This study investigated the regulation of ascorbate and glutathione metabolism by nitric oxide in Agropyron cristatum leaves under water stress. The activities of ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), L-galactono-1,4-lactone dehydrogenase (GalLDH) and γ-glutamylcysteine synthetase (γ-ECS), and the contents of NO, reduced ascorbic acid (AsA), reduced glutathione (GSH), total ascorbate and total glutathione increased under water stress. These increases were suppressed by pretreatments with NO synthesis inhibitors N ^G-nitro-L-arginine methyl ester (L-NAME) and 4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO). However, application of L-NAME and cPTIO to plants sufficiently supplied with water did not affect the activities of above mentioned enzymes and the contents of NO and above mentioned antioxidants. Pretreatments with L-NAME and cPTIO increased the malondialdehyde (MDA) content and electrolyte leakage of plants under water stress. Our results suggested that water stress-induced NO is a signal that leads to the upregulation of ascorbate and glutathione metabolism and has important role for acquisition of water stress tolerance.

We have investigated several factors determining plastid size and number in Peperomia, a genus in the Piperaceae family whose species naturally display great interspecific variation in chloroplast size and number per cell. Using microscopic techniques, we show that chloroplast size and number are differently regulated in the palisade parenchyma and the spongy parenchyma, suggesting that chloroplast division in these cell types is controlled in different ways. Microscopic studies of iodine-stained root cells revealed a correlation between amyloplast size in root cells and chloroplast size in palisade parenchyma cells. However, despite substantial variation in chloroplast number in leaf mesophyll cells, amyloplast number in root cells was very similar in all species. The results suggest that organelle size and number are regulated in a tissue-specific manner rather than in dependency on the plastid type. We also demonstrate that plastid size determines the size but not the number of starch grains in root amyloplasts.

To determine the suitability of micropropagation techniques developed for conserving rare medicinal herb Ungernia victoris we estimated the genetic fidelity of plants produced through direct regeneration from the bulb scale segments and organogenesis from long-term callus culture. Average value of the Jaccard's distances between explant-derived regenerants and maternal plants calculated from RAPD data was 0.5 %, while that of estimated between callus-derived regenerants and maternal cell line was 4.2 %; average distances between the objects among the explant-derived and callus-derived regenerants were 0.7 % and 2.5 %, respectively. The data obtained suggest that conditions for in vitro culture applied in this work provide relatively high genetic stability of the species upon the direct regeneration in vitro and regeneration from the long-term cultured callus.

In the leaves of rice (Oryza sativa), stomatal initials arose from two asymmetric cell divisions and a symmetric division. Guard mother cells (GMCs) and long cells in stomatal files (LCSs) were formed through the first asymmetric division of the precursor cell of GMCs. Subsidiary cells (SCs) were produced by the second asymmetric division of subsidiary mother cells or LCSs. Following SC formation, GMCs divided once symmetrically to generate guard cells and then differentiated terminally to form mature stomata. The developmental patterns of long cells, prickle hairs and short cells (phellem cells and silica cells) were also examined. Interestingly, we found that the different developmental stages of stomata and epidermal cells occurred in the similar location of immature leaves of the same phyllotaxis. In addition, two spacing patterns ("one stoma, one long cell" and "one short cell row") probably exist in rice leaves.

Apomixis represents an alteration of classical sexual plant reproduction to produce seeds that have essentially clonal embryos. In this report, hickory (Carya cathayensis Sarg.), which is an important oil tree, is identified as a new apomictic species. The ovary has a chamber containing one ovule that is unitegmic and orthotropous. Embryological investigations indicated that the developmental pattern of embryo sac formation is typical polygonum-type. Zygote embryos were not found during numerous histological investigations, and the embryo originated from nucellar cells. Nucellar embryo initials were found both at the micropylar and chalazal ends of the embryo sac, but the mature embryo developed only at the nucellar beak region. The mass of the nucellar embryo initial at the nucellar beak region developed into a nucellar embryo or split into two nucellar proembryos. The later development of the nucellar embryo was similar to the zygotic embryo and progressed from globular embryo to heart-shape embryo and to cotyledon embryo.

