A new model "Rhizogenesis in vitro" is proposed to study the biological effects of real and simulated microgravity. Rhizogenesis from leaf explants and callus cultures of Arabidopsis thaliana wild type and a scr mutant has been investigated. The SCARECROW gene is essential for generating the root radial organization as it regulates an asymmetric cell division. It was shown that roots emerged de novo in two ways: 1) from callus dedifferentiated cells (morphogenic loci) and 2) directly from the cambial cells of leaf petioles. It was revealed that roots formed de novo consist of a cap and all growth zones, like to those in embryonal roots. At the same time, the appearance of coalescent roots and roots with diminished growth zones wаs noted in the callus culture. Since roots formed in vitro on the callus surface had some structural abnormalities, roots formed in vitro on leaf explants of A. thaliana wild type, scr mutant, and transgenes were used in the subsequent studies by methods of light, electron and confocal microscopy. No significant differences were found in morphology and anatomy of roots formed in vitro on leaf explants under clinorotation in comparison with embryonal roots. The ultrasructure of cap statocytes in roots formed in vitro in the stationary conditions was typical for graviperceptive cells. A nucleus was located in the proximal part of a cell, ER cisterns - in the cell distal part of a cell and in its corners. Amyloplasts-statoliths were located in the distal part of statocytes in the control. Under clinorotation, amyloplasts were grouped in the cell center or distributed throughout the cytoplasm. Such amyloplasts' position indicates that statocytes do not function as graviperceptive cells. The characteristic features of apical meristem cells in the control were the central nucleus position, a great diversity of a size and a shape of mitochondria and plastids, ER weak development. As cells passed in the distal elongation zone (DEZ), their size enlarged, and a nucleus preserved the central location in the majority of cells. A quantity of ER-cisterns, vacuoles, and ER-bodies increased in comparison with those in meristem cells. Dictyosomes acquired polarity and produced many vesicles. Under clinorotation, the ultrastructure of meristem and elongated cells in main features was similar to that in control cells. No statistically significant differences in the organelle percentage ratio in root meristem cells of A. thaliana wild type were also found between the control and experiments. Cells of the DEZ appeared more sensitive to clinorotation. An increase in the percentage ratio of vacuoles and ER-bodies as well as the presence of numerous smaller mitochondria were found in these cells. The orientation of cortical and endoplasmic actin microfilaments in cells of different root growth zones was similar in control and under clinorotation. Unlike microfilaments, the disorganization of the cortical microtubules in DEZ cells was revealed. As cortical microtubule arrays play an essential role in the cell wall formation, we studied the structure of cell walls, measured their thickness in protoderm and epidermis of DEZ in roots of A. thaliana wild type formed de novo in control and under clinorotation. Although the structure of transverse and longitudinal cell walls under clinorotation was similar to that in control, a large convolution of transverse cell walls in DEZ was observed under simulated microgravity. Simultaneously a tendency towards the cell wall thinning under clinorotation was noted. Auxin-dependent reporter DR5rev green fluorescent protein, that is a marker of auxin localization was revealed in the central cylinder and in the cap of vertically growing roots in vitro. After 2 hours of gravistimulation, DR5rev::GFP fluorescence was observed in the physically low root side. Under clinorotation, DR5rev::GFP signal was noted in cap cells of roots which had no visible bending. Under gravistimulation of roots grown under clinorotation, the DR5rev::GFP localization was also revealed in the cap cells and epidermis. Such DR5rev::GFP position indicates the polar auxin transport, that is the characteristic feature of a gravitropic reaction, the obtained data demonstrate that clinorotated roots in vitro sense a gravitational stimulus and can perceive it. Thus, it was shown that cell differentiation occurs normally in roots of A. thaliana wild type and a scr mutant formed de novo, as in embryonal roots. In simulated microgravity, these roots are gravisensitive, so the investigated model of rhizogenesis on leaf explants is proposed for spaceflight experiments to reveal new structural and functional regularities of plant adaptation to microgravity.
