The regeneration of European linden (Tilia × europaea L.) in vitro was successful. When using axillary buds as primary explants it was possible to induce a new shoot growth. The highest number of shoots per explant (2.13 ± 1.09) was recorded at the presence of 0.2 mg dm^-3 6-benzylaminopurine. Up to 50% of elongated shoots rooted in the presence of 2.0 mg dm^-3 α-naphthaleneacetic acid.

Phenylmethylsulphonyl fluoride (PMSF), a well known inhibitor of both thiol- and serine-type proteases, in aqueous solutions either alone or with the plant growth regulators, methyl ester of jasmonic acid (MeJA) and N⁶-benzyl-aminopurine (BAP), significantly inhibited the growth of excised Cucurbita pepo L. (zucchini) cotyledons. SDS-PAGE analysis of the protein profiles showed that PMSF suppressed the gradual decline of the main 20 - 25 kDa polypeptide group and the low molecular mass polypeptides (below 15 kDa) while leupeptine was not able to affect the electrophoretic pattern of cotyledon proteins. On the other hand, in the presence of PMSF, the content of the polypeptides with higher molecular mass including the 97.4 kDa polypeptide and the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (55 kDa) decreased. Besides, when applied together with MeJA, PMSF prevented the appearance of the jasmonate-induced polypeptides (JIPs; 69, 60 and 43 kDa) thus suggesting that JIPs are synthesized from aminoacids released during the breakdown of cotyledon storage proteins.

Plasmid DNA (pChlCOD), containing the selectable hygromycin phosphotransferase hpt gene for hygromycin B resistance and the Arthrobacter globiformis codA gene for choline oxidase which catalyzes the direct conversion of choline to glycinebetaine, was delivered into rice plants using Agrobacterium-mediated gene transfer via scutellum-derived calli. Southern, Northern and Western blot analyses demonstrated that the foreign gene had been transferred, integrated into rice chromosomal DNA and expressed. Drought test indicated that glycinebetaine acts as an osmoprotectant and its production in transgenic rice plant helped the cells to maintain osmotic potential and increased root growth, and thus enhanced the ability of the plants to tolerate water deficit

Chlorophyll a (Chl a) content and chlorophyllase (Chlase) activity from leaves of wild type (WT) and the ethylene-insensitive mutant (eti 5) of Arabidopsis thaliana (L.) Heynh during temperature stress and plant recovery have been studied. The plants were subjected to temperatures of 4 °C (LT) and 38 °C (HT) for 24 h. Chl a gradually decreased somewhat during stress and in the first day of recovery, especially in HT-treated plants. At the end of the experimental period (1 d stress and 10 d recovery) Chl a content was lower in eti 5 plants than in WT ones. The Chlase in WT was more affected than in eti 5 plants during the temperature treatment and the recovery period.

Relative nuclear DNA contents in cortex parenchyma cells in root segments of 3- and 7-d-old soybean seedlings grown at 25 °C and in plants grown for 3 d at 25 °C, and then for 4 d at 10 °C, were determined with cytophotometry. Measurements revealed that in each variant the cortex cell nuclei with DNA content between 2C and 8C were in all the examined segments and nuclei with 8C - 16C DNA appeared in higher parts of roots. However, in chilled plant cells the number of 8C - 16C DNA nuclei was very low. Therefore, chilling inhibited endoreplication in comparison with plants grown at 25 °C for 7 d, and even reduced endopolyploidy level as compared to the initial seedlings, i.e. 3-d-old plants. DNA contents in root hairs grown at 25 °C (control) and in root hairs emerged at 10 °C were also determined. In controls 4C - 8C DNA nuclei predominated while in chilled plants an additional population of 2C - 4C DNA appeared. Thus a reduction of DNA synthesis was brought about by low temperature. The occurrence of an intermediate DNA contents besides those with full endoreplication cycles suggests the possibility of differential DNA replication. This suggestion seems to be supported by the lack of ³H-thymidine incorporation into root hair nuclei at the examined developmental stage both in control and chilled root hairs. The same number, but larger, chromocentric lumps in polyploid cortex cell nuclei of higher root zones, in comparison to meristematic nuclei, suggests that endoreduplication process occurred.

