ИСТИНА

Development of genetically encoded protein-based nanosensors for in vivo detection of ion fluxes is a promising approach to study physiological processes in plants. However, developing stable transformants is time-consuming being a significant barrier for the analysis of these sensors. The composite plant system (transgenic roots on wild-type shoots) allows rapid “in root” testing of transgenes in the context of a complete plant. We propose this method as a useful tool to study root physiology using nanosensors. We have implemented the protocol of A. rhizogenes mediated hypocotyl dip transformation of Arabidopsis seedlings previously described by Limpens et al. (2004). Composite plants were generated by inoculating shoots of Arabidopsis thaliana (L.) Heynh. ecotype Colombia-0 with Agrobacterium rhizogenes strain MSU440, containing plasmid with a GFP-mRFP1 pH nanosensor, developed previously (Ytting C.K. and Fuglsang A.T. unpublished data) or pRedRoot plasmid with the red fluorescence marker DsRED (Limpens et al. 2004). Seedling age, sucrose content of the media and freshly prepared A. rhizogenes cultures were found to be critical steps affecting transformation efficiency. We have observed 90% root regeneration, but only 30% of the newly formed roots were ageotropic and highly branched showing “hairy-root” phenotype. In the case of the pRedRoot plasmid we observed a callus-like structure from which new adventitious roots were induced. Two to three weeks after inoculation, we have tested hairy roots for the presence of the fluorescent marker. Imaging of DsRED1 or GFP/RFP was done using a Leica MZIII fluorescence stereomicroscope and a Leica SP2 confocal microscope. The most striking observation under high magnification was abnormal root hair morphology in Arabidopsis roots after A. rhizogenes transformation. We have observed twisted, branched and splitting root hairs in transgenic roots regardless of the containing plasmid. We suggest that aux and/or rol genes could be responsible for the altered hair root phenotype. We conclude that this promising method by further improvements will allow efficient and rapid testing and analyzing of transient nanosensor expression in Arabidopsis roots. The detailed analysis of the molecular mechanisms that led to the altered root hair development in transgenic roots of composite plants is a challenge for future work.