Radicle growth of germinated seed of the root parasite O. ramosa is shown to be rapidly accompanied by secretion of proteins including pectinolytic enzymes, polygalacturonase and rhamnogalacturonase. These secretions peaked between 4 to 8 d after induction of germination and remained constant for some further days in the case of polygalacturonases. After 6 d, germinated seeds secreted proteins which exhibit peroxidase activity. The latter may be correlated with expression of OrPOX1, a putative gene encoding for secreted peroxidase. The involvement of these enzymes in host root attack and haustorium formation by the parasite is discussed.

Exogenous salicylic acid has been shown to confer tolerance against biotic and abiotic stresses. In the present work the ability of its analogue, 4-hydroxybenzoic acid to increase abiotic stress tolerance was demonstrated: it improved the drought tolerance of the winter wheat (Triticum aestivum L.) cv. Cheyenne and the freezing tolerance of the spring wheat cv. Chinese Spring. Salicylic acid, however, reduced the freezing tolerance of Cheyenne and the drought tolerance of Chinese Spring, in spite of an increase in the guaiacol peroxidase and ascorbate peroxidase activity. The induction of cross tolerance between drought and freezing stress was observed: drought acclimation increased the freezing tolerance of Cheyenne plants and cold acclimation enhanced the drought tolerance. The induction of drought tolerance in Cheyenne was correlated with an increase in catalase activity.

Epicotyl segments of kumquat (Fortunella crassifolia Swingle cv. Jindan) were transformed with Agrobacterium tumefaciens GV3101 harboring neomycin phosphotransferase gene (npt II) containing plant expression vectors. Firstly, the explants were cultured in darkness at 25 °C on kanamycin free shoot regeneration medium (SRM) for 3 d, and then on SRM supplemented with 25 mg dm^-3 kanamycin and 300 mg dm^-3 cefotaxime for 20 d. Finally, they were subcultured to fresh SRM containing 50 mg dm^-3 kanamycin monthly and grown under 16-h photoperiod. Sixty five kanamycin resistant shoots were regenerated from 500 epicotyl explants after four-month selection. Shoot tips of 20 strong shoots were grafted to 50-day-old kumquat seedlings and survival rate was 55 %. Among the 11 whole plants, 3 were transgenic as confirmed by Southern blotting. This is the first report on transgenic kumquat plants, and a transformation efficiency of 3.6 % was achieved.

Cell lines able to grow on media containing 50, 100, 150 or 200 mM NaCl were established from potato callus cultures by direct recurrent selection or gradual selection. In callus subjected to direct selection only small clusters of cells survived on medium with 150 or 200 mM NaCl, whereas on 100 mM small cell portions appear necrotic. When cell lines were obtained by successive subcultures on media with increased concentrations of NaCl, salt-tolerant calli were more compact and developed a greenish colour free from necrotic areas. The response of calli lines grown on media with NaCl was compared to control line. The NaCl-tolerant calli showed a decrease in relative growth rate and water content, with higher reductions in the 150 mM tolerant callus. Lipid peroxidation was increased in 50 mM and 100 mM NaCl-tolerant calli, while in 150 mM tolerant callus remained similar to 100 mM values. There was a significant increase in ascorbic acid content in 100 mM and 150 mM NaCl-tolerant calli as compared to the 50 mM, that was two-fold the value found in the control. Also, the contents of soluble and insoluble proteins increased in salt-tolerant lines. SDS-PAGE of soluble proteins showed the synthesis of specific polypeptides in the presence of NaCl in culture medium and the synthesis of a new polypeptide.

Two elicitors, termed herein as the botrycin and the cinerein, have been isolated from the crude mycelial cell wall and from culture filtrate preparations, respectively, of a fungal necrotrophic pathogen Botrytis cinerea (Pers. et Fries). In grapevine (Vitis vinifera L. cv. Limberger) both elicitors caused the formation of necrotic lesions that mimic a typical hypersensitive response and apoptosis-related events including protoplast condensation and DNA laddering. Infiltration of minute amounts of the respective elicitors into leaves stimulated a rapid transcriptional activation of genes encoding enzymes of the phenylpropanoid pathway. Cultured grapevine cells respond differentially to respective elicitors. Significant differences were demonstrated in the ability of botrycin and cinerein to induce ion fluxes across the plasma membrane and the production of reactive oxygen species. As demonstrated by immunokinase assays, both botrycin and cinerein activated specific and distinct MAP kinases indicating that grapevine cells that perceived elicitors generated a cascade of signals acting at local, short, and long distances. Using a highly parallel antibody microarray profiling approach, the timing, dynamics, and regulation of the expression of 97 specific genes in elicitor-treated cells of grapevine was analysed.