Artichoke (Cynara scolymus L.) leaf size and shape, glandular and covering trichomes, stomatal density, stomata shape, pore area and epicuticular waxes during micropropagation stages were studied by scanning electron microscopy (SEM) and morphometric analysis with the aim to improve the survival rate after transfer to greenhouse conditions. Leaves from in vitro shoots at the proliferation stage showed a spatular shape, ring-shaped stomata, a large number of glandular trichomes and juvenile covering hairs, but failed to show any epicuticular waxes. Leaves from in vitro plants at the root elongation stage showed a lanceolated elliptic shape with a serrated border, elliptical stomata, decreased pore area percentage, stomatal density, and mature covering trichomes. One week after transfer to ex vitro conditions, epicuticular waxes appeared on the leaf surface and stomata and pore area were smaller as compared to in vitro plants. Artichoke acclimatization may be improved by hormonal stimulation of root development, since useful morphological changes on leaves occurred during root elongation.

Random amplified polymorphic DNA (RAPD) markers were used to analyze genetic fidelity of micropropagated teak (Tectona grandis L.) clones with respect to subcultural passage. Of the twenty primers screened, no variation in RAPD profiles was noticed in the in vitro clones of fifth, tenth, fifteenth and twentieth passage in comparison to the in vivo mother plants. Only one micropropagated plant of twenty-fifth subcultural passage, however, differed from the in vivo ones. It revealed the appearance of a new polymorphic DNA fragment (molecular mass 379 kb) in case of primer OPB-08. This primer, manifesting detectable variation, may be utilized as a diagnostic marker for assessing genetic fidelity of micropropagted teak plants.

Activities of ammonium assimilating enzymes glutamate dehydrogenase (GDH), glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) as well as the amino acid content were higher in nodules compared to roots. Their activities increased at 40 and 60 d after sowing, with a peak at 90 d, a time of maximum nitrogenase activity. The GS/GOGAT ratio had a positive correlation with the amino acid content in nodules. Higher activities of AST than ALT may be due to lower glutamine and higher asparagine content in xylem. The data indicated that glutamine synthetase and glutamate synthase function as the main route for the assimilation of fixed N, while NADH-dependent glutamate dehydrogenase may function at higher NH₄ ⁺ concentration in young and senescing nodules. Enzyme activities in lentil roots reflected a capacity to assimilate N for making the amino acids they may need for both growth and export to upper parts of the plant.

Membrane proteins of purified tonoplast vesicles from leaves of Kalanchoë daigremontiana Hamet et Perrier were solubilized by the non-ionic detergent Triton X-114 and subsequently separated by MonoQ® anion-exchange chromatography. Special attention was given to the range of molecular masses around 30 kDa comprising the central stalk subunit peptides of the H⁺-transporting V-ATPase. Three polypeptides of apparent molecular masses of 32, 33 and 34 kDa were separated. Proteolytic fragments were obtained by trypsin digestion. Analysis by matrix-assisted laser desorption ionization (MALDI) mass spectrometry of tryptic fragments of the 32 and 33 kDa peptides and protein data- bank comparisons showed that they are two different forms of subunit E. N-terminal amino acid sequencing of tryptic fragments of the 34 kDa peptide showed that it is subunit D. This work provides for the first time unequivocal molecular evidence that the central stalk of the V-ATPase of the obligate CAM plant K. daigremontiana includes subunit D and different forms of subunit E.

Five cultivars of Plantago ovata Forsk. (medicinal plant) have been developed by different agricultural universities in India. Genetic variability of these cultivars was estimated using RAPD markers. The data were correlated to morphological characters and a dendrogram was obtained from Jaccard's coefficient.

Eryngium foetidum L. plants were regenerated from mature leaf and petiole explants through direct organogenesis without intervening callus phase. From leaf explants, adventitious multiple shoots raised on Murashige and Skoog (MS) medium supplemented with 4.43 μM benzylaminopurine (BAP) and 0.57 μM indole-3-acetic acid (IAA), whereas in petiole explants shoot regeneration occurred at 8.86 μM BAP and 0.57 μM IAAA. 80% of the leaf explants and 44% of petiole explants produced shoots after four weeks of culture. The regenerated plants were rooted on MS medium supplemented with 2.46 μM indole-3-butyric acid and 2.88 μM gibberellic acid. The plants were successfully established in the soil and showed 70.9% survival in the field.

