At irradiances close to those representing a sunny day, red and green leaves of poinsettia (Euphorbia pulcherrima) showed only minor differences in their photosynthetic capacities despite the strong differences in their pigment composition. However, contrarily to green leaves, red leaves did not show inhibition of photosynthesis at high irradiances, because anthocyanins protected chloroplasts from photoinhibition.

During the entire period of internode growth of Merremia emarginata contents of gibberellic acid (GA₃), phenyl-acetic acid (PAA), indole-3-acetic acid (IAA, free and conjugated) and abscisic acid (ABA, free and conjugated) were estimated by ELISA using polyclonal antibodies raised against each hormones. At the time of internode elongation free auxin content was low and increased with the decrease in the rate of elongation. In contrast, conjugated IAA showed declining trend where free IAA content was remarkably high, suggesting thereby that conjugated IAA might have mobilized during the later phase of internode development. The endogenous GA₃ contents were high as compared to other hormones; however, no significant role of GA₃ was discernible in elongation growth. Conjugated ABA contents remained very low during the elongation growth and increased thereafter.

Withanolides-steroidal lactones, isolated from various Solanaceous plants have received considerable attention due to their potential biological activities. Five selected withanolides (withanone, withaferin A, withanolide A, withanolide B, withanolide E) were identified by HPLC-UV (DAD) - positive ion electrospray ionization mass spectroscopy in Withania somnifera (L.) Dunal cv. WSR plants and tissues cultured in vitro at different developmental phases. Cultures were established from five explants on Murashige and Skoog's medium supplemented with different plant growth regulators. Results suggest that production of withanolides is closely associated with morphological differentiation.

The influence of freezing treatment on plasma membrane (PM) H⁺-ATPase was investigated using plasma membrane vesicles isolated from calluses from Chorispora bungeana Fisch. & C.A. Mey. by the discontinuous sucrose gradient centrifugation. Freezing treatment (-4 °C) for 5 d resulted in significant increases in the ATPase activity and the activity of p-nitrophenyl phosphate (PNPP) hydrolysis, decreases in the K_m for ATP hydrolysis and PNPP hydrolysis, and the shift of optimal pH from 6.5 to 7.0. Also, the activity PNPP hydrolysis was less sensitive to vanadate after freezing treatment compared to control, while the inhibition of ATP hydrolysis by hydroxylamine was more sensitive. In addition, freezing treatment also decreased the activation effects of trypsin on PNPP hydrolysis, but increased the activation effects of lysophosphatidylcholine on ATP hydrolysis. Taken together, these results suggested that PM H⁺-ATPase might play an important role during adaptation to freezing and enhancing the frost hardness in C. bungeana.

A short time effects of 25 and 150 µM Cu²⁺ or 50 µM methyl jasmonate (MJ) on growth of roots and leaves of Phaseolus coccineus, Allium cepa and Zea mays were investigated. Both Cu²⁺ and MJ inhibited root growth. Jasmonate synthesis inhibitors (ibuprofen, IB, salicylhydroxamic acid, SHAM, and propylgallate, PG) partially reversed the inhibitory effect of Cu²⁺ in P. coccineus, but in A. cepa this effect was not clear. Pretreatment with NADPH oxidase inhibitor (20 mM imidazole, IM), and especially ethylene inhibitor (silver thiosulphate, STS) mostly weakened Cu²⁺ effect on root growth in P. coccineus and A. cepa. The growth of P. coccineus leaves also slowed down by Cu²⁺ and this effect was partially ameliorated by IB, PG and IM, and completely by SHAM and STS. In Z. mays the effect of STS was considerably lower than that of PG and SHAM which reversed the effect of Cu²⁺. These results indicate that jasmonate, ethylene and NADPH oxidase activity may be involved in Cu²⁺ inhibitory action on the roots of dicotyledon plants, but in A. cepa only ethylene and NADPH oxidase are involved. However, leaf growth inhibition induced by excess Cu²⁺ is connected in Z. mays especially with jasmonate, and in P. coccineus with ethylene, NADPH oxidase and, to a minor degree, with jasmonate.

We aimed to assess the potential effects of fumigation by methyl salicylate (MeSA) on plant monoterpene production and emissions. We evaluated monoterpene production and emissions both by chromatographic and proton transfer reaction mass spectrometry at the whole plant-and leaf-scales, in MeSa-fumigated (ca. 60 mm³ m^-3 in air) and control (without MeSa fumigation) holm oak (Quercus ilex L.) plants exposed to temperatures ranging from 25 to 50 °C. The MeSa-fumigated plants showed ca. 3-4-fold greater leaf monoterpene concentrations and emission rates than the control plants between the temperatures of 25 to 45 °C.

