Here it shows a ppt file of research work entitled with "Effects of Plasma Technology on Germination and Growth Improvement in Rapeseed (Brassica napus)" which has been submitted to dept. of botany at Rajshahi University, Bangladesh in 2019 by Shariful Islam (session: 2012-'13)

1. Effects of Plasma Technology on Germination and
Growth Improvement in Rapeseed (Brassica napus)
Supervisor
Dr. Ahmad Humayan Kabir
Assistant Professor
Molecular Plant Physiology Laboratory
Department of Botany
University of Rajshahi
Rajshahi-6205, Bangladesh
Presented By
Md. Shariful Islam
M.S. (Thesis) Madar Bux Hall
Dept. of Botany
University of Rajshahi, Rajshahi-6205,
Bangladesh

2. INTRODUCTION
 Rapeseed (Brassica napus) is one of the major oil-yielding crops in
many countries, the production of rapeseed is decreasing due to several
climatic (temperature, humidity, and drought, etc.) and manmade factors.
 Plasma technology showed agronomic importance in rapeseed.
 Plasma is generally described as an ionized gas or as an electrically
neutral medium of positive and negative particles.
Plasma is considered to be the 4th state of matter after solid, liquid &
gas. Solid + Energy = Liquid
Liquid + Energy = Gas
Gas + Energy = Plasma
 Low Pressure Dielectric Barrier Discharge (LPDBD) technique is a
vital source of non-thermal plasma or cold plasma.

3. EFFECTS OF PLASMA ON RAPESEED
Past research showed that:
The effects of Low Pressure Dielectric Barrier Discharge (LPDBD)
produced with Ar/O2 and Ar/Air technique causing biological stimulation
leading to improved germination and growth in rapeseed.
 Plasma treatments take effect on rapeseeds antioxidant enzyme
activities, osmolytes content and genetic sequence.
 Cold plasma treatment is a fast, economic and pollution-free method to
improve seed performance and crop yield (Tong, J. Y., 2014).

4. AIM OF THE STUDY
 To optimize the plasma treatments to enhance germination and growth
in rapeseed.
 Biochemical characterization of traits associated with enhanced
germination and Growth in rapeseed due to plasma treatment.
 Expression analysis of genes underlying seed dormancy breaking and
growth enhancement in rapeseed.
 Evaluate the possibility of environmental-friendly plasma technology
instead of chemical fertilizer for enhanced rapeseed production.
 Delivering message about the effectiveness of plasma technology to
decision makers.

5. METHODES OF PLASMA TREATMENT
Water was treated in a glass tube
by LPDBD plasma with O2 and Ar
to make Plasma Activated
Water(PAW).
The rapeseeds were treated
directly in a glass tube by LPDBD
plasma with the same gasses for 90
seconds.
Fig. : PAW processing
Glass Tube
Water
Spectra
Wire

6. MATERIALS AND METHODS
Measurement of Germination percentage: The seeds were placed in
each 90 mm Petri dish containing two layers of wet filter papers at
the bottom for germination. The germination percentage is recorded
after 3 days.
Plant cultivation: Seedlings were then transplanted to the hydroponic
culture (Hoagland and Arnon, 1950). Plants were cultivated for 5
days once transferred to solution culture.
Chlorophyll (a and b) determination: For chlorophyll analysis the
absorbance of the clear supernatant was recorded according to
Lichenthaler and Wellburn (1985) method by spectrophotometer.

7. MATERIALS AND METHODS
The study conducted with 5 analyzing data:
1. Estimation of total soluble protein
2. Analysis of total soluble sugar
3. Determination of H2O2 (Hydrogen Peroxide)
4. Analysis of antioxidant enzymes
i) SOD – Superoxide Dismutase
ii) APX – Ascorbate Peroxidase
iii) CAT – Catalase
5. Metal Analysis (Fe and Zn)
Root/Shoot Extracts
+
Supernatant
Required reagent
12000rpm
for 5 mins
=
Solution
Spectrophotometer
Root/Shoot
Atomic Absorption Spectroscopy
Determination of Zn and Fe
Washed in CaSO4
(1 mM) for 5 min

8. METHODS FOR GENETIC ANALYSIS
Gene expression analysis: Expression analysis of TaSOD, TaAPX, and
TaCAT transcripts was performed in roots by quantitative qRT-PCR
(reverse transcription PCR).
Grafting of control and Ar/O2 treated seedlings: Reciprocal grafting
between control and Ar/O2 treated plants were performed on post-
germinated young seedlings.
Statistical analysis: Experiments were performed on the basis of
randomized block design (CRBD). Significance of each group data was
analyzed statistically at P≤0.05 by ANOVA one-way followed by Duncan's
Multiple Range Test (DMRT) in SPSS Statistics 20.0.

9. RESULT AND DISCUSSION

10. RESULT OF SEED GERMINATION RATE
Fig.: Seed germination rate of rapeseed treated with LPDBD Ar/Air,
LPDBD Air/O2, PAW O2 and PAW Ar plasmas.
All plasma treatments (LPDBD Ar/Air, LPDBD Air/O2, PAW O2 and
PAW Ar) showed a significant improvement in germination rate in
rapeseeds compared to the seeds germinated without plasma treatment.

