Suspension cultures of Habanero pepper (Capsicum chinense Jacq.) were exposed to salicylic acid or methyl jasmonate to change secondary metabolism. Both treatments led to the accumulation of capsaicinoids and their late biosynthetic intermediate, vanillin. Both elicitors had a positive effect on the activities of phenylalanine ammonia lyase and coumarate O-methyltransferase, but none of them represented the main limiting step for capsaicinoid accumulation since vanillin contents were two orders of magnitude higher than those of capsaicinoids.

Morphological and molecular characteristics were studied in six wild species of Passiflora. There were statistically significant differences among these six species for all characteristics studied. Intra-specific variability was observed for number of flowers, number of fruits, number of seeds, fruit length, fruit width and leaf area. Cluster analysis using morphological data showed three groups: 1) P. palmeri var. sublanceolata, P. morifolia and P. foetida var. foetida, 2) P. coriacea and P. micropetala, and 3) P. suberosa. The dendrogram constructed using randomly amplified polymorphic DNA (RAPD) data showed six different groups for each species. The genetic distances among the 24 accessions ranged from 0.05 (between P. morifolia accessions P1 and P3) to 0.95 (P. coriacea accession 31 and P. palmeri var. sublanceolata accession 49). The species showed high morphological and molecular inter- and intraspecific variability.

In order to investigate the function of chloroplast ascorbate peroxidase under temperature stress, the thylakoid-bound ascorbate peroxidase gene from tomato leaf (TtAPX) was introduced into tobacco. Transformants were selected for their ability to grow on medium containing kanamycin. RNA gel blot analysis confirmed that TtAPX in tomato was induced by chilling or heat stress. Over-expression of TtAPX in tobacco improved seed germination under temperature stress. Two transgenic tobacco lines showed higher ascorbate peroxidase activity, accumulated less hydrogen peroxide and malondialdehyde than wild type plants under stress condition. The photochemical efficiency of photosystem 2 in the transgenic lines was distinctly higher than that of wild type plants under chilling and heat stresses. Results indicated that the over-expression of TtAPX enhanced tolerance to temperature stress in transgenic tobacco plants.

Embryogenic cell suspension cultures of Santalum album were transformed with Agrobacterium tumefaciens harboring pD35SHER plant expression vector having hepatitis B small surface antigen (HBsAg) with a C-terminal ER retention signal. The transformed colonies were selected on culture medium supplemented with kanamycin and subsequently the transgenic nature of these colonies was confirmed by PCR analysis. The expression of HBsAg was confirmed by RT-PCR analysis and Western blot analysis and the expression was quantified using monoclonal antibody-based ELISA. Cell suspension cultures were initiated from the colony with expression of 11.09 μg(HBsAg) g^-1(f.m.). To further increase the expression of HBsAg, transgenic S. album suspensions were cultured on media with various medium additives and cells growing in medium with 30 mM trehalose showed the expression of 19.95 μg(HBsAg) g^-1(f.m.).

Zephyr lily (Zephyranthes grandiflora), an important ornamental plant has been micropropagated in vitro after controlling microbial contamination by a pretreatment with 0.2 % Bavistin and 0.1 % Pantomycin for 4 h before final sterilization with 0.1 % mercuric chloride. In 67 % of the sterile cultures, 11 shoots on average were regenerated directly from basal half of bulb scales in Murashige and Skoog (MS) medium containing 3 % sucrose and 2 mg dm^-3 benzylaminopurine (BAP). Shoots emerged in bunches on a basal achlorophyllous bulbous part. Combination of 2 mg dm^-3 BAP with 1 mg dm^-3 gibberellic acid (GA₃) enhanced shoot growth. Stout roots (maximum of 5-6 per shoot) were developed in presence of 1 mg dm^-3 indole-3-butyric acid (IBA). Micro-bulbs showed potential of regeneration and could be used as secondary explants. The morphologically identical plants derived by in vitro propagation were genetically identical as shown by PCR based ISSR marker analysis of genomic DNA.

