Phleomycin resistance as a dominant selectable marker for plant cell transformation

Tobacco cells are sensitive to bleomycin and phleomycin. The Tn5 and the Streptoalloteichus hindustanus (Sh) bleomycin resistance (Ble) genes conferring resistance to these antibiotics have each been inserted into two plant expression vectors. They are flanked by the nopaline synthase (nos) or the cauliflower mosaic virus (CaMV) 35S promoters on one side, and by the nos polyadenylation signal on the other. These four chimaeric genes were introduced into the binary transformation vector pGA 492, which were thereafter mobilized into Agrobacterium tumefaciens strain LBA 4404. The resulting strains were used to transform Nicotiana tabacum cv. Xanthi nc using the leaf disc transformation procedure. In all cases, phleomycin- and bleomycin-resistant tobacco plants were regenerated from transformed cells under selective conditions; however the highest frequency of rooted plants was obtained when transformation was carried out with the Sh Ble gene under the control of the 35S promoter. Phleomycin resistance was stably transmitted to sexual offspring as a dominant nuclear trait as confirmed by Southern blotting.     

Characterisation of protoplasts from hypocotyls of Helianthus annuus in relation to their tissue origin

Cells and protoplasts isolated from three different tissues of sunflower hypocotyls and cultured either in liquid or agarose medium were compared in terms of their volume, DNA content, division potential and embryoid formation. Epidermal and external cortical cells differ from other tissue cells by their small size, their weak response to plasmolysis and their low DNA content (around 1C). They contribute only very weakly to the dividing protoplast population. In contrast, protoplasts from cortical and medullar cells both have similar division potential, reaching 50%. The nuclear DNA content of these two cell types, as well as their corresponding protoplasts, has a 2C value, taking root tip cells in G0 phase as standard. The culture conditions induce the same specific response in protoplasts isolated from both tissues: exclusively loose colony formation in liquid medium, and mainly production of embryoids in agarose medium.     

Pectolytic enzyme treatment of sunflower explants prior to wounding and cocultivation with Agrobacterium tumefaciens, enhances efficiency of transient β-glucuronidase expression

The genetic transformation of sunflower is very difficult. The protocols published so far combined wounding of meristematic axes by microprojectils or glass beads and gene transfer by Agrobacterium tumefaciens . The yields of transgenic plants were well below 0.1% in all cases. Treatment of meristematic explants of germinating seedlings by a pectinase, before wounding, followed immediately by Agrobacterium tumefaciens cocultivation, enhanced β -glucuronidase (GUS) gene expression in 14-day-old in vitro cultivated explants. Moreover, pectinase-treated explants showed a significant increase in interaction with bacteria compared to non-treated ones. This result suggests facilitated access of the bacteria to competent sunflower cells, due to cell-wall digestion by the pectinases. Furthermore, we show here, using competition experiments, that integrin-like proteins do not seem to be involved in the enhancement of bacterial binding to the cell explants.     

Effect of atmospheric pressure on sunflower (Helianthus annuus L.) protoplast division

Summary Sunflower protoplasts were cultured in liquid medium under high atmospheric pressure (0.2 to 0.6 MPa) and the plating efficiency, cell wall synthesis and microtubule organization were assessed. In 7-day-old cultures under a pressure of 0.4 MPa and above, the division rate was strongly reduced by more than 60% as compared to the control. Although most of the protoplasts had begun to regenerate a new cell wall they were unable to complete this process. Pressure also had an inhibitory effect on microtubule synthesis. The percentage of protoplasts showing a disassembled cortical network of microtubules was significantly increased up to 60% of the population. These effects were reversible: when protoplasts were transferred to normal pressure most of them rapidly recovered their capacity to divide and afterwards developed normally. Culturing protoplasts under a pressurized atmosphere revealed to be a good model system for studying cortical microtubule dynamics.Abbreviations BSA bovine serum albumin - PBS phosphate buffered saline - TBS tris buffer saline - MT(s) microtubule(s)     

Thidiazuron-induced shoot organogenesis from mature leaf explants of scented <Emphasis Type="Italic">Pelargonium capitatum</Emphasis> cultivars

Shoot organogenesis from mature leaf tissues of two scented Pelargonium capitatum cultivars, ‘Attar of Roses’ and ‘Atomic Snowflake’, grown in the greenhouse, were optimized in the presence of thidiazuron (TDZ). The protocol involved preculture of leaf sections on basal Murashige and Skoog (MS) medium supplemented with 10 μM TDZ, 4.4 μM of 6-benzyladenine (BA) and 5.4 μM α-naphtaleneacetic acid (NAA) for a period of 2 weeks and followed by subculture of explants to a fresh medium containing 4.4 μM BA and 5.4 μM NAA. Frequency of regeneration reached approximately 93% for both cultivars, with the induction of more than 100 shoots per explant. Regenerated plantlets were rooted on half-strength MS medium supplemented with 4.4 mM sucrose and 8.6 μM of Indole-3-acetic acid (IAA). All regenerated shoots from both cultivars developed roots when transferred to organic soil mix, acclimatized, and successfully transferred to greenhouse conditions. When regenerated shoots were transferred to hydroponic conditions, frequency of survival was 76.2 and 61.9% for ‘Attar of Roses’ and ‘Atomic Snowflake’, respectively.     

