Establishment And Optimization Of The Regeneration System For Plantago Major L. "Giant Turkish."＆ Yeast Complementation Studies Of SeNHX1 And TtNHX1 Gene

Plantago major L. "Giant Turkish." is not only used as medicinal herb but also an important model plant of ecology. Little work has been reported on the tissue culture of Plantago major L. Establishment of the regeneration system for Plantago major L. "Giant Turkish." is the basis to select for salt tolerance mutants and transform genes. We presented here a protocol for indirect and direct regeneration of Plantago major L. "Giant Turkish." from leaf and seed explants. For indirect regeneration, fragile calli were obtained from leaf explants on MS medium supplemented with 1.0 mg/L NAA. Regenerated plantlets were obtained when these calli were cultured on MS medium containing 4.0 mg/L 6-BA. Multiple shoots were obtained on MS medium containing 0.2 mg/L IAA and 1.0 mg/L TDZ. Random amplified polymorphic DNA (RAPD) analysis of nine regenerated plantlets revealed molecular mutation. An important mechanism of plants for surviving in saline conditions is the accumulation of Na~+ in vacuoles, thereby the Na+ contents in cytoplasm keeps comparatively low and the osmotic potential of the cell decreases. Na~+/H~+ antiporters transport Na~+ from the cytoplasm to the vacuole. SeNHX1 and TtNHX1 genes were cloned from halophyte Salicornia europaea and Tetragonia tetragonioides previously. We tested the ability of TtNHX1 and SeNHX1 to suppress the Na~+ sensitivity of yeast mutants defective in endogenous Na~+/H~+ antiporters. The transformant with p416GPDSeNHX1 showed a higher growth than the wild type when treated with 500 mM, 750 mM NaCl. Expression of TtNHX1 or SeNHX1 increased the tolerance to Na+, Li+ and hygromycin of nhx1 mutant cells to an extent similar to the wild type, confirming the vacuolar localization of SeNHX1, and suggestting that the mechanism by which cations are detoxified in yeast and plants may be similar.