Seventy genotypes belonging to 7 wild and cultivated Vigna species were genetically differentiated using randomly amplified polymorphic DNA (RAPD), universal rice primer (URP) and simple sequence repeat (SSR) markers. We identified RAPD marker, OPG13 which produced a species-specific fingerprint profile. This primer characterized all the Vigna species uniquely suggesting an insight for their co-evolution, domestication and interspecific relationship. The cluster analysis of combined data set of all the markers resulted in five major groups. Most of the genotypes belonging to cultivated species formed a specific group whereas all the wild species formed a separate cluster using unweighted paired group method with arithmetic averages and principle component analysis. The Mantel matrix correspondence test resulted in a high matrix correlation with best fit (r = 0.95) from combined marker data. Comparison of three-marker systems showed that SSR marker was more efficient in detecting genetic variability among all the Vigna species. The narrow genetic base of the V. radiata cultivars obtained in the present study emphasized that large germplasm collection should be used in Vigna improvement programme.

Embryonal axis explants from 2-d-old in vitro germinated seeds were used to induce multiple shoot production. The combination of 4.44 µM BA and 1.59 µM NAA in MS medium triggered the initiation of adventitious shoot buds. The explants with shoot buds produced maximum number of shoots (10.6 per explant) in MS medium supplemented with 4.44 µM BA and 0.065 mM L-glutamine in three successive transfers. The elongated shoots were rooted on MS medium with 4.92 µM IBA. Rooted plants were transferred to soil with a survival rate of 65 %.

Changes in endogenous cytokinin (CK) content and cytokinin oxidase/dehydrogenase activity (CKX) in response to gibberellic acid (GA₃) in two pea cultivars with different life span were assessed. The control leaves of cv. Scinado, which developed faster, had higher initial cytokinin content and lower CKX activity, while opposite trend was observed in cv. Manuela with longer life span. Increased CKX and decreased CK content were detected in leaves of cv. Scinado after treatments with 0.5, 1 and 5 µM GA₃. Changes in CK content and CKX activity in GA₃-treated cv. Manuela leaves were reciprocal to those in cv. Scinado. CK content and CKX activity in roots were not significantly influenced by the application of GA₃. The slight repression of CKX activity in some of the root samples was accompanied by increased isopentenyladenine and isopentenyladenine riboside content. Obtained results suggest that CKX was responsible for the changes in endogenous cytokinin pool in GA₃-treated plants and most probably this enzyme represents an important link in GA/cytokinin cross talk.

In fern (Anemia phyllitidis) gametophytes cellulose in the walls of the antheridial zone cells which was organized in clusters and spots was transformed via dispersed form to fibrillar arrangement (layered in oblique and perpendicular array in relation to the transverse direction of cell expansion) during antheridiogenesis induced by gibberellic acid (GA₃) and/or enhanced by 1-aminocyclopropane-1-carboxylic acid (ACC). In the ACC-treated gametophytes, where antheridia were not induced, the cellulose was arranged in the same manner. Aminooxyacetic acid (AOA), which inhibits antheridiogenesis and development of fern gametophytes, produced in the cell walls both random and longitudinal type of organization of cellulose microfibrils, however, in the GA₃/AOA-treated plants the oblique type was also observed. The total numbers of cells with perpendicular and/or oblique type of cellulose microfibrils in the GA₃-, GA₃/ACC-and GA₃/AOA-treated gametophytes corresponded to the average number of antheridia formed. Moreover, it was found that the extracts from the gametophytes treated with GA₃ or with the mixture of GA₃ and ACC contained significantly less soluble sugars but more α-amylase-and endoglucanase-released sugars than the extracts from the gametophytes of the other series. Thin layer chromatography of the samples from the cell wall extracts hydrolyzed by endoglucanase contained xylose and cellobiose which suggested that these sugars built the xyloglucans, hemicellulose polymers responsible for tethering of walls of fern gametophyte cells like in higher plants.