Nitrate reductase regulation in tomato roots by exogenous nitrate: a possible role in tolerance to long-term root anoxia

The mechanism of nitrate reductase (NR) regulation under long-term anoxia in roots of whole plants and the putative role of nitrate in anoxia tolerance have been addressed. NR activity in tomato roots increased significantly after 24 h of anaerobiosis and increased further by 48 h, with a concomitant release of nitrite into the culture medium. Anoxia promoted NR activation through dissociation of the 14-3-3 protein inhibitor and NR dephosphorylation. After 24 h of anoxia, the total amount of NR increased slightly up to 48 h. However, NR-mRNA levels remained constant between 0 h and 24 h of root anoxia and decreased after 48 h. This is probably due to the inhibition of NR degradation and the accumulation of its native form. NR was slightly dephosphorylated in the absence of oxygen and nitrate. Under anoxia, NR dephosphorylation was modulated by nitrate-controlled NR activity. In addition, the presence of nitrate prevents anoxic symptoms on leaves and delays wilting by 48 h during root anoxia. In the absence of nitrate, plants withered within 24 h, as they did with tungstate treatment, an inhibitor of NR activity. Thus, anoxia tolerance of tomato roots could be enhanced by nitrate reduction.     

EDTA-assisted phytoextraction of lead and cadmium by Pelargonium cultivars grown on spiked soil

The aim of this study was to assess EDTA-assisted Pb and Cd phytoextraction potential of locally grown Pelargonium hortorum and Pelargonium zonale. Plants were exposed to different levels of Pb (0–1500?mg kg^?1) and Cd (0–150?mg kg^?1) in the absence or presence of EDTA (0–5?mmol kg^?1). P. hortorum and P. zonale accumulated 50.9% and 42.2% higher amount of Pb in shoots at 1500?mg kg^?1 Pb upon addition of 5?mmol kg^?1 EDTA. Plant dry biomass decreased 46.8% and 64.3% for P. hortorum and P. zonale, respectively at the combination of 1500?mg kg^?1 Pb and 5?mmol kg^?1 EDTA. In Cd and EDTA-treated groups, P. hortorum and P. zonale accumulated 2.7 and 1.6-folds more Cd in shoots at 4 and 2?mmol kg^?1 EDTA, respectively, in 150?mg Cd kg^?1 treatment. Plant dry biomass of P. hortorum and P. zonale was reduced by 46.3% and 71.3%, respectively, in soil having 150?mg Cd kg^?1 combined with 5?mmol kg^?1 EDTA. Translocation factor and enrichment factor of both plant cultivars at all treatment levels were >1. Overall, the performance of P. hortorum was better than that of P. zonale for EDTA-assisted phytoextraction of Pb and Cd.     

Functional proteomic view of metabolic regulation in "Aromatoleum aromaticum" strain EbN1

The denitrifying "Aromatoleum aromaticum" strain EbN1 utilizes a wide range of aromatic and nonaromatic compounds under anoxic and oxic conditions. The recently determined genome revealed corresponding degradation pathways and predicted a fine-tuned regulatory network. In this study, differential proteomics (2-D DIGE and MS) was used to define degradation pathway-specific subproteomes and to determine their growth condition dependent regulation. Differential protein profiles were determined for cultures adapted to growth under 22 different substrate and redox conditions. In total, 354 different proteins were identified, 199 of which displayed significantly changed abundances. These regulated proteins mainly represented enzymes of the different degradation pathways, and revealed different degrees of growth condition specific regulation. In case of three substrate conditions (e.g. phenylalanine, anoxic), proteins previously predicted to be involved in their degradation were apparently not involved (e.g. Pdh, phenylacetaldehyde dehydrogenase). Instead, previously not considered proteins were specifically increased in abundance (e.g. EbA5005, predicted aldehyde:ferredoxin oxidoreductase), shedding new light on the respective pathways. Moreover, strong evidence was obtained for thus far unpredicted degradation pathways of three hitherto unknown substrates (e.g. o-aminobenzoate, anoxic). Comparing all identified regulated and nonregulated proteins provided first insights into regulatory hierarchies of special degradation pathways versus general metabolism in strain EbN1.     

Production of stable transgenic sunflowers (Helianthus annuus L.) from wounded immature embryos by particle bombardment and co-cultivation with Agrobacterium tumefaciens

Split embryonic axes of 21-day old immature sunflower (Helianthus annuus L.) embryos were bombarded by microparticles and then co-cultured with disarmed Agrobacterium tumefaciens strain EHA105 bearing a binary vector carrying nptII and uidA genes. Apical shoot bud development and organogenesis induced on the explants led T₀ transgenic plants. Southern blot analysis revealed complex integration patterns in T₀ plants. The uidA gene segregated as a dominant trait and single-insertion events were observed in T₁ plants. Patterns similar to those of T₁ plants were observed in T₂ progeny.