Mesophyll protoplasts of Brassica oleracea var. botrytis were successfully transformed using polyethylene glycol (PEG). The success of plant transformation depended on both gene transfer and plant regeneration. Parameters, such as PEG and vector concentrations and heat shock conditions were tested in experiments on transient expression of the beta-glucuronidase (EC 3.2.1.31) gene and the most suitable conditions for DNA uptake were determined. Two antibiotic resistance marker genes for neomycin phosphotransferase (EC 2.7.1.95) and hygromycin phosphotransferase (EC 2.7.1.104), and three vector plasmids with different lengths were used to obtain stable transformants.

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Somatic hybrids were produced by PEGinduced symmetric and asymmetric protoplast fusions in order to transfer resistance to Alternaria brassicicola, A. brassicae, Phoma lingam, Plasmodiophora brassicae and Turnip mosaic virus (TuMV) into Brassica oleracea var. capitata (cv. 'Toskama') and botrytis (cv. 'Korso'). As resistance donors, ten species belonging to several genera of the family Brassicaceae including wild relatives were used. Of 2,189 plants (somatic hybrids, partially in vitro cloned) tested, 1,616 (73.8%) were resistant against at least one of the pathogens, indicating that, mostly, a successful resistance transfer has taken place. Five hundred and twenty-two hybrids showed multiple resistances to two, three and, in a single case, to four pathogens. Irrespective of the donor parents used in the fusion, a broad variability in symptom manifestation ranging from 0 (without symptoms) to 9 (highly susceptible) could be observed. With regard to the Alternaria pathogens, Sinapis alba, B. nigra and B. juncea were the most effective resistance donors, whereas fusions with Raphanus sativus resulted in the most hybrids with resistance to clubroot and TuMV. As could be shown especially in asymmetric fusions with S. alba, Barbarea vulgaris and Hesperis matronalis, transferred resistance to a pathogen may not correspond with resistance exhibited by the donor parent. Some combinations in which both parents were highly susceptible, e.g. R. sativus (?) B. oleracea var. capitata, yielded hybrids that exhibited strong resistance, e.g. to A. brassicicola, revealing that a new type of resistance might be occurring. With regard to the Alternaria pathogens, resistance expression was very unstable. Many hybrids into which (also variable) resistance of some donors, such as B. vulgaris, S. alba and B. carinata, was transferred became as highly susceptible as those of which the fusion parents did not show any resistance reaction (e.g. R. sativus). For reliable characterization of the resistance response, hybrids should be subjected to several resistance tests during growth period of the host, at least until flowering.

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Green fluorescent protein (GFP) was successfully used as a visual reporter at various stages of carrot (Daucus carota L.) transformation. GFPfluorescence was non-invasively observed in protoplasts, callus and plants after the delivery of mgfp5-er gene using two transformation methods: direct DNA transfer into polyethylene glycol (PEG) -treated protoplasts and inoculation of root discs with Agrobacterium rhizogenes. Transient GFP-expression was detected in the treated protoplasts and monitored during the first week of the cell culture until the stable level of expression was observed. It was useful for the comparison of protoplast susceptibility to DNA uptake and the transgene expression as the fluorescence declined with various rates depending on the used carrot genotype and PEG-concentration. GFP-monitoring in callus enabled the selection of stably expressing lines. It also allowed verification of the homogeneous tissue composition with regard to the expression of the transgene. In plants, GFP-performance depended on the assayed tissue and organ despite of the constitutive 35S promoter. The expression was visually detected in both vegetative and generative parts, but particularly strong fluorescence was observed in leaf marginal meristems, petioles, stems, and styles. Those tissues can be convenient for examination of the transgenic plants during their growth. The results encourage that GFP is a valuable reporter and can be routinely used for optimization of transformation protocol, selection of transformants and monitoring transgenic carrot.