Enhancement of Recombinant Protein Production in Transgenic Nicotiana benthamiana Plant Cell Suspension Cultures with Co-Cultivation of Agrobacterium Containing Silencing Suppressors.

We have previously demonstrated that the inducible plant viral vector (CMViva) in transgenic plant cell cultures can significantly improve the productivity of extracellular functional recombinant human alpha-1-antiryspin (rAAT) compared with either a common plant constitutive promoter (Cauliflower mosaic virus (CaMV) 35S) or a chemically inducible promoter (estrogen receptor-based XVE) system. For a transgenic plant host system, however, viral or transgene-induced post-transcriptional gene silencing (PTGS) has been identified as a host response mechanism that may dramatically reduce the expression of a foreign gene. Previous studies have suggested that viral gene silencing suppressors encoded by a virus can block or interfere with the pathways of transgene-induced PTGS in plant cells. In this study, the capability of nine different viral gene silencing suppressors were evaluated for improving the production of rAAT protein in transgenic plant cell cultures (CMViva, XVE or 35S system) using an Agrobacterium-mediated transient expression co-cultivation process in which transgenic plant cells and recombinant Agrobacterium carrying the viral gene silencing suppressor were grown together in suspension cultures. Through the co-cultivation process, the impacts of gene silencing suppressors on the rAAT production were elucidated, and promising gene silencing suppressors were identified. Furthermore, the combinations of gene silencing suppressors were optimized using design of experiments methodology. The results have shown that in transgenic CMViva cell cultures, the functional rAAT as a percentage of total soluble protein is increased 5.7 fold with the expression of P19, and 17.2 fold with the co-expression of CP, P19 and P24

Genetic transformation of Vitis vinifera L. cvs Thompson Seedless and Chardonnay with the pear PGIP and GFP encoding genes

Transgenic plants of Vitis vinifera L. cvs. Chardonnay and Thompson Seedless expressing the β-glucuronidase gene (GUS) and either the pear polygalacturonase inhibiting protein gene (PGIP) or the green fluorescent protein gene (GFP) were produced via somatic embryogenesis. Various media and culture conditions were tested in order to develop an efficient transformation method. Best results were obtained when embryogenic callus was initiated from anthers cultured on PIV medium and maintained in PT medium. Embryogenic lines of the rootstocks Saint George, 110 Richter and Freedom and from inflorescence primordia of Chardonnay and 110 Richter were also established using the same media. Inoculation with 109 cells·ml-1 Agrobacterium resulted in a higher number of selected calli than cultures inoculated with 107 or 108 cells·ml-1. Plants were regenerated in a modified WP medium from up to 46 % of the selected callus. Approximately 80 % of the lines expressed GUS and either PGIP or GFP but a low correlation was found between β-glucuronidase and polygalacturonase inhibiting protein activities.

Molecular markers for grape characterization

Research NoteFive cultivars and 9 Pinot noir clones were used to test the usefulness of RFLP and RAPD markers and determine whether clonal selections could be differentiated

Synteny analysis in Rosids with a walnut physical map reveals slow genome evolution in long-lived woody perennials.

BackgroundMutations often accompany DNA replication. Since there may be fewer cell cycles per year in the germlines of long-lived than short-lived angiosperms, the genomes of long-lived angiosperms may be diverging more slowly than those of short-lived angiosperms. Here we test this hypothesis.ResultsWe first constructed a genetic map for walnut, a woody perennial. All linkage groups were short, and recombination rates were greatly reduced in the centromeric regions. We then used the genetic map to construct a walnut bacterial artificial chromosome (BAC) clone-based physical map, which contained 15,203 exonic BAC-end sequences, and quantified with it synteny between the walnut genome and genomes of three long-lived woody perennials, Vitis vinifera, Populus trichocarpa, and Malus domestica, and three short-lived herbs, Cucumis sativus, Medicago truncatula, and Fragaria vesca. Each measure of synteny we used showed that the genomes of woody perennials were less diverged from the walnut genome than those of herbs. We also estimated the nucleotide substitution rate at silent codon positions in the walnut lineage. It was one-fifth and one-sixth of published nucleotide substitution rates in the Medicago and Arabidopsis lineages, respectively. We uncovered a whole-genome duplication in the walnut lineage, dated it to the neighborhood of the Cretaceous-Tertiary boundary, and allocated the 16 walnut chromosomes into eight homoeologous pairs. We pointed out that during polyploidy-dysploidy cycles, the dominant tendency is to reduce the chromosome number.ConclusionSlow rates of nucleotide substitution are accompanied by slow rates of synteny erosion during genome divergence in woody perennials

