Agrobacterium-mediated transformation in Citrullus lanatus

Agrobacterium tumefaciens-mediated transformation was used to produce transgenic watermelon. Cotyledonary explants of Citrullus lanatus Thumb (cv. Daesan) were co-cultivated with Agrobacterium strains (LBA4404, GV3101, EHA101) containing pPTN289 carrying with bar gene and pPTN290 carrying with nptII gene, respectively. There was a significant difference in the transformation frequency between bacteria strains and selective markers. The EHA101/pPTN289 showed higher transformation frequency (1.16 %) than GV3101/pPTN289 (0.33 %) and LBA4404/pPTN289 or /pPTN290 (0 %). The shoots obtained (633 and 57 lines) showed some resistance to glufosinate and paromomycin, respectively. Of them, the β-glucuronidase positive response and PCR products amplified by bar and nptII specific primers showed at least 21 plants resistant to glufosinate and at least 6 plants to paromomycin. Southern blot analysis revealed that the bar gene integrated into genome of transgenic watermelon. Acclimated transgenic watermelons were successfully transplanted in the greenhouse and showed no phenotypic variation.     

Generation of low copy number and stably expressing transgenic creeping bentgrass plants using minimal gene cassette bombardment

A minimal gene cassette comprised of the ubiquitin (Ubi) promoter + green fluorescent protein (Gfp) gene + Nos terminator DNA sequences, derived from the plasmid vector pPZP201-Gfp was utilized for transformation of creeping bentgrass using particle bombardment. Bentgrass calli bombarded individually with equivalent amounts of the cassette or whole plasmid DNA were compared for Gfp expression and the GFP-positive calli were subsequently regenerated into plants. Percentage of GFP expressing calli and the number of GFP spots/calli were significantly higher in calli that were bombarded with the minimal gene cassette when compared to the whole plasmid. The Gfp expression was stable up to the T₂ generation in minimal gene cassette transformants and there was a lower degree of gene silencing. Southern blot analysis of transgenic plants derived from minimum gene cassette bombardment revealed the presence of single or few copy of the transgene and fairly simple integration patterns. In comparison, whole plasmid transformants had multiple copies and complex integration patterns of the transgene. These results illustrate the advantages of using simple gene cassette for stable plant transformation in bentgrass with possible applications to other plant species.     

Production of recombinant human lactoferrin from transgenic plants

Molecular farming provides a powerful tool for low cost production of recombinant proteins with pharmaceutical value. The use of transgenic plants has been increasingly tested as alternative system for obtaining biologically active human lactoferrin in plants. Precise selection of plant species, transformation techniques and expression cassettes, in addition to conduction of detailed glycosylation and immunogenicity studies, serves as basis of obtaining safe recombinant human lactoferrin in high concentrations for the use of pharmacy. On the other hand, expression of antimicrobial protein lactoferrin in plants is a promising opportunity for crop quality improvement by increasing plant disease resistance.     

Antioxidative response to cadmium in roots and leaves of tomato plants

Treatment of tomato seedlings (Lycopersicon esculentum Mill. cv. 63/5 F1) with increasing CdCl₂ concentrations in the culture medium resulted in Cd accumulation more important in roots than in leaves. Biomass production was severely inhibited, even at low Cd concentration. Cd reduced chlorophyll content in leaves and enhanced lipid peroxidation. An increase in antioxidative enzyme (superoxide dismutase, ascorbate peroxidase, guaiacol peroxidase, glutathione reductase) activities was more pronounced in leaves than in roots, while catalase activity increased only in roots. In addition, changes in isoenzyme composition were observed using the non-denaturing polyacrylamid gel electrophoresis.     

Glutathione and phytochelatin contents in tomato plants exposed to cadmium

The effect of cadmium on growth and contents of glutathione (GSH) and phytochelatins (PCs) were investigated in roots and leaves of tomato plants (Lycopersicon esculentum Mill. cv. 63/5 F1). The accumulation of Cd increased with external Cd concentrations and was considerably higher in roots than in leaves. Dry mass production decreased under Cd treatment especially in leaves. In both roots and leaves, exposure to Cd caused an appreciable decline in GSH contents and increase in PCs synthesis proportional to Cd concentrations in the growth medium. At the same Cd concentration, PCs production was higher in roots than in leaves. The implication of glutathione in PC synthesis was strongly suggested by the use of buthionine sulfoximine (BSO). The major fraction of Cd accumulated by tomato roots was in the form of a Cd-PCs complex.     

