medium, plantlets grown in an ammonium-deficient medium (N1), or plantlets grown in a nitrate-reduced medium (N2). In addition, the plantlets were grown in the presence or absence of 1.5 % (m/v) saccharose as an additional carbon source. Casein hydrolysate, similarly as inorganic N limitation, reduced generally plantlet growth, whereas no significant effects were observed on photosynthetic parameters evaluated by chlorophyll a fluorescence. Although addition of saccharose stimulated the plantlet growth particularly in the MS, it showed a rather negative influence both on the growth and on the photochemical efficiency of photosystem II in the plantlets grown in the CA and N1. The activities of enzymes involved in N assimilation, such as nitrate reductase (NR) and glutamine synthetase (GS), were lower in the plantlets grown in the CA, N1, and N2, both in leaves and in roots. On the other hand, glutamate synthase and glutamate dehydrogenase were employed by the plantlets grown in the CA. The presence of saccharose in the growth medium stimulated mainly NR and GS activities in the MS grown plantlets, whereas enzyme activities of the plantlets grown on the N1, N2, and CA were not significantly influenced. We proved that the tobacco plantlets can utilize casein hydrolysate as the sole source of N particularly during their photoautotrophic cultivation. Contrary to positive effects of photomixotrophic nutrition for the MS grown plantlets, exogenous sugar seemed to diminish the ability of the casein hydrolysate-supplemented plantlets to utilize efficiently the additional C source.

This review summarizes the recent progress made towards the development of transgenic plants with improved tolerance to water stress and salinity. Of the various strategies employed, emphasis has been given to the genes engineered for the biosynthesis of osmoprotectants and osmolytes. This review also briefly discusses the importance of the use of specific stress inducible promoters and the future prospects of transgenic plants with improved agronomic traits.

Three types of transgenic plants of Solanum tuberosum cvs. Kamyk and Oreb, and Nicotiana tabacum cvs. Maryland Mammoth and Trapezond were selected according to intensity of introduced ipt gene expression and resulting amount of synthesised cytokinins (CKs). In comparison with controls, original transgenic regenerants grown in vitro showed a massive increase of CK contents, in tobacco by 379 % and in potato by 159 % (MAS). Potato grown in soil from tubers of transgenic plants demonstrated a moderate increase (44 %) of CK contents (MOD). Transgenic tobacco grown from seeds in vitro did not show any significant change in CK contents (NOT). Initial (RuBPC_i and RuBPO_i) and total (RuBPC_t) activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO), and the activity of phosphoenolpyruvate carboxylase (PEPC) were not significantly affected by the transformation in the NOT plants. In the MOD plants, the RuBPCO activities were stimulated by up to 34 % whereas the PEPC activity was decreased by 17 %. On the other hand, all the measured enzyme activities were 32 - 91 % lower in the MAS. Leaf area, fresh and dry masses, and chlorophyll and soluble protein contents also went down with increasing CK amounts in the transformants. Dependence of RuBPC_i/RuBPO_i and RuBPC_t/PEPC ratios on the relative CK amounts in transgenic plants revealed that the individual enzyme activities were not affected uniformly. Endogenous CK contents in the MAS thus apparently exceeded an optimum needed for positive effects on many physiological traits and became a stress factor for such plants.

Following drought stress at supraoptimal temperature the increase in proline (Pro) content in transgenic (T) soybean [Glycine max (L.) Merr. cv. Ibis] plants overexpressing the gene coding for the last enzyme of Pro biosynthesis, L-Δ¹-pyrroline-5-carboxylate reductase, was much greater than in wild type (W) plants (105-fold versus 19-fold after 7 d). Under control conditions arginine accounted for nearly 60 % of the total free amino acid content. After stress treatment the content of Pro was more than 50 % in both T and W genotypes, and at the end of recovery the γ-aminobutyrate content reached 27 and 53 % in the W and T plants, respectively. Without stress treatment there was only a 2-fold difference between T and W in the tyrosine content. However, during the stress period and the subsequent recovery a similar difference was found for many amino acids. The present results indicate that manipulating of the content of a single amino acid influences the whole free amino acid composition in soybean.

Using 2-D electrophoresis, we analyzed proteins from transgenic rice overexpressing gibberellin acid (GA) catabolic enzyme, GA2-oxidase. These results indicate eight specific proteins differentially expressed in the transformed rice stems of T₁ generation, but non in case of T₂ generation. Proteins isolated from different stages of leaves of T₁ generation showed no significant differences, except one-month-old leaf, where five differentially expressed proteins are visible.

