A protocol for in vitro multiplication of Capparis decidua (Forsk.) Edgew. has been developed from cultured leaves procured from multiplying axillary shoots on the cultured nodal explants. The highest efficiency of shoot formation was observed on Murashige and Skoog (MS) medium containing 2 mg dm^-3 benzyladenine (BA) and 0.5 mg dm^-3 1-naphthaleneacetic acid. The regenerated shoots were transferred to MS medium containing 3 mg dm^-3 BA for growth and proliferation. Shoots above 2 cm in length were transferred to MS medium supplemented with 1 mg dm-3 indole-3-butyric acid plus 0.5 mg dm^-3 indole-3-acetic acid for root induction. No variation was detected among the micropropagated plants by randomly amplified polymorphic DNA (RAPD) markers.

The DNA of fifteen Italian cultivars of durum wheat (Triticum turgidum L. ssp. durum) were analyzed by in fluorescence amplified fragment length polymorphism (fAFLP) in order to obtain the characteristic fingerprintings of genotypes and assess their genetic relatedness. Among 64 combinations of fluorescence labelled primers, three different combinations were chosen as producing a total of 6630 AFLP fragments, 2277 (34.3 %) of them being polymorphic. By using this fAFLP methodology a DNA fingerprinting of each durum wheat cultivar was generated for genotype identification. Analysis of the genetic relationships show the low variability among durum wheat cultivars.

Cadmium (100, 400 and 1000 μM CdCl₂) treatments resulted in the inhibition of root dry biomass, root elongation and increased Cd accumulation in wheat (Triticum aestivum L.) roots. Further, these treatments decreased relative water content, chlorophyll content, ¹⁴CO₂-fixation, activities of phosphoenolpyruvate carboxylase and ribulose-1,5-bisphosphate carboxylase and abscisic acid content while increased malondialdehyde, hydrogen peroxide and free proline contents. Chloroplast and root ultrastructure was also changed. Pretreatment of seeds with SA (500 μM) for 20 h resulted in amelioration of these effects.

Proline (Pro) accumulation under water stress was measured in safflower (Carthamus tinctorius L.) drought tolerant cv. A1 and sensitive cv. Nira. Activities of pyrroline-5-carboxylate reductase (P5C reductase) and pyrroline-5-carboxylate synthetase (P5C synthetase), two enzymes involved in the Pro biosynthetic pathway were also estimated. Water stress resulted in a reduction in the leaf dry mass and chlorophyll content along with a gradual accumulation of Pro. RT-PCR results show higher expression of Δ¹-pyrroline-5-carboxylate synthetase (p5cs) gene in correlation with up-regulated Pro accumulation in cv. A1. P5C reductase was found to be the Pro synthesis rate limiting whereas P5C synthetase did not show any specific response to the drought stress in both cultivars.

The mean effective water self-diffusion coefficient in maize root segments under the effect of aquaporin blocker (mercuric chloride, 0.1 mM) was measured using the spin-echo NMR method with pulsed magnetic field gradient within the temperature range from 10 to 35 °C. HgCl₂ caused the reduction in water diffusion by 30 % as compared to the control samples. Temperature dependences of water self-diffusion coefficients showed two linear regions with different values of Q₁₀ and activation energy, E_a. As the temperature reduced from 20 to 10 °C, E_a values calculated from the Arrhenius plots were close to those of bulk water (20 ± 3 kJ mol^-1) and slightly changed for the sample pretreated HgCl₂. Within the temperature range from 25 to 35 °C the slope of temperature dependences became steeper and E_a values were 31 ± 3 kJ mol^-1 for the control and 40 ± 4 kJ mol^-1 for the treated sample. In the vicinity of 20 °C, the temperature dependence of water diffusion via the mercury-sensitive water channels showed extreme value. In the region, the specific area of the mercury-sensitive aquaporins was 0.004 % of the total cell surface area. The data indicate that water transfer via aquaporins is sensitive to temperature, and the contributions of the transmembrane pathways (aquaporins, lipid bilayer) differ in different temperature ranges.

TaCer6 was firstly cloned by rapid amplification of cDNA ends (RACE) and identified as a tissue-specific gene in wheat. To determine if environmental factors such as drought and low temperature induce TaCer6 transcription, we examined the effects of these factors on TaCer6 in two wheat cultivars. Our results demonstrated that light was essential for TaCer6 transcription, salt stress inhibited TaCer6 expression and application of salicylic acid enhanced TaCer6 transcripts accumulation. In addition, polyethylene glycol (PEG 6000) and abscisic acid increased the expression of TaCer6 more in the drought- and cold-tolerant cultivar Jinmai47 than in non-tolerant cultivar Shi4185. Low temperature increased TaCer6 transcription in Jinmai47 while decreased it in Shi4185.