Multiple shoots of Spilanthes acmella Murr. were induced from hypocotyl segments obtained from 1-week-old seedlings on Murashige and Skoog's (MS) medium containing benzyladenine (BA), isopentenyl adenine, and naphthaleneacetic acid (NAA). High frequency shoot proliferation (95 %) and maximum number of shoots per explant (10 ± 0.6) were recorded with 0.5 mg dm^-3 BA in combination with 0.1 mg dm^-3 NAA. A proliferation was achieved by repeatedly subculturing the nodal segments on shoot multiplication medium. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing indole-3-butyric acid (1.0 mg dm^-3). 95 % of the plantlets were successfully acclimatized and established in soil. Transplanted plantlets showed normal flowering without any morphological variation.

The content of endogenous free abscisic acid (ABA) in the shoots of in vitro cultivated tobacco (Nicotiana tabacum L. cv. White Burley) and its changes during ex vitro acclimation of these plants to the greenhouse or growth chamber were estimated. The content of free ABA significantly increased at the 1^st and/or 2^nd day after plant transfer from in vitro to ex vitro. The ABA content of plants covered with transparent foil to maintain higher relative humidity (RH), did not significantly differ from ABA content of plants cultivated under ambient RH. Transfer to fresh medium also transiently increased the content of endogenous ABA. The ABA content in plants, which had been acclimated for 1 week to ex vitro conditions, decreased to the content found in the in vitro plants. Acclimation to ex vitro conditions affected the stomata on adaxial and abaxial sides differently: stomata on the adaxial side were less open than those on the abaxial one. The exogenous application of 5 μM ABA increased transiently its endogenous concentration in shoots of in vitro plants more than ten fold, but after 1 week the concentration in the shoots decreased.

For most crops growing in polluted areas or treated with agricultural chemicals, no genotoxicity assays are available. We have studied the possibility of using the alkaline protocol of the plant-based molecular assay - the Single Cell Gel Electrophoresis (SCGE) assay (also called Comet assay) as a method for detecting induced DNA damage in 8 agronomic important plants (ordered according to the diameter of the nuclei): sugar beet, alfalfa, tobacco, lentil, maize, potato, hard wheat, and bread wheat. The monofunctional alkylating agent ethyl methanesulphonate (EMS) was applied as a model genotoxic agent on young excised leaves of the tested crops for 18 h at 26 °C in the dark. With increasing concentrations of 2 to 10 mM EMS, the DNA damage, expressed by the averaged median tail moment values, significantly increased in nuclei of all crops studied. No correlation between the diameter of nuclei and sensitivity to EMS treatment was observed. The data obtained demonstrate the feasibility of using the Comet assay for detecting induced DNA damage in crops.

A liquid culture technique has been developed to study lipid metabolism in seeds of Brassica campestris L. grown in vitro from terminal inflorescences detached 4 to 46 days after anthesis. Seeds developed under these conditions exhibited pattern of growth, deposition of storage products and lipid composition similar to those from intact plant.

Kiwifruit plants (Actinidia deliciosa cv. Hayward) were grown in Hoagland nutrient solution with calcium nitrate, potassium nitrate, ammonium nitrate or ammonium chloride as the nitrogen source. Plants grown in the solution with nitrate nitrogen displayed a higher oxalate content, greater shoot length and leaf area, and higher content of ascorbic acid and NO₃^- ions in the leaves. Plants grown in the solution with ammonium nitrate, and particularly with ammonium chloride, showed low oxalate content, low content of ascorbic acid and NO₃^-, high content of Cl^- and Na⁺, low shoot length and leaf area. Oxalate formation appeared to be connected with the assimulation of nitrate, more precisely with nitrate reduction, while ammonium nitrogen assimilation did not induce the synthesis of oxalic acid.