Elevated CO2 concentration enhances the role of the ear to the flag leaf in determining grain yield of wheat

Net photosynthetic rate (P _N) of ear and flag leaf during grain filling stage and grain yield of plants with non-darkened or darkened flag leaf or darkened ear were examined in two different CO₂ concentrations: ambient (AC) and AC+200 μmol mol⁻¹ (EC). Ear showed much higher enhancement (56 %) of P _N than flag leaf (23 %) under EC. Moreover, CO₂ enrichment shortened the photosynthetic duration of flag leaf relative to ear. In this way the ratio of ear to flag leaf contribution to grain yield increased from 1.18 (AC) to 1.39 (EC).     

Somatic embryogenesis from immature zygotic embryos and monitoring the genetic fidelity of regenerated plants in grapevine

Somatic embryogenesis and plant regeneration were successfully established on Nitsch and Nitsch (NN) medium from immature zygotic embryos of six genotypes of grapevine (Vitis vinifera). The optimum hormone combinations were 1.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction and 1.0 mg dm⁻³ α-naphthalene acetic acid (NAA) + 0.5 mg dm⁻³ 6-benzyladenine (BA) for embryos production and 0.03 mg dm⁻³ NAA + 0.5 mg dm⁻³ BA for embryos conversion and plant regeneration. The frequency of somatic embryogenesis varied from 10.5 to 37.5 % among six genotypes and 15.5–42.1 % of somatic embryos converted into normal plantlets. The analysis of DNA content determined by flow cytometry and chromosome counting of the regenerated plantlets clearly indicated that no ploidy changes were induced during somatic embryogenesis and plant regeneration, the nuclear DNA content and ploidy levels of the regenerated plants were stable and homogeneous to those of the donor plants. RAPD markers were also used to evaluate the genetic fidelity of plants regenerated from somatic embryos. All RAPD profiles from regenerated plants were monomorphic and similar to those of the field grown donor plants. We conclude that somaclonal variation is almost absent in our grapevine plant regeneration system.     

MPEC: an important gene in the chlorophyll biosynthesis pathway in photosynthetic organisms

One of the least understood enzymatic steps in chlorophyll biosynthesis is the formation of isocyclic ring, which is catalyzed by the Mg-protoporphyrin IX monomethyl ester (MgPME) cyclase that is involved in the conversion of MgPME to protochlorophyllide. Several genes encoding part of this enzyme have been identified and functional analysis of them has been performed. The enzyme plays important roles in higher plants and photosynthetic bacteria. The review focuses on the current knowledge of MgPME cyclase coding genes, with emphasis on their organization, expression pattern, and functional analysis obtained from mutants.     

Polyamine metabolism in hants : Arginine and omithine decarboxylase activity in ripeningTrlticum durum seeds

The pattern of the activity of arginine decarboxylase (ADC) and omithine decarboxylase (ODC) involved in polyamine synthesis in ripening wheat seeds was examined. The aim was to study the polyamines and the activity of the two enzymes in correlation with the growth processes occurring in the developing wheat seeds. The results obtained showed a very different pattern of polyamine content in the two organs of caryopsis, and that the two enzymes in the embryos have a higher activity than in the endosperms. Moreover, while in the embryos the ADC exhibits higher activity than the ODC, in the endosperms the activity of ODC is about similar to that of ADC. This pattern is discussed in relation to the different histological characteristics of embryo and endosperm tissues during seed development.     