Aspects of plant growth such as height, branch number, leaf number, leaf area, pod area, 100-seed mass, etc., were correlated with biochemical changes such as contents of chlorophyll (Chl), proteins, DNA, and RNA, and protease activity during development and senescent phases in leaves, flowers, and pods of Cajanus cajan L. cv. UPAS-120 after treatments with kinetin (Kn). A significant increase was noticed in branch number, leaf number, leaf area, and seed mass while other growth processes registered a small increase after Kn application. Effectiveness of 5 µM Kn was also noticed in minimizing the loss of Chls, proteins, and nucleic acids as well as reducing the protease activity during maturity and senescence. Chl a/b ratio maintained a high value up to 30-d followed by a decline in leaves while flowers registered much lower ratio at 20-d-age. Pods were unique in having relatively lower ratio of Chl a/b in comparison to leaves.

The relationship between photosynthetic rate and yield formation processes of the newer and older maize hybrids were investigated. Leaf area at flowering (source) and kernel number (sink) of the newer hybrids were greater than the older ones although their light-saturated photosynthetic rate (P_sat) were not greater than the older ones before flowering. After flowering, P_sat and chlorophyll content of the newer hybrids declined more slowly than the older ones. They not only distributed almost all photosynthates produced after flowering to grain but also reallocated some reserved photosynthates produced before flowering to grain. The newer hybrids exhibited greater grain mass than the older ones mostly because they could optimally regulate the photosynthetic rate and yield formation processes to maximize grain mass.

Cytophysiological and cytogenetic characteristics of cell suspension culture of Panax japonicus var. repens were studied in relation to the accumulation of ginsenosides (GSs). The minimal time of cell number doubling was 1.3 ± 0.1 d and cell number increased 7 to 8-fold during growth cycle. The cell culture can be considered as aneuploid with about tetraploid (46-60 chromosomes) modal class. Upon long-term cultivation, the total content of GSs considerably increased and maximal concentration of GSs was 2.2 %(d.m.). The ratio of seven major GSs only slightly altered both over each and different subcultures. The overall amount of GSs of Rg-group significantly exceeded that of Rb-group. Cell volume and the number of large cellular aggregates with the higher proportion (by 20 %) of parenchymal cells increased late in the subculture. In this time the population contained about 20 % of the cells with doubled amount of nuclear DNA and accompanied with elevation in the GS content. These data prompted us to suggest that biosynthesis of GSs has a link with cell differentiation.

Cytokinins N⁶-benzyladenine (BA) and 1-(2-chloropyridin-4-yl)-3-phenylurea (4PU-30) delayed the senescence of detached leaves (3^rd to 7^th leaf node) of wild and ethylene insensitive eti5 mutant of Arabidopsis thaliana. The novel anticytokinins, structural analogues of purine and phenylurea cytokinins also affected the senescence of detached rosette leaves of A. thaliana. They diminished to a significant extent the cytokinin-induced delay of chlorophyll destruction, but without a considerable difference in their action against both types of cytokinins. These results correlated with changes observed in ribonuclease (RNase) activity.

An in vitro method for propagation of Holarrhena antidysenterica Wall. has been developed using nodal explants from mature trees growing in the field. Irrespective of concentrations and combinations of growth regulators used, the axillary and terminal buds sprouted and elongated when inoculated on Murashige and Skoog (MS) medium. The highest numbers of shoots were formed when sprouted shoots were subcultured from MS basal medium onto MS medium containing 2 mg dm^-3 N⁶-benzyladenine (BA) and 0.5 mg dm^-3 α-naphthalene acetic acid (NAA). The shoot number further increased upon subculture on MS medium containing 0.5 mg dm^-3 BA. By repeated sub-culturing of shoots derived from nodal axillary buds, a high frequency multiplication rate was established. The elongated shoots were excised and rooted in auxin free MS basal medium. Ex vitro rooting of in vitro formed shoots was achieved upon dipping the microshoots for 2 min in 2 mg dm^-3 of indole-3-butyric acid solution. Successful field establishment and high (80-90 %) survival of plants was observed.

