Shootlets from Gynostemma pentaphyllum (Thunb.) Makino were grown on MS medium supplemented with hormone BAP and NAA or KN and 2,4D or KN and IAA and GA with concentration of 0.1 – 9 mg/lit for 1 month. The multiple shoots were grown. The best multiple shoots was on MS medium supplemented with BAP, GA and citric acid with the concentration of 0.1 mg/l , 5 mg/l and 150 mg/l respectively and increase the iron in MS medium for equal amount (MS58) in one month. The average of multiple shoots was 4.3 shoots, the average length of shoots was 2.4 cm. When the shootlets were grown on MS medium supplemented with hormone NAA , IAA and IBA with the concentration of 0.1, 1 and 2 mg/l for rooting medium in one to one and a half month. The best medium was MS medium supplemented with IAA 0.1 mg/l having the average percentage of rooting 85 % in one month of culture and 93.3 % in one and a half month of culture. When culture for one and a half month hormone IAA 1 mg/l is better than hormone IAA 0.1 mg/l that has average main shoot length , average shoot higher than 3 cm. and average amount of roots equal to 4.2 cm. 92.8 % and 7.4 roots respectively. There fore the plantlets are more healthy. The average percentage of survival of plantlets when growing in nursery for 1 month was 94.25 %. Total saponincontent of multiple shoots from Chinese and Thai variety on MS58 and MS58+C5 medium were 5.10 % and 5.08 % for Chinese variety, 5.21 % and 5.45 % for Thai variety respectively. The total saponin for three months of Thai variety plants growing in the nursery was 6.67 %.

Key Words: Gynostemma pentaphyllum , Tissue culture , Total saponin