ENHANCEMENT OF STEVIOL GLYCOSIDE PRODUCTION FROM SHOOT CULTURE OF STEVIA (Stevia rebaudiana Bert.) IN THE TIS RITA® BIOREACTOR BY INHIBITION OF GIBBERELLIN BIOSYNTHESIS

• Main Author: Tamania Saptari, Rizka
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/41057
• Institution: Institut Teknologi Bandung

Stevia (Stevia rebaudiana Bert.) is a source of natural sweetener for sugar subtitution. Stevia contains steviol glycosides with nearly zero calorie and glycemic index. Steviol glycosides are a group of diterpenoid secondary metabolites which have the same precursor with Gibberellin (GA), ent-kaurenoic acid. Therefore, inhibition of GA biosynthesis pathway is expected to increase steviol glycoside. The research was conducted to enhance the production of steviol glycosides from the shoot culture of stevia in the TIS RITA® bioreactor, using a GA biosynthesis inhibitor, Daminozide, and to analyse its effect on the expression level of genes encoding enzymes for GA and steviol glycosides biosynthesis from ent-kaurenoic acid. Steps of experiments were determination of concentration range of Daminozide used in in vitro culture using thin layer liquid culture system, application of Daminozide in TIS RITA® bioreactor with the immersion period of 30 minutes every 6 hours for 6 weeks. After that, analysis was conducted for stevia growth, and steviol glycosides content in leaves using High Performance Liquid Chromatography (HPLC). Furthermore, in silico analysis was conducted, including transcriptomics data collection, alignment, annotation, and primer design, followed by analysis of genes expression using Real Time PCR (qRT-PCR) for genes related to steviol glycoside and GA biosynthesis. Data of growth, steviol glycosides content, and genes expression were then analysed using analysis of variance (ANOVA), and followed by Duncan’s Multiple Range Test (DMRT) at the significance level ? ? 0.05. Based on the optimization in the thin-layer liquid culture, 10 ppm and 20 ppm were used for culture in TIS RITA® as these treatments enhanced steviol glycoside content without reducing the plant biomass. Results of the stevia shoot culture in TIS RITA® bioreactor showed that application of Daminozide at 10 ppm and 20 ppm significantly suppressed plantlets height 35,6% and 55,5%, suppressed internodes length 34,3% and 46,2%, but increased leaves area 119,6% and 202,2%. Application of Daminozide at 10 ppm dan 20 ppm also increased leaves fresh biomass at 33,5% and 39,7, and increased biomass ratio of leaves : stem-root, 5:5 and 6:4 (control: 4:6). Moreover, results of HPLC analysis showed that application of 10 ppm Daminozide in the stevia shoot culture in TIS RITA® bioreactors increased stevioside and reb A contents up to 40%, and increased total production of stevioside and reb A two times higher than the control which produced 2,1 ± 0,2 mg stevioside and 3,8 ± 0,6 mg reb A per bioreactor. Furthermore, application of 10 ppm Daminozide increased expression level of synthesis gene, GA3ox, indicating the occurance of negative feedback regulation, and decreased expression level of catabolism gene, GA2ox, indicating the occurance of feed-forward regulation in GA biosynthesis pathway caused by the inhibition on the pathway. Nevertheless, application of 10 ppm Daminozide also increased expression level of genes enroled in steviol glycoside biosynthesis, such as SrKA13H, SrUGT85C2, and SrUGT76G1, indicating more intensive steviol glycoside pathway due to the inhibition of GA pathway. Based on these results, it could be concluded that 10 ppm Daminozide was the optimum concentration for enhancing production of steviol glycoside from the shoot culture of stevia in TIS RITA® bioreactors.