Nodal explants excised from 18 to 20-year-old female plants of Simmondsia chinensis if cultured on Murashige and Skoog's medium supplemented with 20 μM N⁶-benzyladenine (BA) differentiated an average of 2.7 ± 0.4 shoots in 11.5% explants. The percentage of nodal explants inducing multiple shoots enhanced significantly if in vitro raised shoots were used as source of explants. Nearly 100% cultures differentiated an average of 4.7 ± 2.0 shoots per explant on the same medium. Nearly 85% of the shoots induced roots when a pulse treatment of 50 μM indole-3-butyric acid (IBA) was given prior to their transfer to semi-solid MS medium containing 10 μM IBA + 0.5% activated charcoal + 1 μM BA. Plantlets were gradually hardened in Soilrite and acclimatized to soil.

Effects of N⁶-benzyladenine, kinetin, zeatin, N⁶-benzyladenosine, kinetin riboside, zeatin riboside, jasmonic acid, indole-3-acetic acid, indole-3-butyric acid, indole-3-propionic acid, abscisic acid and gibberellic acid on proliferation of hyphae of arbuscular mycorrhizal (AM) fungus Glomus fistulosum were studied under axenic conditions in vitro. The growth of intraradical hyphae of G. fistulosum was fully suppressed by 30 µM indole-3-acetic acid, but a perceptible decrease in the proliferation of the hyphae was observed already at 3 µM. Because such concentration is near the concentrations common in root tissues in vivo, the effect may be biologically significant. Similar effect was also observed for Glomus mosseae. Inhibitory effects of abscisic acid and cytokinins occurred only at very high, non-physiological concentrations. Ribosylated cytokinins showed stronger inhibition effects than their non-ribosylated counterparts. No stimulation of proliferation of hyphae by any plant hormone tested was observed.

Recent investigations on plant molybdenum-containing enzymes that include xanthine dehydrogenase (EC 1.1.1.204) and xanthine oxidase (EC 1.1.3.22), nitrate reductase (EC 1.7.1.1-3), aldehyde oxidase (EC 1.2.3.1), and sulfite oxidase (EC 1.8.3.1) are reviewed. The enzymes belong to closely related protein family and share common structural features. Special attention is being paid to the recently solved crystal structures their implications for the substrate binding and catalytic mechanism.

The plant growth retardant, paclobutrazol at 8.5 or 17.0 μM concentrations effectively inhibited the stem elongation and primary leaf expansion of bean seedlings. Although the retardant reduced the relative water content in well-watered plants, the water and pressure potentials remained high in the primary leaves. K⁺, Na⁺, Mg²⁺ and Ca²⁺ contents in the primary leaves of the paclobutrazol-treated plants were not significantly different from those in the control. The stomatal density increased on both surfaces but the length of guard cells was not reduced significantly on the adaxial epidermes of the paclobutrazol-treated primary leaves. The inhibitory effect of paclobutrazol on the abaxial stomatal conductances became more pronounced with time during the light period but the adaxial surfaces displayed similar or slightly higher conductances than those of the control. The transpiration rate on a unit area basis did not change significantly or increased in the treated leaves thus the reduced water loss of paclobutrazol-treated plants was due to the reduced leaf area. Stomatal conductances of the adaxial surfaces responded more intensively to exogenous abscisic acid and the total leaf conductance decreased faster with increasing ABA concentration in the control than in the paclobutrazol-treated leaves. Paclobutrazol, an effective inhibitor of phytosterol biosynthesis, not only amplified the stomatal differentiation but increased the differences between the adaxial and abaxial stomatal conductances of the primary leaves.

Effective protocol was established for micropropagation of the medicinal plant Eupatorium triplinerve Vahl through rapid axillary bud proliferation and ex vitro rooting. Murashige and Skoog (MS) medium fortified with 8.87 μM benzylaminopurine (BAP) and 2.46 μM indole-3-butyric acid (IBA) was the best for axillary bud proliferation and developed a mean of 8.1 shoots per node. Excision and culture of the node segments of the in vitro shoots on medium supplemented with the same concentration of growth regulators developed more than 30 shoots within 40 d. Shoot multiplication did not exhibit decrease in the number of shoots even at 7^th subculture. Dipping of the basal end of shoots in 2.46 μM IBA solution for 10 d induced roots and its transfer to small pots facilitated the survival of all rooted shoots (100 %). Ex vitro rooting by direct transfer of the shoots from multiplication medium showed 92 % survival.