The changes in the fresh biomass accumulation, photosynthetic and anthocyanin pigments, photosystem 2 (PS 2) activity, ultrastructure of chloroplast, total lipids and fatty acid composition of thylakoid membrane were followed in the aquatic fern Azolla caroliniana grown on medium either deficient or supplied with various phosphorus concentrations. The content of photosynthetic pigments and the anthocyanin/chlorophyll ratio increased significantly with increasing PO₄ ^3- concentration. Phosphate deficiency inhibited growth and PS 2 activity and decreased content of total lipids and phospholipids in isolated thylakoids. This was accompanied with a significant increase in the percentage of galalactolipids.

Leaf gas exchange, chlorophyll fluorescence and activities of antioxidant enzymes were studied in two pepper (Capsicum annuum L.) cultivars grown under drought (D) and heat (H), as well as under drought and heat in combination (HD). The drought-tolerant cv. Zhengjiao 13 exhibited greater net photosynthetic rate (P_N) and cytochrome respiratory pathway activity (R_SHAM), and lower contents of superoxide radical and hydrogen peroxide, as compared to the drought-sensitive cv. Longkouzaojiao. The P_N and R_SHAM decreased and ROS production increased under D and HD in both cultivars. As compared to the Longkouzaojiao, Zhengjiao 13 retained higher non-photochemical quenching (NPQ), photorespiration rate (R_L), and alternative respiratory pathways (R_KCN) under D and HD. Drought increased the superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities in the cytosol, chloroplasts and mitochondria in the two cultivars. Although SOD and APX activities decreased in Longkouzaojiao under HD, SOD activity increased in Zhengjiao 13. There was no H-induced reactive oxygen species production due to increase of R_L, NPQ, R_SHAM, R_KCN and activities of SOD and APX. However, H slightly decreased the P_N. The results indicated that HD was more detrimental than either stress alone.

We quantified inbreeding depression for fruit production, embryo vitality and seed germination in three deceptive orchids, Serapias vomeracea, S. cordigera and S. parviflora, which do not provide any reward to their pollinators, and are predicted to experience high outcrossing. Of the three species examined only S. parviflora was autonomously selfing. Both S. vomeracea and S. cordigera showed highly significant differences in fitness between selfed and outcrossed progenies, resulting in high levels of inbreeding depression, which increased in magnitude from seed set to seed germination. Inbreeding depression may promote outcrossing in Serapias by acting as a post-pollination barrier to selfing. Cumulative inbreeding depression across three stages in S. parviflora was lower that in both outcrossing species. The large difference in germination between selfed and outcrossed seeds is an important issue in conservation biology.

We studied the nutritional modes of the orchid Serapias strictiflora and its mycorrhizal fungus Epulorhiza sp. using the differences in carbon isotopic composition (δ¹³C) of C₃ orchid and C₄ maize tissues. We found that if cultivated in substrate lacking any organic compounds, the mycorrhizal extraradical mycelia (δ¹³C = -26.3 ± 0.2 ‰) developed well, despite being fully dependent on nutrition from orchid roots (δ¹³C = -28.6 ± 0.1 ‰). If the mycorrhizal fungus had additional access to and colonized decaying maize roots (δ¹³C = -14.6 ± 0.1 ‰), its isotopic composition (δ¹³C = -21.6 ± 0.4 ‰) reflected a mixture of biotrophy and saprotrophy. No statistically significant differences in δ¹³C of new storage tubers were found between Epulorhiza-associated orchids with (δ¹³C = -28.2 ± 0.1 ‰) and without access to maize roots (δ¹³C = -28.6 ± 0.2 ‰). We conclude that autotrophy is the predominant nutritional mode of mature S. strictiflora plants and that they supply their mycorrhizal fungus with substantial amount of carbon (69 ± 3 % of the fungus demand), even if the fungus feeds saprotrophically.

The effect of maturation pretreatment on development and growth of Abies numidica De Lann. somatic embryos was studied. The most beneficial was pre-culturing on Schenk and Hildebrandt medium without growth regulator for 2 weeks. Dry mass accumulation of emblings was lower than that of seedlings after 50 d of culturing. Contents of microelements in seedlings were higher than in emblings, but macroelements contents were higher in emblings. Contents of chlorophyll a and chlorophyll b in cotyledons were higher in seedlings than in emblings while no qualitative differences were detected between the protein profiles of seedlings and emblings.

