Simultaneously with the progress in plant biotechnology since the 1980s, new methods in plant pathology have been developed. This review summarizes papers that cover basic research on the effects of selective agents on in vitro cultures of host plants, as well as applications of agents in regeneration systems that result in lines with increased variability in resistance or susceptibility. The first part of the study deals with theoretical aspects of the interactions between plants and toxin-producing pathogens, mode of phytotoxic action, and host-and non-host-selective toxins. The second part lists and describes various agents used for selections in vitro. In the last two decades more than 100 publications focused on these selections for the improvement of resistance to plant pathogens. Over 30 plant species were examined to utilise various selection agents extracted from about 40 plant pathogens. The review covers basic research studies and methods that elucidate the relationships between in vitro and in vivo mechanisms of resistance, but also try to develop practical applications to obtain resistant breeding lines. Such methods often utilise some type of explant cultures of the host plants that are treated with various selective agents (culture filtrates, toxins, elicitors), which then elicit typical reactions that parallel those by the pathogens. Their application successfully resulted in resistant lines in banana, carnation, grapevine, strawberry and wheat. Nowadays, these techniques are an important complement to classical breeding methods.

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Six pea (Pisum sativum L.) cultivars (Adept, Komet, Lantra, Olivín, Oskar, Tyrkys) were transformed via Agrobacterium tumefaciens strain EHA105 with pBIN19 plasmid carrying reporter uidA (β-glucuronidase, GUS, containing potato ST-LS1 intron) gene under the CaMV 35S promoter, and selectable marker gene nptII (neomycin phosphotransferase II) under the nos promoter. Two regeneration systems were used: continual shoot proliferation from axillary buds of cotyledonary node in vitro, and in vivo plant regeneration from imbibed germinating seed with removed testa and one cotyledon. The penetration of Agrobacterium into explants during co-cultivation was supported by sonication or vacuum infiltration treatment. The selection of putative transformants in both regeneration systems carried out on media with 100 mg dm -3 kanamycin. The presence of introduced genes was verified histochemically (GUS assay) and by means of PCR and Southern blot analysis in T 0 putative transformants and their seed progenies (T 1 to T 3 generations). Both methods, but largely in vivo approach showed to be genotype independent, resulting in efficient and reliable transformation system for pea. The in vivo approach has in addition also benefit of time and money saving, since transgenic plants are obtained in much shorter time. All tested T 0 -T 3 plants were morphologically normal and fertile.

The effect of chemical additives (acetosyringone, AS; L-cysteine, CYS; dithiothreitol, DTT; glutathione, GSH; cellulase, CEL; pectinase, PEC) and light regimes (16/8 light/dark photoperiod, 16L/8D; continuous light, 24L; continuous dark, 24D) applied during cocultivation procedure of pea explants with Agrobacterium tumefaciens on transformation efficiency was studied. A hypervirulent strain of A. tumefaciens EHA 105 with two plasmids, namely pGT89 and pBIN19, both carrying reporter gus-int gene, and bar or nptII selectable marker gene, respectively, was used for genetic transformation of cotyledonary node explants of three dry seed pea cultivars Adept, Komet and Menhir. The focus was laid on cocultivation step (48 h) of transformation protocol. After chemical or physical treatments, transient GUS expression was recorded 20 days after cocultivation as a measure of successful transformation, using a four category scale (0 -without GUS expression, 1 -weak, 2 -medium and 3 -strong GUS expression) for calculation of IGE (Intensity of GUS Expression). Of the tested chemical cocultivation additives, 100 lM AS and 50 mg CYS significantly improved GUS expression (IGE value), while DTT, GSH and both macerating enzymes (CEL, PEC used either separately or in combination) either had no positive effect or were even negative. There were no statistically significant differences between the light regimes tested. Nevertheless, cocultivation in 24L, without chemical additives, reproducibly resulted in the highest frequency of explants scored in category 3 of GUS expression (followed by 24D and 16L/8D treatment). However, application of 100 lM AS reverted this trend. Cv. Adept yielded higher transformation frequencies than cvs. Menhir and Komet. Plasmid pGT89 produced a higher IGE value than pBIN19. Based on our results, the improved cocultivation step for pea consists of 48 h cocultivation at 20 ± 2°C, with 50 mg l -1 CYS and 100 lM AS, 16L/8D photoperiod (or without AS in continuous light).

Pea (Pisum sativum L.) somaclones of cultivars Adept, Komet and Bohatýr were obtained after selection in vitro with Fusarium solani filtrate and fusaric acid (FA). R2 regenerants were analyzed by random amplification of polymorphic DNA (RAPD; OPAB4, P-14, UBC-556) and inter-retrotransposon amplification polymorphism (IRAP; Ogre) markers. Marker UBC-556 showed different banding patterns for each cultivar but without specific bands for selected and control plants. Markers OPAB4, P14, and Ogre were useful for clear discrimination between selected and non-selected variants of all three cultivars. Flow cytometry analysis proved the same genome size of selected and non-selected pea lines. Therefore in vitro selection by pathogen derived agents could be the efficient method for obtaining pea somaclones with increased resistance to F. solani.

Optimisation of in vitro regeneration systems of two explant types for low-tannine cultivars of faba bean based on culturing of shoot apices and cotyledonary nodes were provided by usage of various antioxidants -ascorbic acid, citric acid, glutathione and activated charcoal. In subsequent testing, the combined eff ects of antioxidants with transformation co-cultivation compounds acetosyringone and L-cysteine was studied. The application of antioxidants lead to decreased callogenesis, citric acids treatments (50 mg.l −1

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