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Agrobacterium tumefaciens is generally used to achieve genetic transformation of plants. The temperatures that have been used for infection with Agrobacterium in published transformation protocols differ widely and, to our knowledge, the effect of temperature on the efficiency of T-DNA transfer to plants has not been investigated systematically. Agrobacterium tumefaciens strains harbouring a binary vector with the beta-glucuronidase (uidA) gene and either a nopaline-, an octopine- or an agropine/succinamopine-type helper plasmid were tested in two transformation systems at temperatures between 15 and 29 degrees C. One system involved cocultivation of Phaseolus acutifolius callus whereas in the other system Nicotiana tabacum leaves were vacuum-infiltrated. In both situations, irrespective of the type of helper plasmid, the levels of transient uidA expression decreased notably when the temperature was raised above 22 degrees C. Expression was low at 27 degrees C and undetectable at 29 degrees C. We anticipate that the efficiency of many published transformation protocols can be improved by reconsidering the factor of temperature

The accumulation of five murine single-chain variable fragments, binding to dihydroflavonol 4-reductase, was analyzed in transgenic Petunia hybrida plants. The five scFv-encoding sequences were cloned in an optimized plant transformation vector for expression in the cytosol under control of the 35S promoter. In a transient expression assay we found that the scFv expression levels were reproducible and correlated with those in stably transformed petunia. Our results show that accumulation in the cytosol strongly depends on the intrinsic properties of the scFv fragment. Three of the five scFv fragments accumulated to unexpectedly high levels in the cytosol of the primary transformants, but no phenotypic effect could be detected. Experimental results indicate that one of the scFv fragments accumulated in the cytosol to 1% of the total soluble protein as a functional antigen-binding protein in the absence of disulphide bonds. This observation supports the idea that certain antibody fragments do not need disulphide bonds to be stable and functional. Such scFv scaffolds provide new opportunities to design scFv fragments for immunomodulation in the cytosol.Keywords: Agrobacterium-mediated transient expression, disulfide bond, immunomodulation, Petunia hybrida, scFv fragment.There is a growing interest in the expression of antibodies and antibody fragments inside and outside the plant cell to modulate the function of the corresponding antigen [1±6], a strategy called immunomodulation. To obtain immunomodulation, the antibodies have to accumulate as functional proteins at sufficiently high levels. However, achieving high accumulation levels of complete antibodies in the cytosol of the plant cell has proven to be problematic. This difficulty was attributed to bad assembly conditions and subsequent degradation of unassembled heavy and light chains [7]. Therefore, most studies have concentrated on antibody-derived single-chain variable (scFv) fragments, in which the variable domains are joined by a linker peptide avoiding the need for assembling different polypeptides.To study the immunoinhibition of dihydroflavonol 4-reductase (DFR), one of the structural enzymes from the flavonoid pathway of Petunia hybrida [8], we have isolated five genes encoding DFR-specific scFv fragments (A1, A3, A4, G4 and H3) by phage display [9]. Amino acid sequence alignment showed that the scFv fragments A1, A3, and H3 are < 90% similar. Moreover, these fragments contain the same complementarity-determining region 3 (CDR3) of the V H domain, suggesting that they bind the same epitope on DFR. The remaining two scFv fragments, A4 and G4, are more divergent. All five can specifically bind DFR extracted from flower buds on Western blots, whereas ELISA experiments demonstrate that they also recognize native DFR.The dfr gene (dfrA) is expressed in the epidermis and veins of the petals, in the trichomes on the petals and flower stem, in the connectivum, in the flower stem, in the ovaries, and in the seed coat [10]. Cytological and biochemical studies h...