The δ-aminolevulinic acid synthase (ALA-S) is an enzyme which catalyzes the synthesis of δ-aminolevulinic acid (ALA). The Bradyrhizobium japonicum ALA-S coding sequence lacking plastidal transit sequence was introduced into the rice genome (C line). The transgenic lines, C4 and C5, were compared with the transgenic lines expressing TALA-S gene with plastidal transit sequence (P line) to investigate whether the plastidal sequence affects the targeting capacity of B. japonicum ALA-S gene and the ALA-synthesizing capacity in rice plants. The B. japonicum ALA-S mRNA was expressed efficiently in C lines and the protein was localized in the stroma of chloroplasts regardless of the transit sequence as in P lines. The resulting transgenic plants, C line, had similar levels of ALA-S activity, ALA, protoporphyrin IX and chlorophylls, compared to those of P lines. In response to irradiance of 350 μmol m^-2 s^-1, transgenic lines C4 and C5 displayed the characteristic phenotypes of photodynamic damage, i.e., decreases in photosynthetic parameter F_v/F_m, as in P5 and P14 lines, whereas wild type did not. These results indicate that the lack of the plastidal transit sequence influences neither chloroplast translocation of B. japonicum ALA-S nor ALA-synthesizing capacity in the transgenic rice.

Sap flow and potential evapotranspiration rates were analyzed for two coniferous tree species (Douglas-fir and Scots pine) and one broadleaf species (sessile oak) in a mixed Carpineto-Quercetum forest during the growing season 2005. The relationship between sap flow and potential evapotranspiration rates, effective crown area as a measure of the relative transpiration and daily relative proportion of the storage water used for transpiration were used as indicators of the tree water dynamics. These indicators were determined on four consecutive days and all three showed good reliability concerning tree water dynamics.

The major capsid protein L1 of human papillomavirus type 16 (HPV16) was transiently expressed in tobacco (Nicotiana benthamiana and Nicotiana tabacum) and tomato (Lycopersicon esculentum) leaves using Agrobacterium tumefaciens. The expression vector pTV00 was derived from tobacco rattle virus (TRV). The highest L1 expression 15 μg g^-1(f.m.) was achieved when the coding sequence of L1 was optimized for expression in humans that caused an increase of the guanine and cytosine (GC) content from 38.2 % in wild type HPV16 to 64.1 % in optimized sequence. L1 monomers readily self-assembled into capsomeres and further into virus like particles (VLPs). Immunological characterization and electron microscopy showed that 89 % of L1 retained VLP structure also in extracts prepared from freeze-dried leaves. Plant expressed L1 in crude extracts was highly immunogenic without any additional adjuvant as vaccinated mice developed strong humoral and cellular immune response, comparable to that elicited by purified VLPs derived from insect cells. Further, the induced antibodies effectively neutralized infection of 293TT cells with pseudovirions. This finding demonstrates that the TRV expression system is comparable to other plant expression systems and due to the broad host range of TRV is particularly attractive when expression in plants with low content of toxic alkaloids is desired. Moreover, a monoclonal anti-L1 antibody E2 raised in the course of immunization with crude extract from freeze-dried leaves expressing L1 is specific preferentially against HPV VLPs and could be used in direct ELISA for monitoring of VLPs assembly and VLP purification protocols.

The encapsulated shoot tips and nodal segments of Eclipta alba were stored at 4, 12 and 20 °C under irradiance of 1.5 gmmol m^-2 s^-1 and high conversion was observed in synseeds stored at 4 °C for 8 weeks. Duration of storage was extended up to 12 weeks by decreasing sucrose concentration in the alginate matrix from 3 to 1 or 2 % and conversion frequency was 71.2-76.1 %. Synseed-derived plantlets survived by 100 % in ex vitro conditions. RAPD analysis revealed uniform amplification profile in donor and synseed derived plantlets.

To get insight into mechanism by which apple tree (Malus domestica Borkh.) regulates flowering, two apple flowering locus T (FT) homologues, MdFT1 and MdFT2, were isolated from the leaf cDNAs of cultivar Gala. The open reading frames (ORFs) of two MdFTs encoded 174 amino acids. The deduced amino acid sequence of MdFT1 and MdFT2 showed 94.3 % similarity to each other, while 72.6 and 76.0 % to AtFT protein, respectively. Semi-quantitative RT-PCR indicated their specific expression in leaves. Visualization of MdFT2-GFP fusion protein demonstrated its localization on membrane. Ectopic overexpression of either MdFT1 or MdFT2 in Arabidopsis significantly induced early flowering by activating the downstream flowering-related genes.

