We describe the construction of a new plant transformation vector, pBINPLUS, based on the popular pBIN19 vector. Improvements over pBIN19 include location of the selectable marker gene at the left T-DNA border, a higher copy number in E. coli, and two rare restriction sites around the multiple cloning site for easier cloning and analysis of T-DNA insertions in plant genomes.

The effects of subcellular localization on single-chain antibody (scFv) expression levels in transgenic tobacco was evaluated using an scFv construct of a model antibody possessing different targeting signals. For translocation into the secretory pathway a secretory signal sequence preceded the scFv gene (scFv-S). For cytosolic expression the scFv antibody gene lacked such a signal sequence (scFv-C). Also, both constructs were provided with the endoplasmic reticulum (ER) retention signal KDEL (scFv-SK and scFv-CK, respectively). The expression of the different scFv constructs in transgenic tobacco plants was controlled by a CaMV 35S promoter with double enhancer. The scFv-S and scFv-SK antibody genes reached expression levels of 0.01% and 1% of the total soluble protein, respectively. Surprisingly, scFv-CK transformants showed considerable expression of up to 0.2% whereas scFv-C transformants did not show any accumulation of the scFv antibody. The differences in protein expression levels could not be explained by the steady-state levels of the mRNAs. Transient expression assays with leaf protoplasts confirmed these expression levels observed in transgenic plants, although the expression level of the scFv-S construct was higher. Furthermore, these assays showed that both the secretory signal and the ER retention signal were recognized in the plant cells. The scFv-CK protein was located intracellularly, presumably in the cytosol. The increase in scFv protein stability in the presence of the KDEL retention signal is discussed.

Somatic embryogenesis of carrot (Daucus carota L.) is inhibited by the glycosylation inhibitor tunicamycin. This inhibition is reversible by the addition of correctly glycosylated glycoproteins which have been secreted into the culture medium. To identify the proteins responsible for complementation, glycoproteins present in the medium of embryo cultures were purified and tested for their activity in the tunicamycin inhibition/ complementation assay. A 38-kDa glycoprotein was purified that could restore embryogenesis to more than 50% of that in untreated controls. This 38-kDa glycoprotein was identified as a heme-containing peroxidase on the basis of its A405/A280 ratio (Reinheit Zahl or RZ) and enzyme activity. The 38-kDa peroxidase consisted of four different cationic isoenzymes of which only one or possibly two appeared active in the complementation assay. The cationic peroxidase isoenzymes from the carrot medium could be effectively replaced by cationic horseradish peroxidases which depended on their catalytic properties for their ability to restore tunicamycin-inhibited somatic embryogenesis.

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EPI, an extracellular protein from carrot (Daucus carota) cell suspensions, has been partially characterized by means of an antiserum and a cDNA clone. In both embryo and suspension cultures different molecular mass EPI proteins were detected, some of which (31, 32, 52, and 54 kilodaltons) were bound to the cell wall and released into the medium, whereas others (49, 60, and 62 kilodaltons) were more firmly bound to the cell wall and could be extracted with a salt solution. Immunoprecipitation of in vitro translation products revealed a single primary translation product of 45 kilodaltons, suggesting that EP1 heterogeneity is due to differential posttranslational modification. In seedlings organ-specific modification of EP1 proteins was observed, a phenomenon which did not persist in suspension cultures initiated from different seedling organs. In culture EP1 proteins were only found to be associated with vacuolated, nonembryogenic cells, and on these cells they were localized in loosely attached, pectincontaining cell wall material. Purified 52/54 kilodaltons EPI proteins did not alleviate the inhibitory effect of the glycosylation inhibitor tunicamycin on somatic embryogenesis.whereas the extracellular protein patterns of nonembryogenic mutant cell lines deviate from this characteristic pattern (2). Second, the acquisition of embryogenic potential in a newly initiated culture has been shown to be accelerated by the addition of high-molecular weight, heat-labile components from an established embryogenic cell line (3). Third, inhibition of somatic embryogenesis with the glycosylation inhibitor tunicamycin can be complemented by the simultaneous addition of extracellular proteins from an uninhibited embryo culture (2).To obtain more information about the nature of cell suspension extracellular proteins, we screened a carrot Xgtl 1 cDNA expression library with an antiserum raised against total embryo medium proteins. (10) containing 2,4-D and subcultured with 14-d intervals at an initial cell density of 1. 106 cells/mL. Seven days after subculturing embryo cultures were initiated by inoculating a culture fraction enriched for proembryogenic masses at a density of 2.104 cells/mL in B5 medium. Inhibition/complementation assays with tunicamycin were performed as described previously (2). High-density suspension cultures from fennel (Foeniculum vulgare), parsley (Petroselinum crispum), and caraway (Carum carvi) were initiated from seeds obtained locally and maintained under the same conditions as the carrot 10 cell line. Tomato (Lycopersicon esculentum) suspensions were cultured in R3B medium (18). Carrot seedlings were grown from "Flakkese" SG766 Trophy seeds and dissected when the first leaves had fully expanded. Zygotic embryos were ob4Abbreviations: EPI, extracellular protein 1; TBS, Tris-buffered saline; SSC, 0.15 M NaCl/1 5 mM Na citrate (pH 7.0).