We isolated a gene encoding for farnesyl diphosphate synthase (FPS) from Panax ginseng, a species that produces a large quantity of triterpene saponins such as ginsenosides. The deduced amino acid sequence of PgFPS was 77, 84 and 95 % identical to those of Arabidopsis, Hevea, and Centella. Southern blot analysis indicated that P. ginseng contained more than two genes encoding for FPS. When the cDNA of PgFPS was expressed in Escherichia coli, the recombinant enzyme, purified with a His-tag column, was found to possess FPS activity. When cultures of ginseng hairy root were treated with 0.1 mM methyl jasmonate (MJ), PgFPS mRNA was detected within 12 h of the treatment, and achieved maximum after 24 h. Also FPS activity in the hairy root cultures after 12 h of MJ treatment was higher than that of the control.

The transformation of potato (Solanum tuberosum L. cv. Désirée) was extended by the Agrobacterium-mediated biolistic method. Using this approach transgenic shoots could be obtained at a similar frequency to that achieved through conventional biolistics. Leaves from shoot cultures were bombarded with gold particles coated in Agrobacterium tumefaciens cells harboring a binary plasmid encoding three genes of interest in the T-DNA. Nine shoots were obtained from 20 shots, with selection of transgenic shoots on a series of media containing progressively increasing concentrations of hygromycin from 5 to 20 mg dm^-3.

A novel protocol for plant regeneration from cotyledon explants of eggplant (Solanum melongena) reducing concentration of sucrose was established. The most efficient bud induction medium consisted of Murashige and Skoog (MS) medium supplemented with 2.0 mg dm^-3 zeatin, 0.1 mg dm^-3 indoleacetic acid and 10 g dm^-3 sucrose. After 15 d, the shoot buds were fragmented and transferred to the shoot elongation MS supplemented with 1.0-2.0 mg dm^-3 gibberellic acid and 4.0-8.0 mg dm^-3 AgNO₃, which promoted shoots elongation. The genetic stability of the regenerated plants was analyzed by flow cytometry, RAPD and SSR molecular markers. The results indicated that almost no somaclonal variation was detected among the regenerants.

Content of polyamines and activities of antioxidative enzymes in response to stripe rust disease caused by Puccinia striiformis have been studied in two wheat (Triticum aestivum L.) cultivars PBW 343 (resistant) and HD 2329 (susceptible). Various infection stages ranging from traces to 100 % were collected. Infection leads to stimulation of peroxidase (POD), superoxide dismutase (SOD), catalase, diamine oxidase and polyamine oxidase activities along with increase in putrescine, spermidine and spermine content while ascorbate, tocopherol and chlorophyll content decreased in HD 2329 and no visible symptoms appeared in PBW 343. Histochemical localization pattern of POD and SOD activities revealed correlation with lignin deposition in cell walls.

The effect of various hormonal combinations on regeneration of shoots and roots from meristem-derived callus of Crocus sativus L. and activities of antioxidant enzymes have been studied. The most efficient regeneration occurred with 1.0 mg dm^-3 1-naphthaleneacetic acid (NAA) + 1.0 mg dm^-3 thidiazuron and 1.0 mg dm^-3 NAA + 2.0 mg dm^-3 kinetin. For sprouting, regenerated shoot were subcultured on Murashige and Skoog medium containing 1.0 mg dm^-3 NAA + 1.0 mg dm^-3 benzylaminopurine (BAP). Protein content and superoxide dismutase activity decreased in regenerated shoots and roots and increased in sprouting shoots, while catalase (CAT), peroxidase (POX) and polyphenol oxidase (PPO) activities increased during organogenesis and decreased in sprouting shoots. High CAT and PPO activities were detected in regenerated roots, whereas high POX activity was observed in regenerated shoot.