The coat protein (CP) gene of Tobacco streak virus (TSV) from sunflower (Helianthus annuus L.) was amplified, cloned and sequenced. A 421 bp fragment of the TSV coat protein gene was amplified and a gene construct encoding the hairpin RNA (hpRNA) of the TSV-CP sequence was made in the plasmid pHANNIBAL. The construct contains sense and antisense CP sequences flanking a 742 bp spacer sequence (Pdk intron) under the control of the constitutive CaMV35S promoter. A 3.6 kb Not I fragment containing the hpRNA cassette (TSV-CP) was isolated from pHANNIBAL and sub-cloned into the binary vector pART27. This chimeric gene construct was then mobilized into Agrobacterium tumefaciens strain LBA4404 via triparental mating using pRK2013 as a helper. Sunflower (cv. Co 4) and tobacco (cv. Petit Havana) plants were transformed with A. tumefaciens strain LBA4404 harbouring the hpRNA cassette and in vitro selection was performed with kanamycin. The integration of the transgene into the genome of the transgenic lines was confirmed by PCR analysis. Infectivity assays with TSV by mechanical sap inoculation demonstrated that both the sunflower and tobacco transgenic lines exhibited resistance to TSV infection and accumulated lower levels of TSV compared with non-transformed controls.

Sugarcane is cultivated in tropical and subtropical regions where cold stress is not very common, but lower yields and reduced industrial quality of the plants are observed when it occurs. In our efforts to enhance cold tolerance in sugarcane, the gene encoding the enzyme isopentenyltransferase (ipt) under control of the cold inducible gene promoter AtCOR15a was transferred via biolistic transformation into sugarcane (Saccharum spp.) cv. RB855536. Semiquantitative RT-PCR using GAPDH encoding glyceraldehyde-3-phosphate dehydrogenase as the normalizer gene showed the increased expression of the ipt gene under cold stress. The detached leaves of genetically modified plants subjected to low temperatures showed visible reduction of leaf senescence in comparison to non-transgenic control plants. Induced overexpression of ipt gene also enhanced cold tolerance of non-acclimated whole plants. After being subjected to freezing temperature, leaf total chlorophyll contents of transgenic plants were up to 31 % higher than in wild type plants. Also, lower malondialdehyde content and electrolyte leakage indicated less damage induced by cold in transgenic plants. Thus, the expression of ipt driven by the stress inducible COR15a promoter did not affect plant growth while providing a greater tolerance to cold stress.

Transgenic Citrus sinensis (L.) Osb. plants, cvs. Valencia and Hamlin, expressing Citrus tristeza virus (CTV) derived sequences were obtained by genetic transformation. The gene constructs were pCTV-CP containing the 25 kDa major capsid protein gene (CTV-CP), pCTV-dsCP containing the same CTV-CP gene in an intron-spliced hairpin construct, and pCTV-CS containing a 559 nt conserved region of the CTV genome. The transgenic lines were identified by PCR and the transgene integration was confirmed by Southern blot. Transgene mRNA could be detected in most transgenic lines containing pCTV-CP or pCTV-CS transgene. The mRNA of pCTV-dsCP transgene was almost undetectable, with very light bands in most analyzed plants. The transgene transcription appears to be closely linked to the type of gene construct. The virus challenge assays reveals that all transgenic lines were infected. However, it was possible to identify propagated clones of transgenic plants of both cultivars studied with a low virus titer, with values similar to the noninoculated plants (negative control). These results suggested that the transgenic plants present some level of resistance to virus replication. The higher number of clones with low virus titer and where mRNA could not be detected or was presented in a very light band was found for pCTV-dsCP-derived transgenic lines.

