Enhanced Saline Stress Resistance in Threonine and Methionine Overproducing Mutant Cell Line from Protoplast Culture of Rice ( Oryza sativa L.)

Abstract

Summary This study was designed to examine whether saline stress resistance was enhanced in mutant in which free amino acids were overproduced. We selected a mutant cell line (RLTA-2) that overproduced threonine and methionine, and a NaCl resistant cell line (RNaCI-1) through protoplast culture treated with ethylmethane-sulfonate (EMS) with addition of inhibitory concentrations of 1 mM each of lysine plus threonine (1 mM LT) and 200 mM NaCl, respectively. Concentrations of threonine, methionine and the total free amino acids changed largely, being 13.8, 6.2 and 1.6 times higher in RLTA-2 than in the wild type, respectively. Proline concentration increased slightly in RLTA-2 when it was subcultured on the medium supplemented with 200 mM NaCl (RLTA-2-200), i.e. ca. 1.4 times in RLTA-2, while a remarkable increase of proline concentration was observed in RNaCI-1, i.e. 11.5 times as compared with the wild type. Furthermore, calcium accumulation was 3 to 5 times in RLTA-2, but not in RNaCl-1, regardless of NaCl concentration. The selected RLTA-2 calli grew better than the wild type under high concentration of NaCl, but the growth rate of RLTA-2 and RNaCl-1 calli was almost similar above 150 mM NaCl concentration. Almost all RLTA-2 calli survived on N6 medium supplemented with 200 mM NaCl. The capacity to maintain the low level of K + and the high level of Ca ++ in the RLTA-2 cell line under the high level of Na + was highly correlated with free amino acid level as compatible cytoplasmic solute and nitrogen source in cytoplasm. The present results indicate the enhancement of saline resistance in the mutant cell line in which free amino acids are overproduced. The relationship of free amino acid accumulation to enhanced saline resistance is discussed.