An important aspect of heat-shock response of lupin (Lupinus albus cv. Rio Maior) is the formation of cytoplasmic granular aggregates, called heat-shock granules (HSGs). In this study, two-dimensional electrophoresis (2-DE) was used to detect the component proteins of HSG complexes formed in vivo. Evaluation of 2-DE revealed differential expression of several proteins under heat shock conditions when compared with control. Among them, small heat-shock proteins (sHSPs) of 15 to 30 kDa were found to be the major representative proteins along with other proteins of relative molecular mass ranging from 36 to 45 kDa and above.

Bacillus cereus C1L was demonstrated to induce systemic resistance in Lilium formosanum against leaf blight caused by Botrytis elliptica. Suppression subtractive hybridization library of L. formosanum triggered by B. cereus C1L were screened and 3 differentially expressed genes were identified. Based on sequence analysis, these genes encoding putative glycine-rich protein, metallothionein-like protein, and PsbR protein of photosystem 2, were designated LfGRP1, LfMT1, and LfPsbR, respectively. The results of Northern blot analysis showed that expressions of LfGRP1, LfMT1 and LfPsbR increased in response to B. elliptica infection. On the other hand, expression of LfMT1 increased but expressions of LfGRP1 and LfPsbR decreased when the rhizosphere of L. formosanum was drenched with suspension of B. cereus C1L with or without subsequent challenge with B. elliptica on lily leaves. Similar expression profiles of homologues of LfGRP1, LfMT1, and LfPsbR (named LsGRP1, LsMT1, and LsPsbR, respectively) were presented in Lilium oriental hybrid Star Gazer. In addition, application of the photosynthetic inhibitor, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, on the leaves reduced disease severity and expressions of LsGRP1 and LsPsbR just as that in response to B. cereus C1L treatment.

Measurements of stem respiration were conducted for a period of four years (1999-2002) in 14-year old Norway spruce (Picea abies [L.] Karst) trees exposed to ambient (CA) and elevated CO₂ concentration (CE; ambient plus 350 μmol mol^-1). Stem respiration measurements of six trees per treatment were carried out 2-3 times per month during the growing season. Stem respiration in CE treatment was higher (up to 16 %) than in CA treatment. Temperature response of stem respiration (Q₁₀) for the whole experimental period ranged between 1.65-2.57 in CA treatment and 2.24-2.56 in CE treatment. The mean stem respiration rate normalized to 10 °C (R₁₀) in CA and CE treatments ranged between 1.67-1.95 and 2.19-2.72 μmol(CO₂) m^-2 s^-1, respectively. Seasonal variations in stem respiration were related to temperature and tree growth.

An efficient and reproducible in vitro plant regeneration system from shoot apices was developed in Jatropha curcas. Benzylaminopurine (BAP; 2.5 μM) was most effective in inducing an average of 6.2 shoots per shoot apex. Incorporation of gibberellic acid (GA3; 0.5 μM) to basal medium was found essential for elongation of shoots. The BAP-habituated mother explants continuously produced shoots during successive subculture without any loss of morphogenic potential. The shoots rooted efficiently on half-strength MS medium. The rooted plantlets were acclimatized with more than 98 % success and the plants transferred to soil:compost in nursery showed no sign of variation compared to the seed-grown plants. The whole process of culture initiation to plant establishment was accomplished within 5-6 weeks. A genetic transformation system in J. curcas was established for the first time, using bombardment of particles coated with plasmid pBI426 with a GUS-NPT II fusion protein under the control of a double 35S cauliflower mosaic virus (CaMV) promoter. The β-glucuronidase (GUS) activity in J. curcas shoot apices was significantly affected by the gold particle size, bombardment pressure, target distance, macrocarrier travel distance, number of bombardments, and type and duration of osmotic pre-treatment. The proliferating bombarded shoot apices were screened on medium supplemented with 25 mg dm^-3 kanamycin and surviving shoots were rooted on medium devoid of kanamycin. The integration of the transgene into genomic DNA of transgenic plants was confirmed by PCR and Southern blot hybridization. The transgenic plants showed insertion of single to multiple copies of the transgene.