The enzyme 4-coumarate:coenzyme A ligase (4CL) plays an important role in phenylpropanoid metabolism. The 5'-upstream regions of two Sm4CL genes were isolated from danshen (Salvia miltiorrhiza Bunge) and their functions were characterized by promoter-directed GUS gene expression assay in transgenic Arabidopsis. Seedlings containing pSm4CL1 promoter:GUS fusions showed apparent GUS staining in hypocotyl and those harboring pSm4CL2 promoter:GUS fusions were clearly stained in cotyledon vasculars and roots. Mature Arabidopsis transformed with pSm4CL1 promoter:GUS exhibited GUS expression which was weak in the shoots and scarcely in roots and those modified with pSm4CL2 promoter:GUS displayed obvious GUS staining in roots, stigmatic papillae, stamens and sepal veins. Semi-quantitative RT-PCR revealed that Sm4CL2 was transcribed at the highest level in roots which was also shown to be the major accumulation site of salvianolic acid B. The results suggested that Sm4CL2 rather than Sm4CL1 might be responsible for the biosynthesis of salvianolic acid B in danshen roots.

Historically, sugar beets were selected from fodder beets. We used mitochondrial minisatellite loci to analyze cytoplasmic genetic diversity in fodder beet and sugar beet. Among the 8 sugar beet accessions examined we identified 3 multi-locus haplotypes. These 3 haplotypes were a subset of 5 haplotypes identified among the 29 fodder beet accessions examined. All but one haplotype in fodder beet comprised, in turn, a subset of 12 haplotypes identified previously in leaf beets. Such apparent decreases in cytoplasmic genetic diversity must result from genetic bottlenecks associated with domestication and the ensuing breeding processes. We also detected the haplotype associated with the male-sterile Owen cytoplasm of sugar beet in the fodder beet gene pool. Furthermore, the presence of a 39 kDa protein associated with the Owen cytoplasm was confirmed in two fodder beet plants by Western blot analysis. These results lead us to speculate that the Owen cytoplasm may have originated in fodder beet, from which sugar beet was derived.

C-repeat/dehydration-responsive element binding factor (CBF) plays important roles in cold response network in plants. Here, one member of CBF coding gene family in trifoliate orange (Poncirus trifoliata), designated as PtCBF, was isolated. Semi-quantitative reverse transcription-polymerase chain reactions showed up-regulation of PtCBF not only under low temperature but also induced by abscisic acid. Additionally, the CBF genomic fragments in four citrus species including trifoliate orange, sweet orange (Citrus sinensis), pummel (Citrus grandis) and rough lemon (Citrus jambhiri) were isolated with complete open reading frames. According to the results of alignment analysis between full length cDNA and genomic DNA sequences in trifoliate orange, there were no introns in PtCBF. Moreover, the results of multiple sequence alignment analysis and phylogenetic analysis on putative protein sequences suggested that the AP2 DNA binding domains and CBF signature sequences were highly conserved in four citrus CBF proteins. Finally, the CBF promoters in above citrus species were isolated, which provides some information concerning promoter function.

The polyamine (PA) content of in vitro-grown explants of Thymus moroderi Pau ex Martínez has been evaluated during minimal growth storage. The growth restriction was imposed by the combined action of osmotically-active compounds (15 g dm^-3 sucrose and 15 g dm^-3 mannitol) present in the Murashige and Skoog medium and the modification of the physical environment of the culture (4 °C and darkness). In these conditions, cultures were maintained up to 29 weeks without subculture. During this storage period, we analyzed contents of free, perchloric acid (PCA)-soluble and PCA-insoluble conjugated PA. Minimal growth storage brought about an increase in free putrescine (Put) coinciding with a reduction in PCA-soluble conjugated Put occurring during the first weeks of storage. PCA-insoluble conjugated spermidine (Spd) also accumulated in response to storage.

NADP-dependent isocitrate dehydrogenase (NADP-ICDH) catalyses the production of NADPH, which is an essential component in the cellular homeostasis. In Arabidopsis, the kinetic parameters (K _m and V _max) of cytosolic NADP-ICDH were different in leaves and roots. In vitro applied H₂O₂ did not affect the NADP-ICDH activity in either organ, however, the reduced glutathione inhibited the activity in leaves but not in roots. On the other hand, S-nitrosoglutathione (a NO donor) and peroxynitrite depressed NADP-ICDH activity in leaves and roots.