A lot of pepper seeds having 87 % germination were subjected to X-ray inspection using a non lethal dose of radiation. Seeds with less than 2.7 % (on the basis of total seed area) of free space area, i.e. the spaces between embryo and endosperm, were classified as highly viable seeds (97-100 % germination) with the lowest level of abnormal seedlings. Seeds X-ray classified as good were subjected to a computerised image analysis to study seed imbibition and radicle elongation. The patterns of seed area increase, chosen as the most accurate indicator of seed swelling, resembled the triphasic curve of water uptake. The first phase was completed at 9 h followed by a second phase that varied widely in time until completion of germination between 52 and 96 h. The proportion of seeds with radicle protrusion between 52-56 h and 64-72 h assessed with the image analysis was significantly higher than that recorded using a conventional germination test. In addition, the rate of increase of seed area during the third phase of imbibition, mostly due to protrusion of the radicle tip and its growth, was highly correlated with the corresponding radicle elongation rate.

Two vacuolar green fluorescent proteins (GFP) were stably inserted in Nicotiana tabacum and Nicotiana benthamiana genome, with unexpected difficulties, and compared with A. thaliana cv. Wassilewskaja transgenic plants expressing the same constructs. GFP fluorescence was strong in all tissues of A. thaliana but it was barely visible in Nicotiana. Confocal microscopy analysis revealed a variable distribution of the marker in those cells where GFP fluorescence was visible. The role of light dependent proteases was the variable pointing out more inter-species diversity. GFPs degradation was much higher in Nicotiana spp. than in A. thaliana. The version of GFP used appeared not to be a good vacuolar marker for Nicotiana differentiated tissues, although it can efficiently label vacuoles in protoplasts or calli. Nevertheless the sensitivity of the reporter protein can be used as an indicator of hidden characteristics of the plant vacuoles, revealing differences otherwise invisible. One of the markers in our system, GFP-Chi, evidenced a clear morphological difference in the vacuolar system of guard cells of the three species.

We report here the development of transgenic tobacco plants with thaumatin gene of Thaumatococcus daniellii under the control of a strong constitutive promoter-CaMV 35S. Both polymerase chain reaction and genomic Southern analysis confirmed the integration of transgene. Transgenic plants exhibited enhanced resistance with delayed disease symptoms against fungal diseases caused by Pythium aphanidermatum and Rhizoctonia solani. The leaf extract from transgenic plants effectively inhibited the mycelial growth of these pathogenic fungi in vitro. The transgenic seeds exhibited higher germination percentage and seedling survival under salinity and PEG-mediated drought stress as compared to the untransformed controls. These observations suggest that thaumatin gene can confer tolerance to both fungal pathogens and abiotic stresses.

Total protein patterns were studied in the course of development of pea somatic embryos using simple protocol of direct regeneration from shoot apical meristems on auxin supplemented medium. Protein content and total protein spectra (SDS-PAGE) of somatic embryos in particular developmental stages were analysed in Pisum sativum, P. arvense, P. elatius and P. jomardi. Expression of seed storage proteins in somatic embryos was compared with their accumulation in zygotic embryos of selected developmental stages. Pea vegetative tissues, namely leaf and root, were used as a negative control not expressing typical seed storage proteins. The biosynthesis and accumulation of seed storage proteins was observed during somatic embryo development (since globular stage), despite of the fact that no special maturation treatment was applied. Major storage proteins typical for pea seed (globulins legumin, vicilin, convicilin and their subunits) were detected in somatic embryos. In general, the biosynthesis of storage proteins in somatic embryos was lower as compared to mature dry seed. However, in some cases the cotyledonary somatic embryos exhibited comparatively high expression of vicilin, convicilin and pea seed lectin, which was even higher than those in immature but morphologically fully developed zygotic embryos. Desiccation treatments did not affect the protein content of somatic embryos. The transfer of desiccated somatic embryos on hormone-free germination medium led to progressive storage protein degradation. The expression of true seed storage proteins may serve as an explicit marker of somatic embryogenesis pathway of regeneration as well as a measure of maturation degree of somatic embryos in pea.