Short-term treatment of potato (Solanum tuberosum L.) tuber discs with CdCl₂ solution elevated both the StPCS1 transcript level, phytochelatin synthase (PCS) activity and contents of phytochelatin (PC) and abscisic acid (ABA). Similar effects but less marked were noticed after treatment of tuber tissues with ABA solution. Cd-treatment increased also cysteine (CYS) content but did not change glutathione content. If ABA treatment preceded Cd-treatment, the elevation of CYS and PC contents were limited. The data suggest the participation of ABA in the regulation of PCS.

The effects of cadmium and zinc on growth and mineral distribution in Populus tremula × P. alba genotype 717-1B4 were investigated. Exposure to 360 mg(Cd) kg^-1(soil) resulted in accumulation of Cd in all organs and inhibition of primary and secondary growth as well as of the net photosynthetic rate. No growth inhibition occurred under zinc exposure. Cd was mainly stored in the woody parts of stem, whereas zinc was preferentially localized in the leaves. Cd treatment also altered distribution of Zn²⁺, Ca²⁺, Mg²⁺, K⁺, and Fe²⁺ in different organs.

Hydraulic conductance of stem and petioles increased in response to an increase in xylem sap ion concentration, and decreased in response to a decrease in the ion concentration in six temperate deciduous tree species. The ion sensitivity of hydraulic conductance of stem and petioles was higher than the ion sensitivity of the stem alone. The ion sensitivity was lowest in the earliest developmental stages of the xylem, and had a seasonal maximum in the second half of summer. The ion sensitivity was highest in slow-growing species and lowest in fast-growing species. The ion sensitivity correlated negatively with mean radius of xylem conduits, hydraulic conductance of stem and petioles, hydraulic conductance of leaf laminae, and stomatal conductance, and positively with response of the hydraulic conductance of leaf laminae to HgCl₂, and stomatal response to a decrease in leaf water potential or abscisic acid. It was concluded that the high ion sensitivity of xylem hydraulic conductance is a relevant characteristic of slow growth and a conservative water use strategy.

In the present study, we report the cloning and sequence characterization of two isoforms of osmotin, an antifungal PR-5 gene homologue, from a salicylic acid-induced subtracted cDNA library earlier generated in Piper colubrinum. The larger form of the gene is 693 bp long, encoding a 21.5 kDa protein. The smaller form comprises a 543 bp long coding sequence which code for a protein of 16.4 kDa. A notable feature of the smaller form was a prominent internal deletion of 150 bp besides certain point mutations. Cloned isoforms of osmotin from resistant species could be candidates for molecular breeding for the improvement of black pepper as well as candidates for the study of structure based mechanism of antifungal activity attributed to PR-5 family.

The aim of the study was to examine the effect of exogenous 24-epibrassinolide on its uptake and content of endogenous brassinosteroids in wheat seedlings. 24-Epibrassinolide was applied at two concentrations (0.1 and 2.0 μM) and in three different methods: by soaking seeds, by drenching and by spraying plants. Brassinosteroids were determined by high-performance liquid chromatography combined with electrospray mass spectrometry. Three important brassinosteroids, 24-epibrassinolide, brassinolide and castasterone, were detected in the wheat leaves, but their contents varied with leaf insertion and plant age. Increased 24-epibrassinolide content in the leaf tissue was found when this hormone was applied by soaking or drenching. Additionally the seed treatment influenced brassinosteroid balance in seedlings. The growth response of wheat seedlings treated with 24-epibrassinolide has been also investigated.

Susceptibility of C. rubrum to Agrobacterium-mediated transformation was demonstrated by inoculating the petioles of in vitro grown plants with A. rhizogenes strain A4M70GUS. Hairy roots were produced in 8 % of explants. They were isolated and maintained on plant growth regulator-free solid or liquid half-strength Murashige and Skoog medium for two years. Hairy root fresh mass increased 30 - 90 folds when grown in liquid medium, which was superior to solid medium, where most of the hairy roots produced calli. When these calli were grown on medium supplemented with 0.5 mg dm-3 thidiazuron, embryo-like structures were obtained. Transgenic status of long-term callus and hairy root cultures was confirmed by histochemical GUS assay, by PCR specific to the uidA, rolA&B and ags genes and by Southern hybridization.