The oxidative processes and antioxidative system in cucumber (Cucumis sativus L.) fruit were determined during development and senescence. Four distinct developmental stages could be delineated during fruit maturation: immature (3-8 d after anthesis, DAA), mature (9-16 DAA), breaker (17-22 DAA), and yellow (35-40 DAA). The electrolyte leakage, malondialdehyde content, superoxide anion production rate, and hydrogen peroxide content increased continuously during fruit development and senescence. Superoxide dismutase and peroxidase activities consistently increased during fruit maturation, and the catalase activity displayed a single peak at the mature stage. Ascorbate peroxidase and glutathione reductase activities declined during fruit development, but both were activated in yellow fruit. Monodehydroascorbate reductase activity declined and dehydroascorbate reductase (DHAR) activity increased during fruit growth. DHAR was repressed in yellow fruit. Ascorbate dramatically accumulated and its redox state increased, whereas glutathione was degraded and its redox state declined, with fruit maturation.

To explore and isolate genes related to flowering and fruit development, we constructed a cDNA library from floral organs and fruitlets of Ponkan mandarin (Citrus reticulata Blanco). A total of 661 high-quality expressed sequence tags (ESTs) were generated and submitted to GenBank with the accession numbers from GO343532 to GO344192. All these ESTs were assembled into 43 contigs and 296 singletons (totally 339 unigenes). The BLAST2GO software was employed to annotate the unigenes, among which 77 ones had no significant homology with the sequences in NCBI non-redundant proteins database by BLASTX analysis. Additionally, gene ontology (GO) analysis revealed an overview of sequences distribution, which implied some specially expressed genes related to flower and fruit development. Furthermore, some abundantly expressed unigenes involved in several crucial metabolic pathways related to fruit quality were highlighted and three types of homologues of miraculin-like protein2 were analyzed by both semiquantitative RT-PCR and real-time PCR. The results showed different expression profiles of these genes, which meant that they contribute distinctly to fruit development.

Ethylene response factors (ERFs) are involved in many plant development events and stress defenses. In this study, an ERF gene, VvERF3b, was cloned from the leaves of Vitis vinifera. VvERF3b belongs to ERF group VIIIa. Expression of the gene was induced by abscisic acid, ethephon, and salicylic acid, but not by NaCl. Promoter sequence analysis of the VvERF3b gene revealed that there are several potential cis-acting elements that may be potentially recognized and bound by the transcription factors related to hormones and stress responses. Deletion analysis showed that the 5'-flanking sequence of -1047 to -585 from the transcriptional start site is essential to the high expression of the VvERF3b gene, whereas the sequence fragment of -1324 to -1047 revealed suppression effect. The result indicated that the region appears to contain cis-acting elements that can be bound by the proteins in a transcription complex to induce the inhibition of gene expression.

Nucleoside diphosphate kinase (NDPK) operates in the homeostasis of cellular nucleoside triphosphate (NTP) pools and the cytosolic NDPK1 is the main NDPK isoform in plants, accounting for more than 70 % of total NDPK activity in plant. A full length cDNA (697 bp), designated as CsNDPK1 was cloned from Camellia sinensis (L.) O. Kuntze leaves. Sequence analysis of CsNDPK1 shows several motifs, binding and catalytic sites which are highly conserved among other NDPKs. Southern blot analysis revealed that tea genome has two copies of CsNDPK1. Transcription pattern analysis indicated that CsNDPK1 is expressed in all tissues examined, but expressed more in buds than in other organs.