Multiple shoot regeneration from the cut plumular ends of embryo axes of chickpea (Cicer arietinum L.) was evaluated on Murashige and Skoog medium having different concentrations of thidiazuron (TDZ) (0.1 to 10.0 mg dm^-3) 6-benzylaminopurine (BAP) (0.5 and 1.0 mg dm^-3), kinetin (0.5 and 1.0 mg dm^-3) or zeatin (2.0 and 4.0 mg dm^-3). TDZ (0.2 mg dm^-3) was found to be the most effective cytokinin as it produced multiple shoots in 100 % of the explants from genotypes C235, ICC5166, ICC12269, ICC4951, ICC11531, BG256 and a local cultivar. Shoots were elongated on growth regulator-free medium, and rooted on growth regulator-free medium containing 1/4 MS salts + full vitamins + 3 % sucrose. Plantlets formed were acclimatized for 12 - 15 d in MS medium with a gradual reduction in sucrose concentration and transferred into pots filled with soil and kept in the field; this resulted in more than 70 % survival. The plants developed normally and produced fertile flowers and set seeds. Low temperatures, maximum 19.0 °C, and minimum 8.2 °C, during the first 15 d of transfer favoured survival on transfer to pots.

A novel approach for simultaneous localization of two DNA sequences on plant chromosomes is described. The approach is based on a combined use of primed in situ DNA labelling (PRINS) with fluorescent in situ hybridization (FISH). Traditionally, this has been done using FISH with two probes labelled by two different marker molecules. Compared to this method, the combined PRINS-FISH procedure is faster. Furthermore, because one of the DNA sequences is localized by PRINS with specific primers, only one labelled probe is needed.

A greenhouse pot experiment with different phosphorus supply was conducted to study growth, photosynthesis and free polyamine (PA) content in Plantago lanceolata L. plants in relation to arbuscular mycorrhizal (AM) colonization. Inoculum of Glomus fasciculatum (BEG 53) was used. Inoculated plants had high colonization intensities which were related to the P supply. Non-mycorrhizal (NM) plants showed a typical yield response curve for P availability. Dry masses of mycorrhizal (M) plants were higher at the lowest soil P content than those of NM plants, but the opposite was found at the highest P supply. P contents in M plants were always higher. There were no differences in chlorophyll (Chl) concentrations (except the lowest soil P content) and ratios of variable to maximum Chl fluorescence (Fv/Fm) values between M and NM plants, whereas M plants had higher ratios of leaf area to fresh mass (A/f.m.) at low soil P contents and they had significantly higher CO₂ fixation capacities per unit leaf area. Free putrescine (Put), spermidine (Spd) and spermine (Spm) contents in NM plants were usually highest at the lowest P supply. The ratios of Put/(Spd+Spm) were identical in M and NM leaves. They were significantly higher, however, in NM roots at the two low P doses. It is concluded, that a P nutritional status might exist, below which PA concentrations and ratio are increased drastically, possibly indicating P deficiency or a certain state of plant development with a higher demand for AM symbiosis.

Tissue-specific distribution of basic β-1,3-glucanase (Glu2), basic class II chitinase (Ch2), basic class IV chitinase (Ch4), and acidic class III chitinase (SE2) were examined both in leaves and roots of sugar beet treated with salicylic acid (SA), benzothiadiazole (BTH) and glycine betaine. Protein localization was monitored by immunohistological analysis using specific antibodies. BTH, SA as well as glycine betaine induced both Glu2 and chitinase isozymes in leaves and roots of treated plants. The enzymes were accumulated in extracellular space and cell walls. They were mostly deposited in parenchyma cells of leaves and cortex parenchyma and endodermis of roots. In leaf tissues, BTH and SA induced proteins more effectively than glycine betaine but the effect of glycine betaine in roots was as efficient as BTH and SA. Glycine betaine induced the formation of extracellular globuli containing Ch4. Induced proteins were spatially distributed over the whole plant regardless the site of the inducer application.