Tetraploidy was induced in vitro in mat rush (Juncus effusus L.) cultivar Nonglin-4 by exposure to colchicine (0, 50, 100 and 500 mg dm^-3) for 6, 12 and 24 h. Flow cytometric analysis was used to confirm the ploidy level. Anatomical and ultrastructural analyses at cellular and subcellular levels in tetraploid and diploid control plants revealed differences between diploid and tetraploid plants. The leaf epidermis had larger stomata but lower stomatal density in tetraploid plants. In addition, mesophyll cells in tetraploid plants appeared more compact and showed less intercellular spaces along with increased size of vascular bundles. However, a significant reduction of chlorophyll content was observed in tetraploid plants that might be the result of structural modification in the lamellar membranes of chloroplasts.

An efficient protocol for encapsulation of nodal segments of Vitex negundo L. has been developed for the production of non-embryogenic synthetic seeds. The encapsulations of nodal segments were significantly affected by the concentrations of sodium alginate and calcium chloride. A 3 % Na₂-alginate with 100 mM CaCl₂ has been found to be optimum concentration for the production of uniform synthetic seed. For germination, the synseeds were cultured on Murashige and Skoog (MS) basal medium supplemented with kinetin (KIN) and α-naphthalene acetic acid (NAA) either singly or in various combinations. MS medium containing 2.5 μM KIN in combination with 1.0 μM NAA was found to be the optimum for maximum (92.6 ± 3.71 %) plantlet conversion frequency. Well developed regenerated plantlets were hardened, acclimatized and established in field, where they grew well without any detectable variation.

We aimed to study the protection of wheat plasma membrane (PM) under cold stress (0-2 °C) by the overaccumulation of glycine betaine (GB). For this, we used wild-type winter wheat (Triticum aestivum L.) cv. Shi 4185 (WT) and 3 transgenic lines (T1, T4, and T6) expressing the BADH gene isolated from Atriplex hortensis L. Under cold stress, the transgenic lines with higher GB content maintained better membrane integrity and higher plasma membrane H⁺-ATPase activity than WT. In these transgenic lines, ROS production and membrane lipid peroxidation were lower, while antioxidative enzyme activities and compatible solute contents were higher in comparison with WT. This may be attributable to their enhanced cold-stress tolerance mediated by GB overproduction.

Two synthetic populations (Syn-5 and Syn-7), four inbred lines and four landraces of alfalfa (Medicago sativa L.) were studied for genetic relatedness. These forms were characterized by different amount of seeds set and green mass yield. Two primer pairs of specific nucleotide sequences of mitochondrial DNA isolated from soybean were used. The mtDNA revealed the existence of significant polymorphism among the investigated forms of alfalfa. The genetic similarity (Dice coefficient) among studied forms of alfalfa ranged from 20.1 to 96.1. The greatest resemblances were noticed between D₅ inbred line and the population of Syn-5. The lowest resemblances were noticed between Syn-7 and E_1/2. The UPGMA dendrogram split investigated forms of alfalfa into two groups: first group include three landraces, the second consist of the rest analyzed forms. There are two landraces distinct with the highest seed set and yield of green mass: one in the first group, another one in the second group.

The present study describes the assessment of genetic diversity and relationships among 79 Job's tears (Coix lacrymajobi L.) accessions collected from China and Korea using 17 microsatellite markers. A total of 57 alleles were detected with an average of 3.4 alleles per locus. A high frequency of rare alleles (36.3 %) was observed within the collection. Values for observed (H_O), expected heterozygosity (H_E) and Shannon's information index (I) within the analysis ranged from 0.00 (GBssrJT183) to 0.81 (GBssrJT130), from 0.01 (GBssrJT170) to 0.65 (GBssrJT130) and from 0.034 (GBssrJt170) to 1.13 (GBssrJT130), respectively. The locus GBJT130 was the most informative marker with the highest values for observed and effective alleles as well as for H_O, H_E and I. Based on the UPGMA algorithm, the majority of the Chinese accessions grouped in one cluster, whereas all the Korean accessions grouped together in a separate cluster, indicating that Chinese accessions are genetically quite distinct from Korean accessions. No relation between genetic relatedness among Job's tears accessions and their place of collection was observed. Chinese accessions exhibited greater within population polymorphism (P = 95 %, H_E = 0.30, I = 0.52) than the accessions from Korea (P = 68 %, H_E = 0.13, I =0.24), indicating their potentiality as a reservoir of novel alleles for crop improvement. However, in general the low diversity within each population indicates a narrow genetic base within our collection.