Elicitation of cell cultures of Silybum marianum with methyl jasmonate (MeJA) increases the production and release of the secondary metabolite silymarin into the culture medium and this process seems to be dependent on phospholipase D activity and its product phosphatidic acid (PA). However, MeJA did not alter total membrane lipid content or overall fatty acid composition. A progressive increase in some galactolipids was observed with elicitation time. Phospholipids were mainly represented by phosphatidylcholine (PC) followed by phosphatidylethanolamine (PE) and phosphatidylinositol (PI). MeJA caused losses of PC species that contain two unsaturated acyl species, 36:5 and 36:6 and an increase in 36:2 species. A drop in the ratio of compounds with 18:3 in PI and PE was also observed. The presence of the lysophospholipids (LP) LPC (16:0, 18:3, 18:2, 18:1) and LPE (16:0, 18:3, 18:2, 18:1) and the high contents of PA, represented by the molecular species 34:3, 34:2 and 36:5 and 36:4, indicates high basal level of phospholipase activity in cultures and a high phospholipid turnover. MeJA treatment did not quantitatively alter these lipid classes.

Short-term responses of Sedum alfredii roots to Cd exposure was compared in Cd hyperaccumulator (HE) and nonhyperaccumulating ecotype (NHE). Cadmium exposure significantly inhibited root elongation and induced loss of plasma membrane integrity and lipid peroxidation of roots tips in the NHE, whereas these effects were much less pronounced in the HE plants. A strong accumulation of reactive oxygen species with increasing Cd concentration was noted in the NHE root tips, but not in HE. After Cd exposure, a dose-dependent decrease in oxidized glutathione and marked increase in reduced glutathione and non-protein thiols were observed in root tips of HE, but were not seen in the NHE plants. These results suggest that the HE tolerates high Cd in the environment through the differential adaptations against Cd-induced oxidative stress.

Cinnamate 4-hydroxylase (C4H) is the second enzyme in the phenylpropanoid pathway which participates in the synthesis of numerous phenylpropanoid compounds such as flavonoids, lignins, suberins and others. We identified a gene putatively coding for Class I C4H in apricot and plum and we analyzed the expression pattern of this gene under different apricot/plum graft combinations with different degree of compatibility. The full-length cDNA is 1 739 bp with a 1 515 bp open reading frame encoding a protein of 504 amino acids. Like other C4Hs, the predicted C4H polypeptides included conserved domains of cytochrome P₄₅₀. The genomic sequence of the apricot C4H gene was interrupted by two introns 335 bp and 904 bp long. Several regulatory motifs including P-, A-, L- and H-boxes, which were conserved across phenylpropanoid metabolism-related genes in higher plants, were found in a 1 300 bp upstream promoter region of the apricot C4H gene. A phylogenetic analysis showed that all Prunus sequences clustered together and were closely related to Malus and Rubus C4H genes. The transcription of Class I PruC4H was detected in all the examined graft combinations, which suggested its rather constitutive character.

The potential of 5-aminolevulenic acid (ALA) to enhance the salt tolerance of cucumber (Cucumis sativus L.) seedlings was investigated. ALA was applied at various concentrations (0, 1, 10, 25, 50, and 100 mg dm^-3) as foliar spray or root watering. Then the seedlings were exposed to 0 or 75 mM NaCl for 5 d. NaCl stress reduced the root and leaf dry masses, leaf area, and the leaf net CO₂ assimilation rate. These reductions were counteracted by exogenous ALA, and the most efficient was 50 mg dm^-3 concentration via foliar spray. ALA decreased the H₂O₂ contents and increased the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) in NaCl-treated cucumber roots and leaves and the activity of catalase (CAT) in leaves. The ALA application also up-regulated the expressions of CAT and cAPX genes in roots and leaves and the expression of GR gene in roots of the NaCl treated cucumber plants.