Maize (Zea mays L.) seedlings of two cultivars (cv. Bastion adapted to W. Europe, and cv. Batan 8686 adapted to the highlands of Mexico), raised in a glasshouse (19-25 °C), were transferred to 4.5 or 9 °C at photon flux density (PPFD) of 950 µmol m^-2 s^-1 with 10-h photoperiod for 58 h and then allowed to recover at 22 °C for 16 h (14 h dark and 2 h at PPFD of 180 µmol m^-2 s^-1). The ultrastructural responses after 4 h or 26 h at 4.5 °C were the disappearance of starch grains in the bundle sheath chloroplasts and the contraction of intrathylakoid spaces in stromal thylakoids of the mesophyll chloroplasts. At this time, bundle sheath chloroplasts of cv. Batan 8686 formed peripheral reticulum. Prolonged stress at 4.5 °C (50 h) caused plastid swelling and the dilation of intrathylakoid spaces, mainly in mesophyll chloroplasts. Bundle sheath chloroplasts of cv. Batan 8686 seedlings appeared well preserved in shape and structure. Batan 8686 had also higher net photosynthetic rates during chilling and recovery than Bastion. Extended leaf photobleaching developed during the recovery period after chilling at 4.5 °C. This was associated with collapsed chloroplast envelopes, disintegrated chloroplasts and very poor staining.

The organogenic competence of leaf explants of eleven Carthamus species including C. tinctorius on Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ) + α-naphthaleneacetic acid (NAA) and 6-benzyladenine (BA) + NAA was investigated. Highly prolific adventitious shoot regeneration was observed in C. tinctorius and C. arborescens on both growth regulator combinations and the shoot regeneration frequency was higher on medium supplemented with TDZ + NAA. Nodal culture of nine Carthamus species on media supplemented with BA and kinetin (KIN) individually revealed the superiority of media supplemented with BA over that of KIN in facilitating a higher shoot proliferation index. Proliferating shoots from axillary buds and leaf explants were transferred to medium supplemented with 1.0 mg dm^-3 KIN or 0.5 mg dm^-3 BA for shoot elongation. Elongated shoots were rooted on half-strength MS medium supplemented with 1.0 mg dm^-3 each of indole-butyric acid (IBA) and phloroglucinol. The plantlets thus obtained were hardened and transferred to soil.

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.

5-enolpyruvylshikimate 3-phosphate synthase (EPSPS; 3-phosphoshikimate 1-carboxyvinyl-transferase; EC 2.5.1.19) is a critical enzyme in the shikimate pathway. The full-length EPSPS cDNA sequence (CaEPSPS, GenBank accession number: AY639815) was cloned and characterized for the first time from woody plant, Camptotheca acuminata, using rapid amplification of cDNA ends (RACE) technique. The full-length cDNA of CaEPSPS was 1778 bp containing a 1557 bp ORF (open reading frame) encoding a polypeptide of 519 amino acids with a calculated molecular mass of 55.6 kDa and an isoelectric point of 8.22. Comparative and bioinformatic analyses revealed that CaEPSPS showed extensive homology with EPSPSs from other plant species. CaEPSPS contained two highly conserved motifs owned by plant and most bacteria EPSPSs in its N-terminal region. Phylogenetic analysis revealed that CaEPSPS belonged to dicotyledonous plant EPSPS group. Tissue expression pattern analysis indicated that CaEPSPS was constitutively expressed in leaves, stems and roots, with the lower expression being found in roots. The coding sequence of CaEPSPS gene was successfully subcloned in a plasmid-Escherichia coli system (pET-32a), and the cells containing the plasmid carrying the CaEPSPS gene exhibited enhanced tolerance to herbicide glyphosate, compared to the control.

This work focuses on the comparison of field characteristics and amounts of reducing sugars in cold-stored tubers of transgenic plants derived from two potato cultivars. The bacterial gene coding for phosphofructokinase under the tuber-specific promoter was used to support the glycolysis in stored tubers. While the tubers from untransformed control plants steadily accumulated reducing sugars during cold storage, the tubers from transformed plants regardless the genotype were characterized by subsequent decrease in the sugar content. After long period of cold storage the greatest reduction in the reducing sugar content was by more than 60 % compared to control. Before the storage, however, the content of reducing sugars was in 80 % of transgenic lines higher than in control ones. The plants evaluated in field trials for their appearance showed any changes in growth characteristics in about 25 % of the transgenic lines. Despite the introduced modification of sugar metabolism the yield of transgenic plants with normal appearance did not differ significantly from the yield of control plants.

