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.

Shoot and root mass of tobacco plants treated with only 0.05 µM boron was decreased by 25 and 50 %, respectively, when compared to plants sufficiently supplied with B (2 and 5 µM). Leaf B content of 0.05 µM B-treated plants decreased (about 80-90 %) when compared to 2 µM B treated plants; this drop of B content were not as marked (about 25-45 %) in roots. Leaf and root nitrate contents in B-deficient plants were 45-60 % and 35-45 % lower, respectively, than those from 2 and 5 µM B treated plants. It is suggested that B deficiency might decrease nitrate uptake rather than nitrate reductase activity in tobacco plants.

After identifying regions of cDNA conserved between the symbiotic gene DMI1 of the model species Medicago truncatula and the homologous genomic region of Arabidopsis thaliana, universal primers were designed from 8 of 12 exons to allow the routine amplification of plant homologs. As an example, the complete homologous sequence from the pea (Pisum sativum L.) was amplified and sequenced, although the poorly conserved 5'-end and 5'-flanking region of the gene had to be amplified using a modified TAIL-PCR strategy. The identity of this amplified homolog with the SYM8 gene was independently confirmed by the presence of a single nucleotide change in the coding sequence of the mutant line Risnod27 (sym8) that cosegregated with the asymbiotic phenotype. Five insertions in pea introns responsible for increasing the total length of SYM8 by 1443 bp, compared to the M. truncatula homolog DMI1, belong to known transposon and retrotransposon families of pea and legumes in general. In view of the predicted function of SYM8 as an ion channel, the Risnod27 mutation (His309Tyr) appears to be localized in the selectivity filter domain. This finding confirms the essential role of histidine 309 in the symbiotic function of SYM8 and provides a guide to its ionic specificity. In view of the Risnod27 symbiotic phenotype, we hypothesize that SYM8 does not have identical functions in the transduction of rhizobial and mycorrhizal signals. The variability of the N-proximal region of the known legume homologs of DMI1 suggests an interaction with a variable ligand.

The leaves of axenically grown Coleus blumei were inoculated with Agrobacterium rhizogenes strain A4 and hairy root were established. PCR and Southern hybridization analysis confirmed transgenic nature of hairy root clones. Cultures of normal roots, induced by α-naphthaleneacetic acid on leaf explants, and hairy roots were evaluated for growth and rosmarinic acid content. Significantly better growth and up to 2.8 higher amount of rosmarinic acid was detected by HPLC analysis in hairy root clones. Methyl jasmonate stimulated rosmarinic acid accumulation in 6 out of 11 tested clones, while yeast extract induced RA accumulation in two and diminished it in 5 out of 11 tested hairy root clones.

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.

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.

Hypericin, a naphthodianthrone, has been identified as the principal active compound found in St. John's wort (Hypericum perforatum L.). To generate a gene resource for hypericin and other valuable metabolites, we generated expressed sequence tags (ESTs) from H. erectum. Analyses of the ESTs enabled us to select three cDNAs, HeHyp1, HeHyp2, and HeHyp3, evidencing significant sequence homology to Hyp-1 that were involved in hypericin biosynthesis from H. erectum. The deduced amino acid sequence of HeHyp1 cDNA exhibits 95 % identity with Hyp-1. The HeHyp2 and HeHyp3 polypeptides also exhibit 81.1 % identity with Hyp-1. The transcripts of HeHyp1, HeHyp2, and HeHyp3 were detected in the root, stem, leaf, flower, and callus cells. Study using recombinant protein suggests that Hyp-1, HeHyp2, and HeHyp3 may be involved in the biosynthetic of hypericin or other emodin derivatives.

Dihydroflavonol 4-reductase (DFR) is a key enzyme in the anthocyanin biosynthesis. In this study, the localization of 45S rDNA and dfr gene (named as CsDFR-bo) of the blood orange (Citrus sinensis Osbeck cv. Ruby) on chromosomes was investigated by fluorescence in situ hybridization (FISH). A karyotype of C. sinensis was reconstructed based on the length of mitotic metaphase chromosomes. The 45S rDNAs were localized on chromosomes 2p and 7q. The detection ratios of 45S rDNA in the two chromosomes were 69.5 and 77.3 %, respectively. The CsDFR-bo was proved to be a single-copy gene and localized on chromosome 3p. The detection ratio of CsDFR-bo was 8.2 %.

Three genetic similarity coefficients were estimated and compared for their usefulness: simple matching (S _SM), Jaccard's (S _J) and Dice's (S _D), all based on dominant markers data from individuals representing predominantly self-pollinating species. AFLP markers were used to analyze 139 Phaseolus vulgaris L. (common bean) and 67 Lactuca saligna L. (least lettuce) accessions, and RAPD markers were used to analyze 110 Triticum dicoccoides Koern. (wild emmer wheat) accessions. Similar discriminating structure and power based on the three genetic similarity coefficients was found for each of the three species. This discriminating power was high for both P. vulgaris and L. saligna but moderate for T. dicoccoides. With closely related individuals, as in our study, the absence of a band in two individuals should be due to an identical cause inherited from the same ancestor. Accordingly we propose the use of S _SM, which alone out of the three examined coefficients involved shared absence of DNA bands, as contributing to genetic similarity. When RAPDs are employed, inferences about population structure and nucleotide divergence should be made with prudence as the nature of genetic variation uncovered by RAPDs is often unclear.

