USE OF ELECTROMAGNETIC RADIATION & ELECTROPORATION AS EXTERNAL NON-IONIZING STIMULANTS FOR SWITCHGRASS SEED GERMINATION.

Transcription

1 USE OF ELECTROMAGNETIC RADIATION & ELECTROPORATION AS EXTERNAL NON-IONIZING STIMULANTS FOR SWITCHGRASS SEED GERMINATION Matthew Robinson Reginald Devine, M. Tofighi

2 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 uzz.org/kiosks/science -museumminnesota/puttingdown-roots 1

3 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.) 2

4 OBJECTIVE To test and observe the effects of EMR and Electroporation on the Germination percentage of Shelter Switchgrass seeds. Science Daily 3

5 MATERIALS & METHODS EMR DESIGN EMR Gene Pulser Cuvette Fixture layout was designed using ADS(Advance Design Systems) Momentum. Milling Station LPKF Milling Machine 4

6 MATERIALS & METHODS EMR DESIGN Gene Pulser Cuvette connected to Fixture Measuring S11 Parameter of the Gene Pulser Cuvette GENE PULSER CUVETTE CUVETTE FIXTURE GROUND WIRE CONNECTION BETWEEN FIXTURE AND CUVETTE SMA CONNECTOR NETWORK ANALYZER 8720C BY HEWLETT PACKARD NETWORK ANALYZER CABLE FIXTURE+GENE PULSER CUVETTE 5

7 db(s1_1) MATERIALS & METHODS EMR DESIGN Network Analyzer 8720C By Hewlett Packard/ Plot of S11 = db of cuvette at 2.4GHz from ADS display freq, GHz 6

8 MATERIALS & METHODS EMR EXPERIMENT I (2.4 GHz 12.5cm wavelength) ANRITSU MS2721B SPECTRUM ANALYZER ANRITSU MG3691B SIGNAL GENERATOR PASTERNACK PE COUPLER GENE PULSER CUVETTE HEAT SINK 2.4GHz RF LINX AMPLIFIER 7

9 E= (30 P)/r, where r is the separation between the two parallel plates, r=0.004m MATERIALS & METHODS EMR EXPERIMENT I (2.4 GHz) SIGNAL GENERATOR READINGS CUVETTE POWER, P CUVETTE ELECTRIC FIELD, E SPECTRUM ANALYZER READINGS DURATION EXPERIMENT 8 dbm 0.5W = 27 1kV/m 6.89 dbm 10 min dbm EXPERIMENT 8 dbm 0.5W = 27 1kV/m 7.21 dbm 10 min dbm EXPERIMENT 8 dbm 0.5W = 27 1kV/m 6.93 dbm 10 min dbm EXPERIMENT 8 dbm 0.5W = 27 1kV/m 7.27 dbm 10 min dbm EXPERIMENT 8 dbm 0.5W = 27 1kV/m 7.10 dbm 10 min dbm Control 1 10 min 50 NUMBER OF SEEDS P(dBm)= 10 log10p(mw)/1mw EXPERIMENT -2 dbm 0.05W = V/m dbm 10 min dbm EXPERIMENT -2 dbm 0.05W = V/m dbm 10 min dbm EXPERIMENT -2 dbm 0.05W = V/m dbm 10 min dbm EXPERIMENT -2 dbm 0.05W = V/m dbm 10 min dbm EXPERIMENT -2 dbm 00.5W = V/m dbm 10 min dbm CONTROL 2 10 min 55 8

10 MATERIALS & METHODS EMR EXPERIMENT II (0.9 GHz - 33cm wavelength) NRITSU MS2721B SPECTRUM ANALYZER HEAT SINK POWER LOAD RESISTOR ANRITSU MG3691B SIGNAL GENERATOR AMPLIFIER Lzy MHz GENE PULSER CUVETTE POWER DIVIDER ISOLATOR 9