Four rice indica genotypes of local importance were transformed with RC7, rice chitinase cDNA clone through Agrobacterium-mediated gene transfer method using mature seed derived calli as explants. The putative hygromycin resistant calli showed varied level of regeneration efficiency ranging from 2.0 to 7.6 %. The stable integration and expression of RC7 was confirmed through polymerase chain reaction (PCR) and Western analysis. Transformation efficiency ranged from 0.9 to 5.2 %. The expression of RC7 (35 kDa chitinase) in different tissues of transgenic plant (root, sheath and leaf) was proved through Western analysis and in terms of increased chitinase activity. The inheritance of transgene was studied through PCR and Western analysis in transgenic plants of Pusa Basmati 1. Bioassays with transgenic plants of local cultivars exhibited enhanced resistance up to 33.3 % to rice sheath blight pathogen Rhizoctonia solani under glasshouse conditions. Enhanced expression or 3-to 4-fold increased activity of chitinase in transgenic plants was correlated with sheath blight resistance.

Dedifferentiation was monitored in Rubia tinctorum L. leaves over a 14-d period after callus induction using transmission electron microscope (TEM), high performance liquid chromatography (HPLC), spectroscopy and thin layer chromatography (TLC). Photosynthetic pigment loss of leaves took 3-5 d coinciding with the first period of anthraquinone accumulation. Callus cells were discernible in the region of the vascular bundles and wounded edges of leaves after 10-14 d. Characteristic ultrastructural alterations were manifested in vacuolization, appearance of mitochondria, amount of smooth endoplasmatic reticulum and cytoplasm, caryolympha density of nuclei and cytoplasm content of cells. There were special events in the transfer cells: unequal divisions of dedifferentiated plastids and lytic activity in the cell wall. Our results show that mesophyll cells seem to be stopped at a particular level of dedifferentiation, while transfer cells embodied in veins of leaves pass through further alterations and lead to callus formation. Findings suggest that a sort of dedifferentiation drift manifests in the various cells of R. tinctorum leaves during callus induction and depending on their specialized status they achieve different levels of dedifferentiation. Approximately 4 weeks after callus induction, root growth has started from the young calli.

Foliar sprays of water or 1, 10 and 100 µM aqueous solutions of gibberellic acid (GA₃) or kinetin (KIN) were applied to 40-d-old plants of Nigella sativa (L.) to study their effects on net photosynthetic rate, nitrogen metabolism, and the seed yield. 10 µM solutions of both the hormones, especially GA₃, appreciably increased the activities of nitrate reductase and carbonic anhydrase, chlorophyll and total protein contents and net photosynthetic rate in the leaves, along with capsule number and seed yield plant^-1, at harvest.

To evaluate the genetic diversity of some Vicia species, seed proteins of 160 accessions (30 of Vicia faba, 15 of V. narbonensis, 82 of V. sativa and 25 of V. ervilia and 8 accessions of other Vicia species) were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The dendrogram showed that the two outcrossing species V. faba and V. villosa were the most distant among all species (average percent disagreement value PDV 0.47 and 0.45, respectively). The tree was divided into small clusters of two species each. V. narbonensis fell in one cluster with V. michausai (at PDV = 0.35) and V. lutea (var. hirta) fell in one cluster with V. serococorpes (at PDV = 0.32) whereas, V. ervilia fell in one cluster with V. sativa (at PDV = 0.27). The V. sativa subspecies, however, were closely related (PDV < 0.1). In general, this study did not prove any relationship between the studied storage proteins and the geographical distribution or ecological needs of the studied accessions.

Embryogenic cultures of Dactylis glomerata L. were subjected to NaCl stress by culturing for 4 passages in Schenk and Hildebrandt (SH) medium containing 30 µM dicamba and 200 mM NaCl. Ultrastructural studies indicated invaginations and disruptions of the plasma membrane. Membrane-bound vesicles were observed in the cytoplasm of NaCl treated cells and their occurrence were increased with the culture age.

Wild-type and the handshake (has) mutants in Arabidopsis thaliana were analyzed. Compared to the wild-type, has mutants display a number of morphological alterations, which can largely be traced back to altered meristem function. Analyses of apical meristem of mutant plants showed that mutation affected meristem structure and patterns of STM expression.