In vitro cultures of Azadirachta indica A. Juss. were raised by first culturing the root segments on modified Murashige and Skoog (MS) medium supplemented with 8.88 μM 6-benzylaminopurine (BAP), 9.84 μM N⁶-(2-isopentenyl) adenine (2iP), 5.71 μM indole-3-acetic acid (IAA), 81.43 μM adenine hemisulphate and 2.27 μM putrescine for 2 d followed by their transfer to the same medium except containing one-tenth of the initially used concentrations of BAP, 2iP and IAA. The regenerated shoots sustained proliferation in the basal medium supplemented with 1.11 μM BAP, 1.43 μM IAA and 135.72 μM adenine hemisulphate. The isolated shoots were rooted to produce plantlets in the presence of 2.46 μM indole-3-butyric acid (IBA). The plantlets showed uniform luxuriant growth under field conditions. True-to-type nature of the field-grown root-regenerated plants was ascertained by random amplified polymorphic DNA (RAPD) analysis.

The effects of NiSO₄, calcium, and L-histidine (His) on the components of ascorbate-glutathione cycle, antioxidant enzymes and lipid peroxidation in a tomato cultivar Early Urbana Y was investigated. The activities of enzymes including catalase (CAT), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), superoxide dismutase (SOD), glutathione reductase (GR), lipoxygenase (LOX), and phenylalanine ammonia lyase (PAL) were measured. In addition, the content of H₂O₂, ascorbate (ASC), dehydroascorbate (DHA), reduced glutathione (GSH), chlorophyll (Chl) a+b, carotenoids, proteins, malondialdehyde (MDA), membrane aldehydes, and electrolyte leakage (EL) were determined. Results suggest that the excess of Ni increased the content of H₂O₂, MDA, membrane aldehydes and proteins in roots as well as GPX, LOX, APX activities, and EL in leaves, whereas Ca and His ameliorated these effects. Moreover, decreasing leaf GSH and DHA content and GR activity were observed under the Ni stress, but these parameters were raised by Ca plus His treatment. However, no improvement in leaf protein, ASC, root GSH content, and activities of PAL and CAT were observed by using Ca or His under Ni stress.

Sunflower plants were transformed via co-cultivation of previously bombarded hypocotyl explants with Agrobacterium tumefaciens harboring the plasmid pNGL that contains the human lysozyme gene. The transformed shoots were selected using kanamycin and regenerated plants were analyzed using histochemical β-glucuronidase assay. Southern, Western and Northern blot analyses indicated the transfer, expression and stable integration of the foreign DNA into the sunflower genome. Resistance against the phytopathogenic fungus Sclerotinia sclerotiorum, which causes white mold disease, was confirmed using a phytopathogenic test and microscopic observation of the infection process.

Polygalacturonases are enzymes involved in plant cell wall growth and reorganization. Transgenic Arabidopsis thaliana plants with a Saccharomyces cerevisiae endopolygalacturonase gene (PGU1) were obtained. The yeast gene was properly expressed in the plants as it has been shown by RT-PCR as well as by the increase in the endopolygalacturonase activity. The transgenic plants showed conspicuous malformations in early stages of development probably due to a weak cell adhesion. On the other hand, adult plants exhibited almost no phenotypic differences as compared to the wild type plants, this suggesting the appearance of some mechanisms on the plant side to counteract the effect of the overexpressed polygalacturonase.

The present study evaluated the use of γ-radiation to physically remove selective marker genes previously introduced into the soybean genome. Homozygous seeds from a transgenic soybean line carrying the gus and ahas transgenes were irradiated with γ-rays. Six plants presenting a deleted gus gene were analyzed by Southern blot to confirm removal of both ahas and gus genes. Line 1A presented an absence of the gus gene cassette and presence of the ahas gene cassette.

The pattern and expression level of β-glucuronidase (gus) reporter gene regulated by six heterologous promoters were studied in transgenic Populus tremula × P. alba plants obtained by Agrobacterium-mediated transformation. Binary vector constructs used contained the following promoter sequences: the CaMV35S from cauliflower mosaic virus; its duplicated version fused to the enhancer sequence from alfalfa mosaic virus; CsVMV from cassava vein mosaic virus; ubiquitin 3 from Arabidopsis thaliana (UBQ3); S-adenosyl-L-methionine synthetase (Sam-s) from soybean; and the rolA from Agrobacterium rhizogenes. Histochemical staining of root, stem and leaf tissues showed phloem and xylem-specific gus expression under rolA promoter, and constitutive expression with the other putative constitutive promoters. Quantitative GUS expression of 10 - 15 independently transformed in vitro grown plants, containing each promoter, was determined by fluorimetric GUS assays. The UBQ3-gus fusion induced the highest average expression level, although an extensive variation in expression levels was observed between independent transgenic lines for all the constructs tested.