In vitro studies of salt and drought tolerance at the physiological, cellular and molecular levels in Medicago truncatula

My aim was to acclimate calli of Medicago truncatula to be NaCl tolerant and discover the extent to which various traits could act as markers of improved NaCl tolerance in vitro. I developed a new tissue culture protocol incorporating multi-step up acclimation (Phase I) followed by two rounds of culture ± NaCl (Phases II and III) over 0 to 350 mM exogenous NaCl. Following Phases I, II and III, calli were tolerant at 150 mM and embyrogenically competent at 100 mM NaCl. Remarkably, in Phase III, calli grew faster in the 100 and 150 mM NaCl treatments compared with the 0 mM controls. There were positive and negative linear relationships between Na+ and K+ uptake and exogenous NaCl concentration, respectively that intercepted each other at 160 mM suggesting a Na+/K+ homeostasis at this NaCl concentration. Proline level and vacuolar size peaked at 100/150 mM whilst highest osmolarity and lowest water content were at 250/350 mM NaCl. The concentration of water soluble sugars was positively related to 0 to 250 mM NaCl. Flow cytomerty was used to examine the extent to which cells underwent to endoreduplication. From 50 to 250 mM NaCl, cells were observed in G1 (2C), G2 (4C) or a state of endopolyploidy (8C). Hence, callus growth and embryogenesis occurred regardless of endoreduplication. Expression of genes linked to growth (WEE1), in vitro embryogenesis (SERK), salt tolerance (SOS1), proline synthesis (P5CS) and ploidy level (CCS52 and WEE1) peaked at 100/150 mM NaCl. Hence, these genes and various physiological traits except sugar levels, served as useful markers for NaCl tolerance. To my knowledge, this is the first report of multi-step acclimation conferring salt tolerance in leaf-derived calli of Medicago truncatula. A pilot study of drought tolerance, mimicked by polyethylene (PEG), demonstrated tolerance to 10% PEG increases in gene expression of WEE1, with proline and sugar accumulation