Hairy roots of Rhamnus fallax Boiss. were induced using Agrobacterium rhizogenes strain A4M70GUS. The culture established on Woody plant media (WPM) showed a typical hairy root phenotype: rapid growth, reduced apical dominance and root plagiotropism. Seven clones of R. fallax were selected on the basis of their differences in colour and the root branching. The growth of hairy root culture, measured through gain in fresh mass, was done under 16-h photoperiod or in the dark. An increase in anthraquinone (AQ) content was obtained in clones with yellow and less branched roots, like clone 1 [16.43 mg g^-1(d.m.)] and clone 7 [14.21 mg g^-1(d.m.)], compared with other analysed transformed and non-transformed tissue. This study presents the first report of successful transformation of any species from family Rhamnaceae by A. rhizogenes and analysis of AQ production in transformed tissue.

A mathematical computer-aided model CELLOP was constructed in which the desirability functions in a three-dimensional experimental design can be used to find the optimal growing conditions for plant cells. CELLOP is intended for the optimisation of 3 to 6 physical, chemical, or biological variables in the cultivation conditions of plant cell cultures. The model was used to optimise the culturing conditions (calcium, inorganic nitrogen, and sucrose concentrations) for coumarin-producing, spontaneously embryogenic cell lines of angelica Angelica archangelica L. subsp. archangelica and hogs fennel Peucedanum palustre (L.) Moench. For A. archangelica the overall optimum concentrations were 0.47 mM Ca²⁺, 5.06 mM NO₃ ^-, 0.40 mM NH₄ ⁺, and 96.25 mM sucrose. The dry mass was 24.7 % higher and the coumarin content 40.5 % higher than those achieved in the standard 75 % Gamborg B5 medium. For A. archangelica the highest embryogenic activity was reached in the media containing 1.25 mM Ca²⁺. In the case of P. palustre the overall optimum concentrations were 1.60 mM Ca²⁺, 2.84 mM NO³ _-, 0.23 mM NH₄ ⁺, and 85.10 mM sucrose. For P. palustre the dry mass production increased by 61.8 % and the coumarin content by 58.1 % compared to the values achieved in the Gamborg B5 medium. For P. palustre the highest embryogenic activity was reached in the presence of 50.0 mM NO₃ ^- and 4.01 mM NH₄ ⁺.

The present work investigated the effect of NaCl and seawater salinity (SWS) on accumulation of proline and glycinebetaine (betaine) and expression of two key enzymes of their biosynthetic pathway, betainealdehyde dehydrogenase (BADH) and Δ¹-pyrroline-5-carboxylate synthetase (P5CS) in two aquatic macrophytes, salt-tolerant Najas gramenia and moderately-salt-tolerant Najas indica. N. gramenia did not show any accumulation of betaine or expression of BADH in response to NaCl or SWS treatments. The content of proline increased significantly in response to the salt treatments. High expression of P5CS was also observed, however, the transcript abundance did not correlate with the amount of proline. N. indica, exhibited significantly greater accumulation of proline in response to SWS, which contained Ca²⁺ in addition to NaCl, than in response to NaCl alone.

Ultraviolet-B radiation (biologically effective dose 2.6 kJ m^-2 d^-1) had negative influence on morphology and physiology of the young triticale plants. Plants exposed to UV-B were of lower height than control plants, their leaves were narrow, and the rate of net photosynthesis was decreased. The line RAH 336, which wax cover is lesser than that of traditional cultivar Magnat, was more susceptible to UV-B radiation, considering primary photosynthesis reactions, recorded by chlorophyll a fluorescence. An activation of protective mechanisms was observed: plants responded to UV-B by an increase of the content of UV-B absorbing compounds, and changes of antioxidant enzyme activities.

A protocol for induction of direct somatic embryogenesis, secondary embryogenesis and plant regeneration of Dendrobium cv. Chiengmai Pink was developed. Thidiazuron (TDZ) at 0.3, 1 and 3 mg dm^-3 induced 5-25 % of leaf tip segments of in vitro grown plants to directly form embryos after 60 d of culture, and 1 mg dm^-3 TDZ was the best treatment. Somatic embryos mostly formed from leaf surfaces near cut ends, and occasionally found on leaf tips. Higher frequency of embryogenesis was obtained in light than in darkness. During subculture, secondary embryos developed from outer cell layers of primary embryos. All combinations of NAA (0, 0.1, 1 mg dm^-3) and TDZ (0, 0.3, 1, 3 mg dm^-3) increased the multiplication rate of embryos. It takes about 8 months from embryo induction, plantlet formation to eventually acclimatization in greenhouse.