2007 Annual Report - Central Queensland University

2007 Centre for Plant & Water Science Annual ReportSTUDIES ON STEVIASUMMARYThe physiological role of steviol glycosides (SG) in S. rebaudiana was explored by monitoringchange in SG content in response to environmental conditions. Roles initially explored arecarbohydrate reserve and osmoregulator. Variability of SG levels in leaves of a single plantwas also monitored. SG leaf content was determined through HPLC analysis of leaf extracts.Additional biological functions to be investigated in the future are insect-deterency andherbivore attraction. Near Infrared (NIR) spectroscopy will also be explored for non-invasiveanalysis of SG leaf content.Method development for filtration and and HPLC analysis of leaf extractsSG content in leaf extracts was optimized by changing pre-load and wash solvents in SPEfiltration. The protocol for leaf extract filtration includes the following steps:(1) Load 2 mL of leaf extract to XC cartridge,(2) Pre-condition NH2 cartridge with 3 mL ethanol (100%),(3) Load 2 mL filtrate from the XC cartridge in step 1,(4) Wash with 2 mL acetonitrile,(5) Vacuum dry NH2 cartridge,(6) Elute with 3 mL ethanol (70%)The following recovery rates for XC filtration (step 1) and NH 2 filtration (step 2-6) apply:Standard Solution(1, 0.5, 0.25 & 0.125 g/L)Recovery rate %XC cartridge NH2 cartridgeStevioside only 100 100Rebaudioside A only not tested 100Stevioside and Rebaudioside A not tested 83 - stevioside100- rebaudioside AHPLC parameters used in SG calibration (stevioside and rebaudioside A) and leaf extractanalysis are:1) Stationary column – Zorbax NH 2 column (250 mm x4.6 mm, 5μm)2) Mobile phase – 80% acetonitrile (pH 5)3) Flow rate – 1.00 mL min -14) Injection volume – 10 μL5) UV wavelength detection – 210 nmHPLC for sugar analysis was also explored in a refractive index detector in the set-up. The nextstep involves detector calibration using standard sugar solutions (sucrose, glucose and fructose)and sugar analysis in S. rebaudiana leaf extracts.Initial investigations on physiological significance of steviol glycoside in steviaSG as a carbohydrate reserve was investigated in 3 S. rebaudiana plants placed in different lighttreatments (continuous light, continuous dark and normal daylight cycle). Leaves from themiddle section of the stem (n=5) were sampled in the morning and in the afternoon for four45