The present study reports an efficient protocol for indirect shoot organogenesis and plantlets regeneration of Withania somnifera (L.) Dunal. Leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of 6-benzylaminopurine (BAP) and indole-3-acetic acid (IAA). The highest callus induction rate (89.5 %) and shoot regeneration rate (92 %) were obtained when 2 mg dm^-3 BAP was combined with 0.5 mg dm^-3 IAA. Three major withanolides (withaferine A, 12-deoxywithastramonolide and withanolide A) were investigated in different plant organs from in vitro and greenhouse grown plants. Leaves contained higher contents of withanolides and phenolics than roots or stems, whereas roots contained the highest contents of flavonoids and polysacharides. In vitro grown plants contained greater contents of phenolics, flavonoids and polysaccharides while lower contents of withanolides than greenhouse grown plants.

Four different markers [random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP), and selective amplified microsatellite polymorphism length (SAMPL)] were applied for evaluating somaclonal variation of micropropagated genotypes of stone pine (Pinus pinea L.). The total number of primers tested was 130, with 223 combinations assayed. A high number of them amplified successfully (178), representing 79.82 % of the total, and the average number of amplified fragments ranged from 2.47 (ISSR) to 65.76 (SAMPL). Based on internal controls, no problem of reproducibility was detected. Almost no somaclonal variation was detected within the clones. Of the tested markers, ISSR, AFLP, and SAMPL showed monomorphic amplification profiles, with only RAPD markers showing some interclonal variation.

Both morphological characteristics and amplified fragment length polymorphism (AFLP) markers were used to validate the genetic fidelity of 1 080 field-grown Echinacea purpurea plants regenerated from leaf explants of donor T5-9. Morphological diagnosis revealed that 1 067 out of 1 080 regenerants were normal, while 13 regenerants were aberrant. AFLP analysis was further performed to assess DNA variations among donor, 43 sampled normal regenerants and all 13 aberrant regenerants. Seven primer combinations generated 471 fragments among donor and normal regenerants, of which 9 fragments were polymorphic. The same primer pairs generated 484 fragments for aberrant regenerants, of which 417 fragments were polymorphic. UPGMA clustering indicated that 42 normal regenerants and donor fell into same cluster at similarity scale of > 0.99, while all 13 aberrant regenerants and one morphologically normal regenerant comprised the other clusters. AFLP analysis indicated that these 14 regenerants are off-types.

CDPKs (calcium-depended protein kinases) are of great importance for the activation of defense reactions in plants. In this study, we aimed to find a connection between CDPK expression and increased salt tolerance in Panax ginseng. Treatment of P. ginseng cell cultures with W7 (CDPK protein inhibitor) showed that CDPK proteins were necessary for salt tolerance. Expression of PgCDPK1c, PgCDPK2c and PgCDPK4a was significantly increased in the cells treated with 60 mM NaCl compared to control cells, whereas expression of PgCDPK1b and PgCDPK3a was decreased. In the NaCl-treated cells, new CDPK transcripts also appeared (PgCDPK3c, PgCDPK4as). We also used rolC and rolB transformed cultures and the effects of the rol genes on CDPK expression were similar to the effects of salt stress: they caused a significant increase in the expression of PgCDPK1c, PgCDPK2c, and PgCDPK4a and decreased expression of PgCDPK3a, in addition to the appearance of the "short" CDPK transcripts.