Using in vitro culture, we determined the effect of photoperiod during growth of Chenopodium rubrum mother plants on vegetative and reproductive development of offspring. Photoperiod during flowering induction of mother plants (the first 6 d after the germination) has the key influence on seed germination and offspring growth, while offspring flowering and seed maturation is determined by photoperiod their mothers experienced during, and shortly after, flowering induction. The mechanism can be through changes in seed protein pattern which we found dependent on photoperiod experienced by mother plants.

The genetic relationship of 36 Dendrobium species in China was determined based on sequence analysis of the internal transcribed spacer (ITS) region of ribosomal DNA. Aligned sequences of the complete ITS region obtained from the 36 Dendrobium species and 2 outgroup species (Epigeneium amplum and Epigeneium nakaharaei) by using PCR amplification and direct DNA sequencing. The nrDNA ITS1 of Dendrobium was 225-234 bp and ITS2 was 239-248 bp. Phylogenetic tree was constructed, and seven main clusters were generated among the 36 Dendrobium species. From the results, D. moulmeinense was not grouped in the classification of Dendrobium and E. amplum and E. nakaharaei were shown to be divergent from Dendrobium species. The phylogenetic relationships revealed by ITS DNA analysis partially supported previously published morphological data.

The effects of NaCl stress on growth, water status, contents of protein, proline, malondialdehyde (MDA), various sugars and photosynthetic pigments were investigated in seedlings of Salicornia persica and S. europaea grown in vitro. Seeds were germinated under NaCl (0, 100, 200, 300, 400, 500 and 600 mM) on Murashige and Skoog medium for 45 d. The shoot growth of both species increased under low NaCl concentration (100 mM) and then decreased with increasing NaCl concentrations. In contrast to S. persica, root length in S. europaea reduced steadily with an increase in salinity. Proline content in S. persica was higher than in S. europaea at most NaCl concentrations. Proline, reducing saccharide, oligosaccharide and soluble saccharide contents increased under salinity in both species. In contrast, contents of proteins and polysaccharides reduced in both species under salt stress. MDA content remained close to control at moderate NaCl concentrations (100 and 200 mM) and increased at higher salinities. MDA content in S. europaea was significantly higher than S. persica at higher salinities. Salt treatments decreased K⁺ and P contents in seedlings of both species. Significant reduction in contents of chlorophylls and carotenoids due to NaCl stress was also observed in seedlings of both species. Some differences appeared between S. persica and S. europaea concerning proteins profile. On the basis of the data obtained, S. persica is more salt-tolerant than S. europaea.

This study examined the impacts of elevated CO₂ or O₃ on indole-3-acetic acid (IAA) content, activities of IAA oxidase (IAAO) and peroxidase (POD) in Ginkgo biloba leaves. Plants grown in open-top chambers were exposed to ambient atmosphere (control; C), elevated CO₂ and elevated O₃ from 1 June to 30 September. An increase in IAA content and decrease in IAAO and POD activities were observed in plants exposed to elevated CO₂ compared with C. Elevated O₃ had no significant effect on IAA content and IAAO activity, but increased POD activity during the early days. When trees pre-exposed to elevated CO₂ were transferred to elevated O₃ or C, the increase in IAAO activity resulted in the decrease in IAA content. When trees pre-exposed to elevated O₃ were transferred to elevated CO₂ or C, IAA content, IAAO and POD activities showed no significant changes. The influence of POD activity on the IAA activity was low.

Two Coffea arabica - Hemileia vastatrix incompatible interactions (I₁: coffee cv. Caturra - rust race VI and I₂: coffee cv S4 Agaro - rust race II) and a compatible interaction (coffee cv. Caturra - rust race II) were compared in relation to the infection process and chitinase activity. In the two incompatible interactions the fungus ceased growth in the early infection stages, while in the compatible interaction no fungus growth inhibition was observed. A high constitutive level of chitinase activity was detected in the intercellular fluid of healthy leaves. Upon infection, chitinase isoforms were more abundant in incompatible interactions than in the compatible interaction. Immunodetection showed that class I chitinases are particularly relevant in the incompatible interactions and might participate in the defence response of the coffee plants.

Plantago algarbiensis and P. almogravensis are endemic Al tolerant species from the Western-centre of the Algarve region (South of Portugal) and Portuguese Southwest coast, respectively, which are in risk of global extinction. The aim of this work was to establish an efficient protocol to in vitro propagate these species using shoots obtained from in vitro germinated seeds. The best results in terms of multiplication response were afforded in Murashige and Skoog's (MS) medium supplemented with 6-benzyladenine (8.5 and 9.2 shoots per explant in P. algarbiensis and P. almogravensis, respectively). Shoots of both species showed a great rooting capacity (100 and 80 % for P. algarbiensis and P. almogravensis, respectively) that was not significantly influenced by the concentration of MS macronutrients or auxins. Plants were acclimatized to ex vitro conditions, exhibited normal development (survival rate of 95 and 80 % in P. algarbiensis and P. almogravensis, respectively), and were successfully reintroduced in their natural habitat.

