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Vacuum Infiltration (vacuum + infiltration)
Selected AbstractsRelationship between potato seed tuber contamination by Erwinia carotovora ssp. atroseptica, blackleg disease development and progeny tuber contaminationPLANT PATHOLOGY, Issue 2 2003I. K. Toth The relationship between contamination of potato seed tubers with Erwinia carotovora ssp. atroseptica (Eca), blackleg disease development, and the incidence and level of progeny tuber contamination in field grown crops was studied in 1998, 1999 and 2000. Seed tubers were inoculated by vacuum infiltration at three levels (low, intermediate and high) with a streptomycin-resistant marker strain of Eca (SCRI1039Str) and planted in the field. Blackleg disease development was directly related to the level of seed tuber contamination. The higher the level of seed tuber contamination, the earlier in the season blackleg disease appeared and the greater the final level of disease, which continued to rise as the season progressed. High and low levels of seed tuber contamination were related to high and low incidences of progeny tuber contamination, respectively, at all sampling times. However, an intermediate degree of seed tuber contamination tended to be associated with a low level of blackleg disease, a variable incidence of progeny tuber contamination early in the season but a high incidence later in the season. The level of progeny tuber contamination, derived from seed tubers inoculated at the three different levels of Eca, was categorized into four contamination classes (< 102, 102,103, 103,104 and > 104 marker strain colony-forming units/mL peel extract). At the lowest level of seed tuber contamination, progeny tuber contamination tended to be in the two lower categories. However, as seed tuber contamination increased, the proportion of contaminated progeny tubers in the two higher categories also increased. Overall, the results suggest that progeny tuber contamination is related to seed tuber contamination and blackleg disease, and that the threshold level of seed tuber contamination remains an important factor in predicting both blackleg disease and tuber health. [source] Arabidopsis ecotypes and mutants that are recalcitrant to Agrobacterium root transformation are susceptible to germ-line transformationTHE PLANT JOURNAL, Issue 1 2000Kirankumar S. Mysore Summary Germ-line transformation (vacuum infiltration) is frequently used to transform Arabidopsis thaliana using Agrobacterium tumefaciens. We have recently identified several Arabidopsis ecotypes and T-DNA-tagged mutants that are recalcitrant to Agrobacterium -mediated transformation of cut root segments. Some of these ecotypes and mutants are deficient in their ability to bind bacteria. Some are deficient in T-DNA integration. We report here that using a germ-line transformation protocol we transformed these ecotypes and mutants, including attachment- and integration-defective Arabidopsis plants, with a frequency similar to that of highly susceptible wild-type plants. However, we could not transform otherwise highly susceptible Arabidopsis plants by germ-line or root transformation using several vir and attachment-deficient Agrobacterium mutants. These results indicate that certain plant factors important for transformation may exist in germ-line tissue but may be lacking in some somatic cells. [source] Optimization of the bioprocessing conditions for scale-up of transient production of a heterologous protein in plants using a chemically inducible viral amplicon expression systemBIOTECHNOLOGY PROGRESS, Issue 3 2009Michael A. Plesha Abstract Use of transient expression for the rapid, large-scale production of recombinant proteins in plants requires optimization of existing methods to facilitate scale-up of the process. We have demonstrated that the techniques used for agroinfiltration and induction greatly impact transient production levels of heterologous protein. A Cucumber mosaic virus inducible viral amplicon (CMViva) expression system was used to transiently produce recombinant alpha-1-antitrypsin (rAAT) by co-infiltrating harvested Nicotiana benthamiana leaves with two Agrobacterium tumefaciens strains, one containing the CMViva expression cassette carrying the AAT gene and the other containing a binary vector carrying the gene silencing suppressor p19. Harvested leaves were both infiltrated and induced by either pressure or vacuum infiltration. Using the vacuum technique for both processes, maximum levels of functional and total rAAT were elevated by (190 ± 8.7)% and (290 ± 7.5)%, respectively, over levels achieved when using the pressure technique for both processes. The bioprocessing conditions for vacuum infiltration and induction were optimized and resulted in maximum rAAT production when using an A. tumefaciens concentration at OD600 of 0.5 and a 0.25-min vacuum infiltration, and multiple 1-min vacuum inductions further increased production 25% and resulted in maximum levels of functional and total rAAT at (2.6 ± 0.09)% and (4.1 ± 0.29)% of the total soluble protein, respectively, or (90 ± 1.7) and (140 ± 10) mg per kg fresh weight leaf tissue at 6 days post-induction. Use of harvested plant tissue with vacuum infiltration and induction demonstrates a bioprocessing route that is fully amenable to scale-up. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] | ||||||||||