Seedlings of Sorghum bicolor (L.) Moench were subjected to 180 mM NaCl with or without 0.25 mM spermine (SPM) for 7 d. NaCl treatment resulted in the inhibition of growth and increased the content of free proline, soluble protein and malondialdehyde (MDA). Additionally, it also enhanced the activity of catalase (CAT), peroxidase (POX) in both shoots and roots, while decreased that of glutathione reductase (GR). When exogenous spermine was added to the test solution, the growth of sweet sorghum seedlings was improved, and a smaller increase in the free proline and MDA contents was observed. The addition of spermine also partially increased the activities of POX and GR, but had no effects on soluble protein content or the activity of CAT.

Rapid micropropagation was achieved in Chlorophytum borivilianum Santapau and Fernandes using shoot base as explants. Multiple shoots were induced on Murashige and Skoog's (MS) medium supplemented with 3.0 mg dm^-3 6-benzylaminopurine, 0.1 mg dm^-3 1-naphthaleneacetic acid, 150 mg dm^-3 adenine sulphates and 3 % saccharose. Rooting was readily achieved upon transferring the shoots onto half strength MS medium supplemented with 0.1 mg dm^-3 indolebutyric acid and 2 % saccharose. Micropropagated plantlets were hardened in the greenhouse and successfully established in soil. Random amplified polymorphic DNA (RAPD) markers were used to evaluate the genetic stability of the micropropagated plants. Thirty one arbitrary decamers were used to amplify genomic DNA from in vitro and in vivo plant material to assess the genetic stability. All RAPD profile analysis from micropropagated plants was genetically similar to mother plants.

Direct somatic embryogenesis of Frittilaria meleagris L. was induced using leaf base explants excised from in vitro grown shoots. Somatic embryos occurred at the basal part of leaf explants 4 weeks after culture on a Murashige and Skoog (MS) medium supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or kinetin (KIN). The highest number of somatic embryos (SEs) were formed (9.74) from leaf explant on MS medium supplemented with 0.1 mg dm^-3 2,4-D after 4 weeks of culture initiation. An initial exposure to a low concentration of KIN in the medium also enhanced SEs induction. Our observations by light and scanning electron microscopy revealed that SEs originate directly from the epidermal and subepidermal layers of leaf explant. The developmental stages of somatic embryogenesis from the first unequal cell division through the meristematic clusters, multi-cellular globular somatic embryos to the fully formed cotyledonary embryos were determined. After 4 weeks on MS medium without plant growth regulators, SEs developed into bulblets.

Primary leaf explants of aseptically grown seedlings of moth bean [Vigna aconitifolia (Jacq.) Marechal] immersed in water or not were treated in microwave oven (2450 MHz, 800 W cm^-2) for 1, 3, 5 and 7 s before culturing. Callusing and shoot emergence from these explants were enhanced up to microwave exposure lasted 5 s while longer treatment of water-immersed explants delayed callusing. One polypeptide (26.6 kD) was up regulated in the callus derived from microwave treatment in water-immersed explants. RAPD analysis detected alteration in DNA sequences due to microwave treatment in water-immersed explants for 7 s. The frequency of mutation was 1.6 % (4 bands out of 248) over all the cultures analyzed and the same was 13 % (4 bands out of 31), if amplicons generated at 7 s treatment alone were considered.

High rates of homologous recombination (HR) in comparison to other plants make the moss Physcomitrella patens an attractive model organism for genetic studies as well as biotechnological applications. We describe a simple protocol for the efficient biolistic transformation of protonemal tissue with minimum tissue handling steps. The transformation efficiency depends on the biolistic conditions. The bombardment of tissue with 1 μm gold particles yielded between 20 and 40 stable transformants per 1 μg of DNA. Transformation with circular plasmids generates higher frequencies of random transgene integration, whereas linear plasmids are more efficient in generating gene-targeted insertions.

We used a random amplified polymorphic DNA (RAPD) amplification method to identify molecular markers associated with high quercetin accumulation in the leaves of Psidium guajava L. trees, selected from four different Mexican agronomic regions. We identified six polymorphic RAPD fragments of 620, 590, 370, 690, 480 and 460 bp among individuals of P. guajava. Genetic linkage disequilibrium analysis revealed that three RAPD profiles considered as DNA markers (620/590 bp, 370 bp and 480/460 bp) had a positive, direct association with quercetin content. These informative molecular markers can be used for selective identification of plants with the highest accumulation of flavonoids.