The present study investigated whether, depending on the abscisic acid (ABA) concentration, phospholipase C (PLC) would be implicated within a Ca²⁺ mobilizing pathway that would regulate stomatal aperture under standard watering conditions. Among Al sensitive mutants the als1-1 mutant of Arabidopsis thaliana (L.) Heynh. (Columbia-4 ecotype) was selected for a pharmacological approach of stomatal closing in leaf epidermal peels induced by 3, 20 or 30 µM ABA. Comparison with the wild type (WT) revealed that, exclusively in the als1-1 mutant, the stomatal response to 3 or 20 µM ABA was inhibited by about 40 %, whereas the stomatal response to 30 µM ABA and the wilting response to drought were unaffected. In WT, the Ca²⁺ buffer EGTA and the PLC inhibitor, 1-[6-[[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), specifically inhibited by about 70 and 40 %, respectively, the response to 3 or 20 µM ABA, while the Ca²⁺ buffer 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) inhibited by about 70 % the response to 3, 20 or 30 µM ABA. EGTA, BAPTA and U73122 did not inhibit the part of the response to 3 or 20 µM ABA that was unaffected by the als1-1 mutation. Together, these results showed that ABA closes the stomata through two different Ca²⁺ mobilizing pathways. Since PLC could be indirectly deactivated in the als1-1 mutant, these results might suggest that, under sufficient water supply, PLC-mediated Ca²⁺ mobilization is needed for the regulation of stomatal aperture by endogenous ABA resting at concentrations below a drought-specific threshold value.

The freezing sensitivity in the gi-3 mutant (an allele of the gigantea mutant) was associated with a constitutive reduction in soluble sugar content. Although sugar accumulation was evident in wild-type plants in response to cold treatment, the gi-3 mutant showed a constitutive reduction in soluble sugar content. There were no significant differences in the proline content and the transcript levels of cold-responsive gene RD29A and abscisic acid-responsive gene RAB18 between the wild type and the gi-3 mutant in response to cold treatment. These results suggest that freezing sensitivity in the gi-3 mutant is associated with sugar deficiency.

A simple and reliable protocol for regeneration of okra through somatic embryogenesis from suspension cultures has been developed. Embryogenic callus was obtained from hypocotyl explants cultured on media with Murashige and Skoog (MS) salts, Gamborg (B5) vitamins, 2.0 mg dm^-3 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 mg dm^-3 naphthaleneacetic acid (NAA), 25 mg dm^-3 polyvinylpyrrolidone and 30 g dm^-3 sucrose. More number and high frequency of healthy embryoids appeared individually in suspension culture containing MS salts, B5 vitamins, 2.0 mg dm^-3 2,4-D and 1.0 mg dm^-3 kinetin. Formation of cell clusters from the single cells was clearly noticed during ontogeny. Matured embryos at the cotyledonary stage were transferred to agar solidified medium for germination. The best conversion of embrya into plantlets (67.3 %) was recorded on media with half strength MS salts, B5 vitamins, 0.2 mg dm^-3 benzylaminopurine (BAP) and 0.2 mg dm^-3 gibberellic acid (GA₃). The plantlets were transferred to soil and hardened in the plastic pots. After proper acclimatization, the plantlets regenerated through somatic embryogenesis were compared to seed grown plants to observe any variation.

The effect of elevated carbon dioxide concentration on the changes in the biomass, photosynthesis and nutrient composition was investigated in two leafy vegetables. Spinach (Spinacia oleracea L.) and fenugreek (Trigonella foenum-graecum L.) plants were grown in open top chambers under either ambient (ACO₂, 350 ± 50 µmol mol^-1) or elevated (ECO₂, 600 ± 50 µmol mol^-1) CO₂ concentration and analyzed 40, 60 and 80 days after exposure. The plants grown in ECO₂ had higher net photosynthetic rate and lower stomatal conductance when compared with the plants grown in ACO₂. ECO₂ also changed the nutrient composition: a lower N, Mg and Fe contents and higher C and Ca contents were observed in the leaves of plants exposed to ECO₂ than in those grown at ACO₂.

Shoot apices of Pinus roxburghii Sarg were cultured on Murashige and Skoog's medium (MS) supplemented with cytokinins [6-benzyladenine (BA), kinetin and N-benzyl-9-(2-tetrahydropyranyl) adenine (BPA)] alone and in combination with auxin, α-napthaleneacetic acid (NAA). Of the three cytokinins tested at varying concentrations, medium supplemented with 10 µM BA was found optimal in respect of explant responsiveness (97.22 %) and average number of buds induced per explant (7.42). The concentration of cytokinins in the induction medium had a profound effect on rate of elongation of induced buds on MS basal medium containing 0.5 % activated charcoal. Further, shoots induced on lower concentrations of BA increased up to 2.4 times in length in 4 weeks. Decapitation of the explant enhanced the rate of axillary bud elongation. Proliferating shoot cultures were established by sub-culturing the axillary shoots on MS supplemented with 10 µM BA. Shoots 2-3 cm in length were suitable for culturing as more buds were induced on them compared to longer or shorter shoots. Root primordia were induced on 70.83 % shoots when transferred to 1/2 MS medium supplemented with 5.0 µM NAA. Elongation of root primordia (60 %) was achieved in liquid 1/2 MS basal medium. The plantlets were successfully transferred to soil after hardening; the time period from initiation of shoot buds to transplantation being 20-22 weeks.