11 MATERIALS & METHODS EMR EXPERIMENT II (0.9GHz) CUVETTE POWER SPECTRUM ANALYZER POWER SIGNAL GENERATOR POWER CUVETTE FIELD INTENSITY DURATION NUMBER OF SEEDS EXPERIMENT W= ~17 dbm (-2.78 dbm) (-8.5 dbm) ~300 V/m 5 min. 55 EXPERIMENT W= ~17 dbm (-3.04 dbm) (-8.5 dbm) ~300 V/m 5 min. 55 EXPERIMENT W= ~17 dbm (-3.18 dbm) (-8.5 dbm) ~300 V/m 5 min. 55 Control min. 55 EXPERIMENT W= ~27 dbm 8.05 dbm 1.5 dbm ~1.0 kv/m 5 min. 55 EXPERIMENT W= ~27 dbm 7.73 dbm 1.5 dbm ~1.0 kv/m 5 min. 55 EXPERIMENT W= ~27 dbm 7.64 dbm 1.5 dbm ~1.0 kv/m 5 min. 55 Control min. 55 EXPERIMENT W= ~37 dbm dbm 11.5 dbm ~3.0 kv/m 5 min. 55 EXPERIMENT W= ~37 dbm dbm 11.5 dbm ~3.0 kv/m 5 min. 55 EXPERIMENT W= ~37 dbm dbm 11.5 dbm ~3.0 kv/m 5 min. 55 Control min

12 MATERIALS & METHODS ELECTROPORATION DESIGN BIO-RAD MICRO PULSER GENE PULSER CUVETTE CHAMBER SLIDE ELECTROPORATION BUFFER 11

13 MATERIALS & METHODS ELECTROPORATION EXP. VOLTAGE NUMBER OF PULSES DURATION OF PULSE NUMBER OF SEEDS Exp kV 15 1ms 55 Exp kV 15 1ms 55 Exp kV 15 1ms 55 Control 1 55 Exp kV 30 1ms 55 Exp kV 30 1ms 55 Exp kV 30 1ms 55 Control 2 1ms 55 12

14 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) 13

15 RESULTS 10 th Day EMR 2.4GHz Exp. Experiment Set Cuvette Power # of Seeds Sprouted # of Seeds Plated Percent Germinated Exp W= 27dBm % Exp W= 27dBm % Exp W= 27dBm % Exp W= 27dBm % Exp W= 27dBm % Exp 1 Avg % Exp W= 17 dbm % Exp W= 17 dbm % Exp W= 17 dbm % Exp W= 17 dbm % Exp W= 17 dbm % Exp 2 Avg % Control % Control % Control Avg % 14

16 RESULTS 10 th Day EMR 0.9GHz Exp. Experiment Set Cuvette Power # of Seeds Sprouted # of Seeds Plated Percent Germinated Exp W= 17 dbm % Exp W= 17 dbm % Exp W= 17 dbm % Exp 1 Avg % Exp W= 27dBm % Exp W= 27dBm % Exp W= 27dBm % Exp 2 Avg % Exp W= 37dBm % Exp W= 37dBm % Exp W= 37dBm % Exp 3 Avg % Control % Control % Control % Control Avg % 15

17 RESULTS 10 th Day Electroporation Exp. Experiment Set Volatge # of Seeds Sprouted # of Seeds Plated Percent Germinated Exp kv/cm % Exp kv/cm % Exp kv/cm % Exp 1 Avg % Exp kv/cm % Exp kv/cm % Exp kv/cm % Exp 2 Avg % Control % Control % Control Avg % 16

18 RESULTS & DISCUSSION 17

19 RESULTS & DISCUSSION -Germination percentages for experiments were generally very close. -Power settings 0.05w, and 5.0w GHz EMR experiments -Experiment 1 for electroporation with 3.0kv/cm and 15 pulses EXPERIMENTS CONT 18

20 ACKNOWLEDGEMENTS 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 19

21 REFERENCES Monti, A., Barbanti, L., Zatta, A. and Zegada-Lizarazu, W. (2012), The contribution of switchgrass in reducing GHG emissions. GCB Bioenergy, 4: 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 Christian J.E., Simone B., Michael P., Holger P. and Wolfgang F. (2009)Effects of Nanosecond Pulsed Electric Field Exposure on Arabidopsis thaliana pp What is electromagnetic radiation. (n.d.). Retrieved from Electroporation facts. (2004, Dec. 28). Retrieved from University of Nebraska-Lincoln (2008, January 14). Biofuel: Major Net Energy Gain From Switchgrass-based Ethanol. ScienceDaily. 20