The effects of precursors,metabolic intermediate of theanine synthesis and the NO donor SNP （sodium nitroprusside）on the synthesis of theanine in tea callus were investigated by using HPLC (high performance liquid chromatography) to detect theanine contents,and an accumulation of theanine in the roots of tea plants with different cultivation time after addition of (NH4)2SO4 into cultivation liquid was also discussed.As a result,25 mmol/L ZtNH2·HCl (ethylamine hydrochloride) could increase the content of theanine in callus significantly,especially in the callus grown from the 6th day to the 12th day.The theanine concentration in callus which grown for 12 days with addition of 25 mmol/L ZtNH2·HCl reached 108.96 μmol/g in dry weight basis,7.72 times of that in the control; addition of glutamine,alanine or sodium glutamate into medium with low concentrations could also increase theanine in callus,but the increasing amount was not very obvious compared to that with addition of 25 mmol/L ZtNH2·HCl with the exception of 5 mmol/L glutamine,which could boost the theanine to 7 times of that in the control.Addition of SNP with concentration of 0.05 or 0.075 mmol/L could increase the content of theanine in callus,but the increase amount was low in general.With addition of (NH4)2SO4 to 100 mg/L into cultivation liquid,the theanine content in tea plants was 2.25 times of that in the control on the 3rd day of the cultivation.The materials obtained with different theanine contents in this study could be used as materials for discovery of the genes correlated with theanine metabolism using digital gene expression profile in the future.