USE OF ELECTROMAGNETIC RADIATION & …electroporation design 11 bio-rad micro pulser electroporation buffer chamber slide gene pulser cuvette . 12 materials & methods electroporation

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

4

LPKF Milling Machine

Gene Pulser Cuvette connected to Fixture

Measuring S11 Parameter of the Gene Pulser Cuvette

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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


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



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



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



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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Documents

USE OF ELECTROMAGNETIC RADIATION & …electroporation design 11 bio-rad micro pulser electroporation buffer chamber slide gene pulser cuvette . 12 materials & methods electroporation