Photosynthetic behaviour of Arabidopsis plants with a Cap Binding Protein 20 mutation under water stress

Under non-stressed conditions the net photosynthetic rate (P _N) of the mutant plants cbp20 of Arabidopsis was similar to that of the wild type (WT). In response to water deprivation, however, P _N started to decrease later in the mutants and remained substantially higher. Thermoluminescence measurements showed that the lipid peroxidation induced by severe water stress was also less pronounced in the mutant than in the WT. Both soil gravimetric and plant water potential data showed that cbp20 mutants lose water more slowly than the WT plants. The drought-induced decline in F_v/F_m, the quantum efficiency of photosystem 2, and photochemical quenching parameters also started later in the cbp20 mutants than in the WT plants. Thus the restricted gas exchange in the cbp20 mutants does not impair the photosynthetic performance of the plant; however, under drought improved water retention provides significant protection for the photosynthetic apparatus.     

Genetic diversity assessment in Portugal accessions of <Emphasis Type="Italic">Olea europaea</Emphasis> by RAPD markers

Eighty seven olive (Olea europaea ssp. sativa L.) cultivar accessions from Portugal were characterized by means of randomly amplified polymorphic DNA (RAPD) markers. Of the 11 arbitrary 10-mer primers tested a total of 92 polymorphic bands were obtained, representing 87.6 % of the total amplification products. Twenty nine different genotypes were clearly discriminated. Differences were not found among the amplification profiles from different individuals of the same cultivar. All the genotypes could be identified by the combination of three primers: OPR-1, OPK-14 and OPA-1, seven genotype-specific markers being detected. Genetic relationships were estimated by the unweighted pair-group method with arithmetic averaging (UPGMA). The genetic analysis of the results showed a gradual distance between the various cultivars, making it difficult to identify well-differentiated phylogenetic groups, although two clusters were distinguishable with 35 % similarity, in addition to three independent branches with lower similarity: Galega, Tentilheira and Redondal. The dendrogram reflect some relationships for most of the cultivars according to the use of the fruit and ecological adaptation.     

Plant regeneration from mesophyll protoplasts of Agrobacterium rhizogenes-transformed Astragalus melilotoides

Plant regeneration from mesophyll protoplasts of Agrobacterium rhizogenes-transformed Astragalus melilotoides Pall. was here developed. The protoplasts were isolated directly from the leaves of the hairy root-induced plants. The highest yield of protoplasts was obtained from fully expanded leaves of young plants. Their viability was up to 72 ± 2.3 %. The highest division frequency (32.4 ± 0.13 %) and sustained divisions were obtained in Durand, Potrykus and Donn (DPD) medium supplemented with 2.0 mg dm⁻³ 2,4-dichlorophenoxyacetic acid, 0.2 mg dm⁻³ 6-benzylaminopurine, 0.3 M mannitol, 2 % sucrose and 500 mg dm⁻³ casein hydrolysate at the plating density of 3.0 × 10⁵ cm⁻³. The frequency of shoot differentiation from protocalli reached to 91.75 ± 3.1 %. Opine synthesis and polymerase chain reaction analysis confirmed that T-DNA still existed in the protoplast regenerated plants.     

Lead uptake,toxicity and accumulation in <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> plants

The effects of lead were investigated in bean plants (Phaseolus vulgaris L. cv. Zlota Saxa) grown hydroponically in nutrient solution and exposed to Pb(NO₃)₂ (0.1, 0.5, 1 mM) with or without equimolar concentrations of chelator ethylenediaminetetraacetic acid (EDTA). The roots treated only with Pb(NO₃)₂ accumulated up to 25 g(Pb) kg⁻¹(d.m.), during 4-d exposure. However, in bean plants exposed to 0.5 mM Pb + 0.5 mM EDTA or 1 mM Pb + 1 mM EDTA 2.5 times less Pb was determined. In bean plants treated only with Pb, less than 6 % of total lead accumulated was transported to the aboveground parts, while in the case of plants grown with Pb + EDTA, around 50 % of total Pb was transported to the shoots.     

