Physiological and biochemical studies towards an understanding of production and quality assessment of Stevia rebaudiana L. under tropical conditions
David Midmore, Kerry Walsh, Andrew Rank, Geeta Gautam, Ria Reyes, Phul Subedi, Nanjappa Ashwath, Mousumi Debnath. Central Queensland University
Stevia and its origin
Stevia and its potential
FSANZ Approval
Australian production of SG • Commercially non-existent • Imports mainly from China • We need to do something about this..!
Outline of presentation • Chemical analyses with HPLC and NIR (and LCMS/MS) • Reasons for accumulation of SG in Stevia • Selecting for photoperiod insensitivity • Nutrient requirements • pH requirement • Water excess and deficit • Salinity • Overall conclusions
HPLC modifications to Subedi and Hearn (2008) • • • •
Extraction at 70oC in Milli-Q water Physical filter 0.45 µm Omit SPE XC and NH2 cartridge clean-up phase 5 μL sample injection to avoid overlapping of adjacent broadened peaks in water • 80% acetonitrile mobile phase in HPLC, flow rate 1 mL/min Detection limit of Stevioside 0.061% and Reb-A 0.140% leaf dry weight (de Guzman, 2011)
NIR for non-invasive prediction of SGs • Testing over 1100 to 2400 nm, best region start 1450 and end 2060 nm, best model for Reb-A solution obtained for the region 1600 – 1726 nm. R of 0.995 and RMSECV of 0.049% w/v. • But NIR overlay of water peaks in fresh materials, varies with leaf moisture content, temperature • NIRS 6500 calibration statistics for total SG content – ground stevia leaves (R = 0.87, RMSECV = 1.63%) – dry intact leaves (R = 0.79, RMSECV = 2.07%) and – fresh leaves (R = 0.67, RMSECV=2.53%).
• OK to select low, medium and high SG using NIR
Stevia and NIR
But hand-held 700-1100 nm fresh leaves R=0.31 RMSEC = 3.15% Shame for Si detectors are cheaper than for longer λ
LC-MS/MS • Much more precise than HPLC, ppb not ppm, and can identify chemicals in peaks and check for purity • Mobile phases consisted of: A: 10 mM ammonium acetate in water; B: 10 mM ammonium acetate in 95:5 acetonitrile: water used at a flow rate 0.4 mL/min with the column temperature at 30°C and an injection volume of 1 µL • Stevioside A, Rebaudioside A, Rebaudioside C, Rebaudioside D, Dulcoside and Rubusoside • >1000 runs
So, we can measure accurately, so now we ask why do Stevia plants have high steviol glycoside concentration?
• Role as an energy reserve – no effect of extended dark or extended light periods on SG concentration, slow SG turnover • Role as osmo-regulators for drought tolerance – no SG concentration change in short-term drought studies • Role in insect-deterrence - grasshopper avoidance, and starvation, spider mite not related to SG concentration • Role in influencing mammalian herbivore feeding – 10% SG increased feed intake by 39% in guinea pigs, commercial application? • Other reasons?? We don’t know.
But we have other issues to attend to.. • Precocious flowering – try to delay genetically • Macro and micro nutrient requirements – testing in hydroponics • pH of medium in hydroponics • Drought and waterlogging • Salinity All above in pots/hydroponics • Weeds (tomorrow) In field and in pots
Some studies on flowering – mass selection for later flowering under short days
Flowering attributes under natural daylengths of F1 stevia variety selection for maturity class Midmore et al., 2012
Effects of different constant photoperiods on number of days to first flowering of F1 progeny from parents selected for early, medium or late flowering
Effects of different constant photoperiods on % of plants flowering for F1 progeny from parents selected for early, medium or late flowering
Photoperiodic effect is not unusual for tropical species.