The antioxidant defense system in three ecotypes of reed (Phragmites communis Trin.), swamp reed (SR), dune reed (DR), and heavy salt meadow reed (HSMR), from northwest China were investigated. The HSMR possessed the highest ratio of ascorbate (ASC)/dehydroascorbate (DHA) and activities of superoxide dismutase (SOD) and catalase among the three reed ecotypes, whereas, the DR exhibited the highest ratio of glutathione/glutathione disulfide and activities of ASC peroxidase (APX) and DHA reductase. Malondialdehyde and hydrogen peroxide contents were highest in HSMR, intermediate in SR, and lowest in DR. In addition, different isoenzymes of glutathion reductase, APX, SOD and DHA were also observed in three reed ecotypes.

Inter simple sequence repeat (ISSR) marker assay was employed to validate the genetic fidelity of Swertia chirayita plantlets multiplied in vitro by axillary multiplication upto forty-two passages. Sixteen ISSR primers generated a total of 102 amplicons among the tissue-cultured plants. Forty-eight amplicons were amplified in the outlier (a Swertia species). The outlier (negative control) was employed to rule out the possibility that the invariant fingerprint was due to chance alone and that the ISSR technique employed was not discriminatory enough to detect the off-types. A homogenous amplification profile was observed for all the micropropagated plants. The results confirmed the clonal fidelity of the tissue culture-raised S. chirayita plantlets and corroborated the fact that axillary multiplication is the safest mode for multiplication of true to type plants.

The heat stress-induced dehydrin proteins (DHNs) expression and their relationship with the water relations of sugarcane (Saccharum officinarum L.) leaves were studied. Sugarcane seedlings were subjected to heat stress (day/night temperature of 40/35 °C) under relative humidity 60/65 % to avoid aerial desiccation and determinations made at 4, 12, 24, 36, 48, 60 and 72 h. The leaves showed a sharp decline in the water and osmotic potentials, and relative water content during first 12 h of heat stress, but a regain in their values in 24 h. The pressure potential (ψ_p) decreased initially but increased later and approached control leaves. The increase in ψ_p was tightly correlated to the accumulation of free proline, glycinebetaine and soluble sugars, indicating their possible involvement in the osmotic adjustment under heat stress. Immunological detection revealed the expression of three DHNs with an apparent molecular mass of 21, 23 and 27 kDa under heat stress (48 to 72 h) and their expression was independent of the changes in the water relations of leaves.

We examined the effects of repeated inbreeding on fitness components of the long-lived perennial Succisa pratensis (Dipsacaceae). Plants from six populations differing in size were used to establish lines with expected inbreeding coefficients f of 0, 0.5 and 0.75. The effects of different inbreeding levels were measured for seed set, seed mass, percentage germination and seedling relative growth rate. Seed set decreased following one generation of inbreeding and seedling growth rate decreased after two generations of inbreeding. Our study indicated that the mutational load is difficult to purge and that continued inbreeding tends to affect important traits in S. pratensis. Although the partial dominance hypothesis for inbreeding depression seems to account for the results, the overdominance hypothesis cannot be ruled out completely. Overall, we conclude that the response of a long-lived plant, such as S. pratensis, to repeated inbreeding does not differ from that of other plant species with shorter life spans, surely because the mechanisms that account for inbreeding depression are universal for all plant species.

Wheat (Triticum aestivum L. Sakha 61) grains were exposed to microwave radiation of a wavelength 2.85 cm and frequency 10.525 GHz for 15, 45, or 75 min. The exposed grains were germinated and then harvested after 7 and 14 d. While 15-and 45-min exposure of the grains stimulated seedling shoot length and fresh and dry masses, the exposure for 75 min had no pronounced effects. 15 and 75-min irradiation increased succulence and pigment contents in 7-and 14-d-old seedlings. While the ratios chlorophyll (Chl) a/b, carotenoids (Car)/Chl were higher in 7-d-old seedlings than in the control ones for all doses, they decreased in 14-d-old seedlings. The microwave radiation increased protein and amino acid contents, but decreased the contents of saccharides, nucleic acids, and phenolic compounds. Low dose (15 min) stimulated proline synthesis, whereas the other doses showed negative effect on its production.

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.