11. PHENOTYPIC RESULT FOR RAPESEED
Fig.: Phenotype of rapeseed seedlings after cultivation for 5 days in nutrient
medium with different plasma treatments.
Control Air/O2 Air/Ar PAW/O2 PAW/Ar
Phenotypic result shows a significant growth improvement for
the treatment of Plasma Activated water of O2, Ar both and
LPDBD plasmas.

12. Fig.: Root length of rapeseed Fig.: Root dry weight of rapeseed
In case of Root length it did not show any significant changes when
LPDBD Ar/Air and LPDBD Air/O2 plasmas were treated.
In case of dry weight, it significantly increased when seeds were treated
by PAW/O2 and PAW/Ar during germination stage.
PHENOTYPES: ROOT LENGTH and DRY WEIGHT

13. Fig.: Shoot dry weight of rapeseedFig.: Shoot height of rapeseed
PHENOTYPES: HEIGHT and DRY WEIGHT
 Shoot height of rapeseed significantly increased due to all type of plasma
treatments compared to controls.
 In case of shoot dry weight, plants showed a significant increase only when
seeds were treated with PAW/Ar.

14. RESULT: CHLOROPHYLL CONCENTRATIONS
Fig.: Total Chlorophyll concentrations (chll. a and chll. b) in leaves.
Total chlorophyll concentrations (a and b) showed no significant changes in
comparison with controls when treated with LPDBD Ar/Air.
LPDBD Air/O2, PAW Ar, and PAW O2 caused a significant increase in total
chlorophyll (a and b) concentration in leaves of rapeseed compared to controls.

15. Fig.: Total soluble protein of rapeseed
Total soluble protein showed no significant changes in the roots of
rapeseed under different plasma treatments compared to controls.
In addition, application of LPDBD Air/O2 caused a significant increase
in total soluble protein in the shoot of rapeseed compared to controls.
RESULT: TOTAL SOLUBLE PROTEIN

16. RESULT OF TOTAL SOLUBLE SUGAR
Fig.: Total soluble sugar of rapeseed
Total soluble sugar in roots showed significant increase when plants were
grown from the seeds treated with LPDBD Air/O2, PAW O2 and PAW Ar
plasmas.
In shoots, total soluble sugar showed no significant variations either treated
with LPDBD Ar/Air or PAW O2. On the other hand, both LPDBD Air/O 2 and
PAW Ar caused a significant increase in total soluble sugar in shoot compared
to controls.

17. RESULT OF HYDROGEN PEROXIDE
Fig.: H2O2 of rapeseed treated with LPDBD Ar/Air, LPDBD Air/O2, and
PAW O2 and PAW Ar plasmas.
H2O2 concentration showed no significant variations in either root or shoot
among the LPDBD Ar/Air, LPDBD Air/O2, PAW O2 and PAW Ar plasma
treatments .

18. RESULT OF ENZYMATIC ACTIVITIES
Fig.: Antioxidant enzyme activities in roots of rapeseed treated with LPDBD
Ar/Air, LPDBD Air/O2, PAW O2 and PAW Ar plasmas.
Analysis of antioxidant enzymes showed that none of the plasma treatment
caused significant changes in SOD activity in either root or shoot of rapeseed
compared to controls.
 APX activity showed a significant increase only under LPDBD Air/O2 plasma
compared to controls in roots of rapeseed but APX no significants change in shoots.
 In case of CAT activity, PAW O2 and PAW Ar caused a significant increase in
CAT activity in both root and shoot of rapeseed.

19. GENE EXPRESSION IN ROOTS
Fig.: Expression of genes related to antioxidant activities in rapeseed
treated with LPDBD Ar/Air, LPDBD Air/O2, PAW O2 and PAW Ar
plasmas.
Real-time PCR analysis showed no significant changes in BnSOD
expression in roots of rapeseed plants. However, expression of BnAPX and
BnCAT significantly increased in roots when treated with LPDBD Air/O2 and
PAW O2, respectively.

20. CONCLUSIONS
After this study we found that:
 Remarkable increase in germination rate.
 Morphological improvement.
 Chlorophyll content that leads to the better photosynthesis.
 Significant increase in total soluble protein.
 The elevation of CAT activity in seeds kept the production of H2O2 at
optimum level due to Ar/O2 plasma.
 SOD activity and its corresponding TaSOD gene significantly increased in
roots of plants derived from Ar/O2 plasma.

21. ACKNOWLEDGEMENT
First of all, I would like to thank my supervisor, Dr. Ahmad Humayan
Kabir, for giving me the opportunity to complete my M.S. thesis.
I am grateful to Professor Mamunur Rashid Talukder, Department of
Applied Physics and Electronic Engineering, University of Rajshahi for
helping me with the plasma treatment in his laboratory.
I like to convey my deepest gratitude and thank to Md. Azizul Bari (PhD
fellow) and Professor Dr. Abu Reza for his precious advice and guidance
during research.
I would also like to show my appreciation to the Department of Botany,
University of Rajshahi, for the research facilities.