The ABC1 protein family (ABC1P), a new family of putative kinases, widely existed in procaryote and eucaryote. A comprehensive genome-wide analysis was carried out in this study to find all ABC1Ps in rice (Oryza sativa subsp. japonica). We identified 15 ABC1P genes in rice. All the ABC1Ps contained an ABC1 domain of about 120 conserved amino acid residues and conserved kinase motifs-VAIK (VAVK, VAMK) and DFG (DEG). The phylogenetic analysis showed that all the ABC1Ps were grouped into three subgroups, and formed a total of 12 sister pairs. Conserved motifs analysed by MEME program indicated that almost all ABC1Ps contains motifs 1, 3, 7, 8 and 9. Predictably, the ABC1Ps were localized in mitochondria or chloroplasts, which implied that the ABC1Ps might be involved in energy metabolism in plants. RT-PCR assays demonstrated that all 15 ABC1P genes were active, and some of them were affected by abiotic stresses (NaCl, high temperature, methyl viologen, abscisic acid and cadmium).

We compared stomatal ammonia compensation point (χ_s) and its metabolic regulation in tobacco (Nicotiana tabacum) leaves of a quick-leaf-senescence phenotype ZY90 and a slow-leaf-senescence phenotype NC89. Compared with NC89, ZY90 had significantly higher χ_s values between 40 and 60 d after leaf sprouting in spite of its lower nitrogen content. During the same time, a steeper decline in glutamine synthetase activity was detected in ZY90 leaves, simultaneously with a steep increase in χ_s. These results suggested that the quick leaf senescence phenotype exhibited high NH₃ emission potential due to efficient nitrogen recycling and remobilization, and glutamine synthetase played a key role in regulating χ_s in ZY90.

We found that cadmium promoted flowering in Arabidopsis and suppressed nitric oxide accumulation in leaves. Supplementation with NO donor SNP delayed flowering, whereas application of NO scavenger cPTIO further promoted the transition from vegetative to reproductive stage under Cd stress. Semi-quantitative RT-PCR showed that Cd treatment up-regulated the expression of CONSTANS and FLOWERING LOCUS T, whereas down-regulated the expression of FLOWERING LOCUS C.

Brassinosteroids (BRs) have been proposed to increase the resistance of plants to drought stress. The effect of foliar application of 0.1 μM 24-epibrassinolide (EBR) on chlorophyll (Chl) content, photosystem 2 (PS 2) photochemistry, membrane permeability, lipid peroxidation, relative water content (RWC), proline content, and the antioxidant system in drought-stressed Chorispora bungeana plants was investigated. The results showed that polyethylene glycol (PEG) induced water stress decreased RWC, Chl content and variable to maximum Chl fluorescence ratio (F_v/F_m) less in plants pretreated with EBR than in non-pretreated plants. In addition, lipid peroxidation, measured in terms of malondialdehyde content, membrane permeability and proline content in drought-stressed plants were less increased in EBR pretreated plants, while antioxidative enzyme activities and reduced ascorbate and glutathione contents were more increased in EBR pretreated than in non-pretreated plants. These results suggested that EBR could improve plant growth under drought stress

The differentiation of metaphloem sieve element (MSEs) in the developing caryopsis of wheat (Triticum aestivum L.) was a programmed cell semi-death process. We studied the changes of microtubules and polysaccharide contents during MSEs development. Some significant features are presented in MSEs, such as cell wall non-uniform thickening, chromatin condensation and so on. During the period of MSEs differentiation, numerous microtubules are distributed in the vicinity of the cell wall, but finally they vanished in mature MSEs. Large glycoconjugates in the cell wall and small glycoconjugates in the Golgi apparatus were observed in the developing MSEs. Programmed cell death (PCD) ceased in the mature MSEs after 6 d after flowering and higher aggregation of glycoconjugates appeared in the cytoplasm. All of these processes were in tight contact with the cell wall non-uniform thickening during PCD.

Somatic embryogenesis (SE) in Arabidopsis was induced using various systems, including auxin treatment of in vitro cultured explants (immature zygotic embryos, IZEs) and transgenic plants overexpressing embryogenesis-related transcription factors, e.g. LEC2 together with the GUS reporter gene under control of the auxin-induced DR5 promoter. The study indicated that the SE-systems used gave different embryogenic capacities for the production of true embryos. The highest ratio of true embryos (75 %) was found among embryo-like structures in transgenic seedlings overexpressing LEC2. Analysis of in vitro induced SE systems indicated that in somatic embryos produced in response to exogenous auxin treatment the formation of root poles is frequently disturbed. A lack of a properly formed root meristem was observed in 35-80 % of in vitro induced somatic embryos, in dependence on auxin concentration and duration of treatment.

