Matthew Robinson Reginald Devine, M. Tofighi
USE OF ELECTROMAGNETIC RADIATION &
ELECTROPORATION AS EXTERNAL NON-IONIZING
STIMULANTS FOR SWITCHGRASS SEED
GERMINATION
INTRODUCTION
Switchgrass Panicum virgatum L.
Biofuel Feedstock
GHG Emissions
Ability to be grown on Marginal Land
Erosion Control
Non-Food biofuel
Problem: Very low germination percentage
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http://www.sciencebuzz.org/kiosks/science
-museum-minnesota/putting-
down-roots
BACKGROUND INFORMATION BIO-STIMULATION
Electromagnetic Radiation (EMR)
- a stream of energy-bearing particles that comes out of an electromagnetic source.
Electroporation
-The process in which a cell is exposed to an electrical current surge (pulse).
-The displacement current can charge intracellular membranes and affect cell organelles. (C.J. Eing et al.)
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OBJECTIVE
To test and observe the effects of EMR and Electroporation on the Germination percentage of Shelter Switchgrass seeds.
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Science Daily
MATERIALS & METHODS EMR DESIGN
EMR Gene Pulser Cuvette Fixture layout was designed using ADS(Advance Design Systems)
Momentum. Milling Station
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LPKF Milling Machine
Gene Pulser Cuvette connected to Fixture
Measuring S11 Parameter of the Gene Pulser Cuvette
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MATERIALS & METHODS EMR DESIGN
CUVETTE FIXTURE
SMA CONNECTOR
GENE PULSER CUVETTE
WIRE CONNECTION BETWEEN FIXTURE AND CUVETTE
NETWORK ANALYZER 8720C BY HEWLETT PACKARD
FIXTURE+GENE PULSER CUVETTE
NETWORK ANALYZER CABLE GROUND
MATERIALS & METHODS EMR DESIGN
Network Analyzer 8720C By Hewlett Packard/
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2 4 6 8 10 12 14 16 18 200 22
-25
-20
-15
-10
-5
-30
0
freq, GHz
dB
(s1
_1
)
Plot of S11 = -22.667 dB of cuvette
at 2.4GHz from ADS display
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MATERIALS & METHODS EMR EXPERIMENT I (2.4 GHz – 12.5cm wavelength)
ANRITSU MS2721B SPECTRUM ANALYZER ANRITSU MG3691B
SIGNAL GENERATOR
2.4GHz RF LINX AMPLIFIER
GENE PULSER CUVETTE
PASTERNACK PE2210-20 COUPLER
HEAT SINK
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MATERIALS & METHODS EMR EXPERIMENT I (2.4 GHz)
E=√(30×P)/r, where r is the separation between the two parallel plates, r=0.004m
P(dBm)= 10×log10P(mW)/1mW
SIGNAL
GENERATOR
READINGS
CUVETTE
POWER, P
CUVETTE
ELECTRIC
FIELD , E
SPECTRUM
ANALYZER
READINGS
DURATION NUMBER OF
SEEDS
EXPERIMENT
1-1
8 dBm 0.5W = 27
dBm
1kV/m 6.89 dBm 10 min 50
EXPERIMENT
1-2
8 dBm 0.5W = 27
dBm
1kV/m 7.21 dBm 10 min 50