An integrated approach has been developed for targeted retrieval of microsatellite markers from selected regions of the field bean (Vicia faba L.) genome. The procedure is based on a combination of advanced physical and genetic mapping techniques and includes the following steps: 1) flow-sorting of metaphase chromosomes, 2) construction of microsatellite-enriched chromosome-specific DNA libraries, 3) isolation of polymorphic microsatellite sequences from the libraries, 4) testing chromosome specificity of the microsatellites using polymerase chain reaction with purified fractions of individual chromosome types, and 5) integration of chromosome-specific markers into a genetic map. Several strategies for isolation of microsatellite clones were tested, including direct screening of non-enriched libraries with single or mixed probes and screening of the libraries after one or two rounds of enrichment. Finally, the usefulness of this approach was demonstrated by the retrieval of novel markers from a selected portion of the largest field been chromosome (No. 1).

The suitability of the fluorescent dye rhodamine 123 for qualitative and quantitative determinations of the electrical potential difference (ΔΨ) in isolated pea (Pisum sativum L.) stem mitochondria was evaluated. A fluorescence quenching of rhodamine 123, as a consequence of dye uptake, occurred following mitochondria energization by both external and internal substrates. This quenching was associated to the generation of ΔΨ, because it was completely released by uncouplers and respiratory inhibitors. The conversion of the proton gradient (ΔpH) into ΔΨ, induced by nigericin or a permeant weak acid (phosphate), increased the quenching. The uptake of the probe was accompanied by 40 % of unspecific binding in coupled, but not in uncoupled, mitochondria. Rhodamine 123 quenching varied linearly with a K+-diffusion potential. ADP induced a transient and cyclic change of fluorescence which was associated to ATP synthesis. Consequently, rhodamine 123 did not influence oxygen consumption by mitochondria in both state 4 and 3, thus indicating that, at the concentrations assayed, the probe was not toxic. It is concluded that rhodamine 123, followed by fluorescence quenching, is a suitable probe to study the energetics of isolated plant mitochondria.

The present investigation has been performed to evaluate nitrate reductase (NR) and nitrogenase activities as well as growth and mineral nutrition of wheat plants grown under drought stress and inoculated with different Azospirillum strains (NR^- and NR⁺). Fresh, dry mass and water content decreased with decreasing soil moisture content, which was accompanied with low soluble sugars and soluble protein content and increase in the total amino acids content. Azospirillum inoculation with either bacterial strain (NR^- and NR⁺) significantly increased the above characteristics even at 40 % moisture content. NR activity decreased in both the shoots and roots by decreasing soil moisture content. NR⁺ strain exhibited increased root NR activity compared with uninoculated plants or inoculated with NR^- strain. However, plants inoculated with NR^-strain increased NR activity in the shoot more than in the root of the same plant and in the shoot of control plants. Inoculation with either NR^- and NR⁺ Azospirillum strains gave higher nitrogenase activity than uninoculated control plants. The low N supply (0.5 mM) did not affect nitrogenase activity. NR^-strain was less effective than NR⁺strain in promoting total N-yield, spike numbers and their mass per pot. Azospirillum inoculation exhibited no significant changes in wheat Mg²⁺ content. However, K⁺ and Ca²⁺ have shown significantly increased values. Azospirillum beneficial effect on plant N balance and growth are most probably composed of multiple mechanisms and beneficial NR is one of them. The importance of Azospirillum NR⁺strains for increasing wheat resistance to water stress is also supported by the obtained data.

Effect of environmental conditions on formation of Nicotiana tabacum L. megagametophyte was studied. It was established, that unfavorable temperatures can specifically modify the structure of embryo sacs (ES). At low temperature (9/5 °C), ES with a reduced number of cells or with egg-like synergide(s) can be formed; at high variable (40/25 °C) or constant (37 °C) temperatures, ES with excessive numbers of cells or with synergide-like egg cells arise. Total frequencies of the changed ES patterns varied from 8 up to 35 % per plant and depended on the plant genotype and conditions of exposure.