The objective of this study was to examine the role of nitrate reductase, nitric oxide and non-symbiotic hemoglobin in imparting waterlogging tolerance in mung bean genotypes. Experiment was conducted with two cultivated mung bean [Vigna radiata (L.) Wilczek] genotypes T 44 (tolerant) and Pusa Baisakhi (susceptible) and a highly tolerant wild species Vigna luteola (Jacq.) Benth. The content of nitric oxide increased up to 6 d of waterlogging in Vigna luteola and T 44, and up to 4 d of treatment in Pusa Baisakhi. Increase in nitrate reductase (NR) activity was observed only up to 4 d of waterlogging in Vigna luteola and T 44, and up to 2 d of treatment in Pusa Baisakhi, and thereafter the activity decreased in all the genotypes. The increase in NO content and NR activity was greater in Vigna luteola and T 44 than in Pusa Baisakhi. Non-symbiotic hemoglobin (NSHb) and cNR mRNA expressions were observed only in waterlogging treated roots of Vigna luteola and T 44, while very little expression was observed in control plants of Vigna luteola and T 44, and in control and waterlogged plants of Pusa Baisakhi. PCR bands of Hb and cNR were cloned, and nucleotide and deduced amino acid sequences were obtained and conserved regions and domains were identified using database.

Harpin, an elicitor molecule of bacterial origin induces hypersensitive response (HR) in non-host plants. In an attempt to induce male sterility, harpin was tagged with a signal peptide and expressed downstream to tapetum-specific TA29 promoter resulting in extracellular secretion, subsequent degeneration of tapetum and development of male sterility in tobacco. Putative transgenics were analyzed by PCR amplification of transgene, semiquantitative RT-PCR analysis from total RNA extracts from anther tissue with transgene specific probe, Western blotting using polyclonal antibody raised against harpin, by transmission and scanning electron microscopy, and by confocal microscopy of anthers and pollen at various stages of development. Varying degrees of male sterility (30-100 %) was observed with plants showing complete and partial male sterility as well as several morphological variations were seen especially in leaves and flowers. Further, some of the transgenics showed un-induced of HR-like local lesions in the vegetative tissues. Harpin_Pss got deposited on the pollen grains upon tapetal degeneration resulting in significant alterations in the morphology of pollen cell wall. However, megagametogenesis was not affected in complete and partial male sterile plants and female gametes were completely fertile. The complete male sterility was attributed to premature tapetal cell death due to sufficient extracellular harpin_Pss accumulation whereas insufficient protein content might be the reason for partial male sterility. These findings indicate the possible use of cytotoxic harpin_Pss for the development of male sterile plants.

To induce virus resistance in tobacco and rice we constructed hairpin RNA expression system harbouring inverted repeat fragments of coat protein cDNA of Potato virus Y (PVY) or Rice stripe virus (RSV). These structures were driven by three promoters [cauliflower mosaic virus 35S (CaMV 35S), polyubiqutin gene of maize (Ubi), and Pharbitis nil leucine zipper gene (PNZIP)] which have different tissue-specific activity. PVY resistance ratios were 65.18, 24.33 and 83.54 % in transgenic tobacco plants harboring p35S-PVY, pUbi-PVY and pPNZIP-PVY. RSV resistance was 16.21, 28.61 and 29.33 % in transgenic rice plants harboring p35S-RSV, pUbi-RSV and pPNZIP-RSV. Northern blotting and GUS assay demonstrated that virus resistance levels were related to promoter activity. Therefore, choice of the more effective and tissue-specific promoter to reinforce transcription of hpRNAs will favour the cultivation of highly virusresistant transgenic plants.