EXPERIMENT
1-3
8 dBm 0.5W = 27
dBm
1kV/m 6.93 dBm 10 min 50
EXPERIMENT
1-4
8 dBm 0.5W = 27
dBm
1kV/m 7.27 dBm 10 min 50
EXPERIMENT
1-5
8 dBm 0.5W = 27
dBm
1kV/m 7.10 dBm 10 min 50
Control 1
__ __ __ __ 10 min 50
EXPERIMENT
2-1
-2 dBm 0.05W = 17
dBm
300V/m -2.16 dBm 10 min 55
EXPERIMENT
2-2
-2 dBm 0.05W = 17
dBm
300V/m -1.96 dBm 10 min 55
EXPERIMENT
2-3
-2 dBm 0.05W = 17
dBm
300V/m -2.24 dBm 10 min 55
EXPERIMENT
2-4
-2 dBm 0.05W = 17
dBm
300V/m -1.98 dBm 10 min 55
EXPERIMENT
2-5
-2 dBm 00.5W = 17
dBm
300V/m -2.18 dBm 10 min 55
CONTROL 2
__ __ __ __ 10 min 55
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MATERIALS & METHODS EMR EXPERIMENT II (0.9 GHz - 33cm wavelength)
ANRITSU MS2721B SPECTRUM ANALYZER
ANRITSU MG3691B SIGNAL
GENERATOR
HEAT SINK
GENE PULSER CUVETTE
AMPLIFIER Lzy-2+
500-1000 MHz
POWER DIVIDER ISOLATOR
POWER LOAD RESISTOR
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MATERIALS & METHODS EMR EXPERIMENT II (0.9GHz)
CUVETTE POWER SPECTRUM
ANALYZER
POWER
SIGNAL
GENERATOR
POWER
CUVETTE
FIELD
INTENSITY
DURATION NUMBER OF
SEEDS
EXPERIMENT 1-1 .05W= ~17 dBm (-2.78 dBm) (-8.5 dBm) ~300 V/m 5 min. 55
EXPERIMENT 1-2 .05W= ~17 dBm (-3.04 dBm) (-8.5 dBm) ~300 V/m 5 min. 55
EXPERIMENT 1-3 .05W= ~17 dBm (-3.18 dBm) (-8.5 dBm) ~300 V/m 5 min. 55
Control 1 - - - - 5 min. 55
EXPERIMENT 2-1 0.5W= ~27 dBm 8.05 dBm 1.5 dBm ~1.0 kV/m 5 min. 55
EXPERIMENT 2-2 0.5W= ~27 dBm 7.73 dBm 1.5 dBm ~1.0 kV/m 5 min. 55
EXPERIMENT 2-3 0.5W= ~27 dBm 7.64 dBm 1.5 dBm ~1.0 kV/m 5 min. 55
Control 2 - - - - 5 min. 55
EXPERIMENT 3-1 5.0 W= ~37 dBm 17.64 dBm 11.5 dBm ~3.0 kV/m 5 min. 55
EXPERIMENT 3-2 5.0 W= ~37 dBm 17.58 dBm 11.5 dBm ~3.0 kV/m 5 min. 55
EXPERIMENT 3-3 5.0 W= ~37 dBm 17.65 dBm 11.5 dBm ~3.0 kV/m 5 min. 55
Control 3 - - - - 5 min. 55
MATERIALS & METHODS ELECTROPORATION DESIGN
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BIO-RAD MICRO PULSER
ELECTROPORATION BUFFER
CHAMBER SLIDE
GENE PULSER CUVETTE
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MATERIALS & METHODS ELECTROPORATION EXP.
VOLTAGE
NUMBER
OF PULSES
DURATION
OF PULSE
NUMBER
OF SEEDS
Exp 1-1 3.0kV 15 1ms 55
Exp 1-2 3.0kV 15 1ms 55
Exp 1-3 3.0kV 15 1ms 55
Control 1 55
Exp 2-1 2.5kV 30 1ms 55
Exp 2-2 2.5kV 30 1ms 55
Exp 2-3 2.5kV 30 1ms 55
Control 2 1ms 55
MATERIALS & METHODS Plating of Seeds
• After EMR & Electroporation seeds were: I. Taken out of Cuvette and dried
II. Transported to the Greenhouse
III. 25 seeds were plated in each
petri dish on filter paper (50 seed Max for each trial)
IV. Watered and lids were
placed on top (fresh water was given
everyday)
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RESULTS 10th Day EMR 2.4GHz Exp.