Cadmium-induced oxidative damage and antioxidant responses in Brassica juncea plants

Indian mustard (Brassica juncea L. cv. Vitasso) plants exposed to 10, 30, 50 and 100 μM of Cd for 5 d in hydroponic culture were analysed with reference to the distribution of Cd²⁺, the accumulation of biomass and antioxidants and antioxidative enzymes in leaves. Cd induced a decrease in plant biomass. The maximum accumulation of Cd occurred in roots followed by stems and leaves. Cd induced a decrease in catalase (CAT) and guiacol peroxidase (GPX) activities but an increase in ascorbate peroxidase (APX) and monodehydroascorbate reductase (MDHAR) activities. Enhancement in dehydroascorbate reductase (DHAR) activity was also at 10 μM Cd. Glutathione reductase (GR) activity showed pronounced stimulation after all treatments, but glutathione S-transferase (GST) and glutathione peroxidase (GPOX) activities decreased. The effectiveness of ascorbate-glutathione cycle (AGC) was determined by the ratio of ascorbate to H₂O₂. This ratio decreased in the Cd-treated leaves which indicated that the cycle was disordered.     

Assessment of genetic diversity of pigeonpea cultivars using RAPD analysis

In our present study assessment of genetic diversity and identification of pigeonpea cultivars has been done by employing 76 random amplified polymorphic DNA (RAPD) primers. Out of 796 amplified products, 587 showed polymorphism (73.7 %) and an average of 10.47 bands were amplified per primer. Cluster analysis based on Jaccard’s similarity coefficient using UPGMA grouped all the cultivars into three clusters. The cluster I consists of 7 cultivars, cluster II of 11 cultivars in 4 sub-clusters and cluster III 4 cultivars. Two cultivars were not included in any cluster. The clustering was strongly supported by high bootstrap values. Furthermore, high values of the average heterozygosity (H_av) and marker index (MI) also indicated the efficiency of RAPD as a marker system.     

Modelling the dynamics of the electron transport rate measured by PAM fluorimetry during Rapid Light Curve experiments

We propose a dynamic model specifically designed to simulate changes in the photosynthetic electron transport rate, which is calculated from fluorescence measurements when plants are exposed, for a short time, to a series of increasing photon flux densities. This model simulates the dynamics of the effective yield of photochemical energy conversion from the maximum and natural chlorophyll fluorescence yields, taking into account a cumulative effect of successive irradiations on photosystems. To estimate a characteristic time of this effect on photosystems, two series of experiments were performed on two benthic diatom culture concentrations. For each concentration, two different series of irradiations were applied. Simplified formulations of the model were established based on the observed fluorescence curves. The simplified versions of the model streamlined the parameters estimation procedure. For the most simplified version of the model (only 4 parameters) the order of magnitude of the characteristic time of the residual effect of irradiation was about 38 s (within a confidence interval between 20 and 252 s). The model and an appropriate calibration procedure may be used to assess the physiological condition of plants experiencing short time-scale irradiance changes in experimental or field conditions.     

Induction of <Emphasis Type="Italic">in vitro</Emphasis> flowering in the orchid <Emphasis Type="Italic">Dendrobium</Emphasis> Sonia 17

In this study, Dendrobium Sonia 17 plantlets were used to induce in vitro flowering. Inflorescences were induced and rooting was inhibited in the half-strength Murashige and Skoog medium containing 20 μM N ⁶-benzyladenine (BA). The medium with high P and low N contents was effective to induce inflorescences while the medium with low P and high N contents was only effective to promote forming of shoots. In addition, the induced in vitro inflorescences were able to multiply and maintain without exhibiting a distinctive vegetative phase. Different morphologies of in vitro flowers such as incomplete flower structures, abnormal and unresupinated in vitro flowers were observed.     

Cadmium effects on the organization of microtubular cytoskeleton in interphase and mitotic cells of Allium sativum

We have investigated the effects of cadmium on the microtubular (MT) cytoskeleton in the root tip cells of Allium sativum L. using indirect immunofluorescence microscopy. Cd affected the mechanisms controlling the organization of MT cytoskeleton, as well as tubulin assembly/disassembly processes. Cd induced the formation of abnormal MT arrays, consisting of discontinuous wavy MTs or short MT fragments at the cell periphery. Cadmium caused irregular nuclear disorder in cells where the MT organization and function was disturbed. Furthermore, with increased Cd concentration and duration of treatment the MTs depolymerized more severely, the frequency of abnormal cell increased and the mitotic index decreased progressively. The above findings showed that MT cytoskeleton is one of target sites of Cd toxicity in root tip cells.