The present study investigated whether Ca²⁺ mobilization independent of phosphoinositide-specific phospholipase C (PI-PLC) would delay wilting in Arabidopsis thaliana (L.) Heynh. cv. Columbia through mediating stomatal closure at abscisic acid (ABA) concentrations rising beyond a drought-specific threshold value. In wild type (WT) epidermis, the PI-PLC inhibitor (U73122) affected the stomatal response to 20 µM ABA but not to 30 µM ABA. Disruption in GTP-binding protein ά subunit 1 (GPA1) affected the stomatal response to 30 µM ABA, but not to 20 µM ABA. In the gpa1-4 mutant, the inhibitory effects of the Ca²⁺ buffer, 1,2-bis(0-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), the inactive mastoparan analogue, mas17 and the antagonist of cyclic ADP-ribose synthesis, nicotinamide, were differentially attenuated on 30 µM ABA-induced stomatal closure. By contrast, the NADPH oxidase atrbohD/F double mutation fully suppressed inhibition of 20 µM ABA-induced stomatal closure by BAPTA or U73122 as well as inhibition of 30 µM ABA-induced stomatal closure by BAPTA, mas17 or nicotinamide. On the contrary, The Al resistant alr-104 mutation modulated ABA-induced stomatal closure by a stimulatory effect of U73122 and an increased sensitivity to mas17, nicotinamide and BAPTA. Compared to WT, the atrbohD/F double mutant was more hypersensitive than the gpa1-4 mutant to wilting under the tested water stress conditions, whereas wilting was delayed in the alr-104 mutant. Since the atrbohD/F mutation breaks down ABA-induced Ca²⁺ signalling through fully preventing apoplastic Ca²⁺ to enter into the guard cells, these results showed that a putative guard cell GPA1-dependent ADP-ribosyl cyclase activity should contribute to drought tolerance within PI-PLC-independent-Ca²⁺-mediated ABA signalling.

Diurnal changes of photosynthesis in the leaves of grapevine (Vitis vinifera × V. labrusca) cultivars Campbell Early and Kyoho grown in the field were compared with respect to gas exchanges and actual quantum yield of photosystem 2 (Φ_PS2) in late May. Net photosynthetic rate (P_N) of the two cultivars rapidly increased in the morning, saturated at photosynthetic photon flux density (PPFD) from 1200 to 1500 µmol m^-2 s^-1 between 10:00 and 12:00 and slowly decreased after midday. Maximum P_N was 13.7 and 12.5 µmol m^-2 s^-1 in Campbell Early and Kyoho, respectively. The stomatal conductance (g_s) and transpiration rate changed in parallel with P_N, indicating that P_N was greatly affected by g_s. However, the decrease in P_N after midday under saturating PPFD was also associated with the observed depression of Φ_PS2 at high PPFD. The substantial increase in the leaf to air vapour pressure deficit after midday might also contribute to decline of g_s and P_N.

Agrobacterium-mediated genetic transformation is the most widely used technology to obtain overexpression of recombinant proteins in plants. Molecular events that occur within Agrobacterium during interactions with host plants have been studied extensively, and now we have a reasonable understanding the key factors involved in the regulation of T-DNA nuclear import and genomic integration. By contrast, very little is known about the events that take place in the host cells during genetic transformation by Agrobacterium. Here, we describe the plant-related factors including genotype, genes, proteins, competency of target tissues and phenolic compounds that participate in Agrobacterium-mediated genetic transformation and discuss their possible roles in this process. Because Agrobacterium probably adapts existing cellular processes for its life cycle, identifying the processes in host cells during Agrobacterium infection might contribute to better understanding of basic biological processes as cell communication, intracellular transport and DNA repair and recombination as well as to expanding the host range of Agrobacterium as a genetic engineering tool.

The genetic variability of Leucojum valentinum Pau (Amaryllidaceae), a vulnerable endemic species restricted to a small area in the region of Valencia (Eastern Spain), has been studied using random amplified polymorphic DNA (RAPD) markers. A total of 197 individuals from eleven populations were studied using 13 RAPD primers. Our results show high variability for the species, low differentiation among populations and uncorrelated levels of genetic variability and population size. Four groups in which three populations (SAG, PUG and COL) are separated from all the others were found, but without connection to geographical location.