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Experiment Set Cuvette Power # of Seeds Sprouted # of Seeds Plated Percent Germinated
Exp 1-1 0.5W= 27dBm 8 40 20.0%
Exp 1-2 0.5W= 27dBm 4 37 10.8%
Exp 1-3 0.5W= 27dBm 15 38 39.5%
Exp 1-4 0.5W= 27dBm 0 39 0.0%
Exp 1-5 0.5W= 27dBm 5 39 12.8%
Exp 1 Avg - 32 193 16.6%
Exp 2-1 .05W= 17 dBm 5 39 12.8%
Exp 2-2 .05W= 17 dBm 7 40 17.5%
Exp 2-3 .05W= 17 dBm 2 34 5.9%
Exp 2-4 .05W= 17 dBm 3 40 7.5%
Exp 2-5 .05W= 17 dBm 2 40 5.0%
Exp 2 Avg - 19 193 9.7%
Control 1 - 3 38 7.9%
Control 2 - 12 40 30.0%
Control Avg - 15 78 18.9%
Experiment Set Cuvette Power # of Seeds Sprouted # of Seeds Plated Percent Germinated
Exp 1-1 .05W= 17 dBm 19 48 39.6%
Exp 1-2 .05W= 17 dBm 22 50 44.0%
Exp 1-3 .05W= 17 dBm 25 50 50.0%
Exp 1 Avg - 66 148 44.5%
Exp 2-1 0.5W= 27dBm 20 50 40.0%
Exp 2-2 0.5W= 27dBm 19 50 38.0%
Exp 2-3 0.5W= 27dBm 22 50 44.0%
Exp 2 Avg - 61 150 40.7%
Exp 3-1 5.0 W= 37dBm 22 49 44.9%
Exp 3-2 5.0 W= 37dBm 22 52 42.3%
Exp 3-3 5.0 W= 37dBm 29 51 56.9%
Exp 3 Avg - 73 152 48.0%
Control 1 - 24 50 48.0%
Control 2 - 24 49 49.0%
Control 3 - 15 43 34.9%
Control Avg - 63 142 44.0%
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RESULTS 10th Day EMR 0.9GHz Exp.
Experiment Set Volatge # of Seeds Sprouted # of Seeds Plated Percent Germinated
Exp 1-1 3.0 kV/cm 29 52 55.8%
Exp 1-2 3.0 kV/cm 31 50 62.0%
Exp 1-3 3.0 kV/cm 24 49 49.0%
Exp 1 Avg - 84 151 55.6%
Exp 2-1 2.5 kV/cm 19 50 38.0%
Exp 2-2 2.5 kV/cm 30 49 61.2%
Exp 2-3 2.5 kV/cm 29 49 59.2%
Exp 2 Avg - 78 148 52.8%
Control 1 - 22 48 45.8%
Control 2 - 30 50 60.0%
Control Avg - 52 98 52.9%
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RESULTS 10th Day Electroporation Exp.
RESULTS & DISCUSSION
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RESULTS & DISCUSSION
-Germination percentages for experiments were generally very close. -Power settings 0.05w, and 5.0w - 0.9 GHz EMR experiments -Experiment 1 for electroporation with 3.0kv/cm and 15 pulses EXPERIMENTS CONT…
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• Special thanks to : – NSF-REU program
– Reginald Devine and
Dr. Tofighi in the Engineering Dept.
– Ernst Seed Company
– Dr. Shobha, Alison Shuler, Dr. Sairam
– Central Pennsylvania Laboratory for Biofuels
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ACKNOWLEDGEMENTS
REFERENCES
• Monti, A., Barbanti, L., Zatta, A. and Zegada-Lizarazu, W. (2012), The contribution of switchgrass in reducing GHG emissions. GCB Bioenergy, 4: 420–434.
• Fike J.H., Parrish D.J., Wolf D.D., Balasko J.A., Green Jr. J.T., Rasnake M., Reynolds J.H. Long-term yield potential of switchgrass-for-biofuel systems (2006) Biomass and Bioenergy, 30 (3), pp. 198-206.
• Christian J.E., Simone B., Michael P., Holger P. and Wolfgang F. (2009)Effects of Nanosecond Pulsed Electric Field Exposure on Arabidopsis thaliana pp. 1322- 1328
• What is electromagnetic radiation. (n.d.). Retrieved from http://www.wisegeek.org/what-is-electromagnetic-radiation.htm
• Electroporation facts. (2004, Dec. 28). Retrieved from http://www.macwormlab.net/index.php?option=com_content&view=article&id=74&Itemid=88
• University of Nebraska-Lincoln (2008, January 14). Biofuel: Major Net Energy Gain From Switchgrass-based Ethanol. ScienceDaily.
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