Chinese waterchestnut: Li, Kleinhenz and Midmore, 2000
What to do about early flowering? • Seems as though under short tropical days there is little expression of lateness • There is expression of lateness at longer days • Only one cycle of selection so need to set up a longer term trial to test for shifting of population flowering time/% under short tropical days
N
Micro nutr
Distinct Visual Symptoms
Ca
Cu
Mg Fe B
Effects of nutrient deficiencies on biomass in stevia, harvest at 14 wks
1.8
1.4
1.2
1.0
0.8
0.6
0.4
25
20
15
a a
4
abc abcabcd
3
bcd
2
cd cd
1
10
Treatment d d d d d d
0
1.6
a ab abc abcabc abcd
bcde bcde cde de de
0.2
de e e e e
0.0
a abab abc abcd abcde bcdef cdef defgefg efg fg lsd=7.7
fg fg fg g
5
0 4
3
1
6.0
0.6
0.4
no Mo complete no Cu no K no Cl no Mn no Zn no B no Mg no Fe no Ca no S no micro no P no N no NPK
5
Leaf dry weight (g/plant)
a
Root dry weight (g/plant)
complete no Mo no Cu no Cl no Mn no K no Zn no B no Fe no Mg no Ca no S no P no micro no N no NPK
Total biomass dry weight (g/plant)
a
no Cl no Mo no Fe no Zn complete no Cu no Mn no S no P no N no B no K no Mg no NPK no Ca no micro
complete no Cl no Cu no Zn no Mn no Mo no B no K no Fe no Mg no Ca no S no P no micro no N no NPK
Stem dry weight (g/plant) 6.0 5.0 4.0 3.0
complete no Cl no Zn no Cu no Mo no Mn no B no Fe no K no Ca no Mg no S no P no micro no N no NPK
Plant height (cm) 6
lsd=2.7 6
5
lsd=2.0
a a a a ab ab abc
2
abcd bcdbcdbcd cd
lsd=0.7
a a a a a a a a a a a a
0.2
30
Treatment
d d d d
0
3.0
lsd=0.3
1.8
1.6
1.4
1.2
1.0
0.8
a b b b
0.0
Biomass in plant parts followed similar trends as for total biomass as affected by deficiencies
Effects of nutrient deficiencies on %SG and SG content per plant
Effects of pH in hydroponics on biomass production and distribution
Effects of pH in hydroponics on %SG and SG content per plant
Soil moisture treatments in pots % of field capacity
120
gravimetric %H2O
38.4 32.2 25.6 19.2 16.1
100
80
• 8 week treatment • soil permanent wilting point = 14.7% w/w.
60
50
Effects of soil moisture on rates of photosynthesis and transpiration
Effects of soil moisture on %SG and content of SG per plant
Salinity and soil moisture effects on canopy temperature, chlorophyll content, stomatal conductance, and osmotic potential in pots. T0 = control T1 = 50 mM NaCl T2 = 100 mM NaCl T3 = 50% field capacity T4 = 80% field capacity T5 = water logging C1 = Shoutian C2 = Fengtian
Salinity and soil moisture effects on stevioside and Reb-A concentrations • • • • • •
T0 = control T1 = 50 mM NaCl T2 = 100 mM NaCl T3 = 50% field capacity T4 = 80% field capacity T5 = water logging
Effects of salinity in hydroponics on Reb-A and stevioside concentrations
T0: control; T1 50 mM; T2 100 mM; T3 200 mM; T4: 300 mM NaCl; C1: Shoutian and C2 : Fengtian
Some conclusions • Lab analyses for SG accurate with HPLC and LC-MS/MS • NIR up to 85% predictability for dry ground leaf • Poor predictability for fresh leaf Working with populations and their natural variability: • Some advance in selecting for lateness in flowering • But effect only evident in longer days • Copper deficiency reduces SG concentration • Deficiency in Fe, Mg, Ca, S, P, N severely reduce biomass • Alkaline pH doesn't favour growth, no effect on SG% • Drought reduces biomass but not SG%, waterlogging has no consistent significant effect • Salinity increases stevioside concentration (but reduces biomass) Future work with clonal materials
Acknowledgements • • • •
RIRDC, Australia Sanitarium Health Food Company CQUniversity Organising committee for this meeting
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