Progress toward the production of transgenic grapevines by Agrobacterium-mediated transformation

Grape possesses the basic prerequisites for Agrobacterium-mediated transformation it is a host for Agrobacterium and plant regeneration can be induced from cultured grape explants. Leaf explants were cocultivated with disarmed Agrobacterium vectors carrying kanamycin resistance and GUS genes and cultured on shoot-inducing medium containing kanamycin. After 21 d, intense and sharply-defined blue regions were observed, including some blue organized meristematic structures, consistent with plant-driven GUS gene expression. No GUS activity was detected in control explants. Among single leaf tips excised from over 200 regenerated shoots, one was GUS positive. The recovery of transgenic shoots might be improved by increasing the frequency or modifying the site of transformation and/or regeneration

Analysis of salinity tolerance of Vitis vinifera 'Thompson Seedless' transformed with AtNHX1

Several transgenic plant species expressing AtNHX1, coding for a vacuolar Na+/H+ antiporter from Arabidopsis thaliana, have shown their ability to cope with salinity. The aim of this study was to analyze the response of Vitis vinifera cv. 'Thompson Seedless' transformed with AtNHX1 to salt stress, using soil substrate or hydroponic media, and to compare the response with untransformed 'Thompson Seedless' and allegedly tolerant 'Criolla' cultivars: 'Pedro Giménez' and 'Criolla Chica'. 'Thompson Seedless' embryogenic calli were transformed with Agrobacterium tumefaciens carrying AtNHX1 under the control of CaMV 35S promoter. Transgenic and untransformed plants were grown in a greenhouse under hydroponics or in pots with soil, and were subjected to increasing concentrations of sodium chloride (NaCl) up to 150 mM for a period of 7 weeks. Growth and toxicity symptoms were less affected in transgenics as compared to the untransformed grapevines, and transgenic lines had higher shoot length, leaf area and dry weights at the end of the experiment. Root concentrations of Na in transgenics were similar or lower than that observed in untransformed genotypes. Growth impairment and toxicity symptoms were observed in all genotypes under both conditions, but effects were more severe in plants growing with hydroponic culture. Potassium content and shoot to root dry weight ratio decreased with NaCl in hydroponics but not in pots. 'Criolla' cultivars grew less than the other genotypes, although 'Pedro Giménez' always exhibited highest shoot/root ratios

Expression, Purification, and Biophysical Characterization of a Secreted Anthrax Decoy Fusion Protein in Nicotiana benthamiana.

Anthrax toxin receptor-mediated drug development for blocking anthrax toxin action has received much attention in recent decades. In this study, we produced a secreted anthrax decoy fusion protein comprised of a portion of the human capillary morphogenesis gene-2 (CMG2) protein fused via a linker to the fragment crystallizable (Fc) domain of human immunoglobulin G1 in Nicotiana benthamiana plants using a transient expression system. Using the Cauliflower Mosaic Virus (CaMV) 35S promoter and co-expression with the p19 gene silencing suppressor, we were able to achieve a high level of recombinant CMG2-Fc-Apo (rCMG2-Fc-Apo) protein accumulation. Production kinetics were observed up to eight days post-infiltration, and maximum production of 826 mg/kg fresh leaf weight was observed on day six. Protein A affinity chromatography purification of the rCMG2-Fc-Apo protein from whole leaf extract and apoplast wash fluid showed the homodimeric form under non-reducing gel electrophoresis and mass spectrometry analysis confirmed the molecular integrity of the secreted protein. The N-glycosylation pattern of purified rCMG2-Fc-Apo protein was analysed; the major portion of N-glycans consists of complex type structures in both protein samples. The most abundant (>50%) N-glycan structure was GlcNAc₂(Xyl)Man₃(Fuc)GlcNAc₂ in rCMG2-Fc-Apo recovered from whole leaf extract and apoplast wash fluid. High mannose N-glycan structures were not detected in the apoplast wash fluid preparation, which confirmed the protein secretion. Altogether, these findings demonstrate that high-level production of rCMG2-Fc-Apo can be achieved by transient production in Nicotiana benthamiana plants with apoplast targeting