1. EFFECTS OF SIMULATED ACID RAIN (SAR) ON
SUNFLOWER (Helianthus annuus L.) GROWTH,
PHOTOSYNTHETIC PIGMENTS AND YIELD
For the award Degree of
DOCTOR OF PHILOSOPHY
IN
BOTANY
Submitted By
HEM LATA SINGH
M.Sc. (Botany), B. Ed.

2. INTRODUCTION
• Pollution is an undesirable change in the physical, chemical and biological characteristics of
soil, water and air.
• Pollution is the result of industrial technological revolution and speedy exploitation of
every bit of resources.
• The progress in agriculture, industry, transportation, technology and industrial
agglomerations bring about severe adverse side effects which are often hard to predict and
mostly remain uncontrolled.
• Air pollution is indeed of great immediate concern than any other aspect of pollution.
Generally air pollution is created by all over the crust in the form of oil and coal burned to
run factories, machinery and all forms of transportation.
• The discharge of industrial effluents, emissions and automobile gases into natural air
reservoir, water and soil exerts detrimental effects on natural ecology of air, soil and other
life supporting systems.
• The automobile discharges contain many toxic organic and inorganic pollutants, which
affect the biochemistry of living organisms.
• The damages caused by acid precipitation include corrosive action damaging building,
wood, steel and cements concrete structures, release of toxic metals and trace element
drastic effects on aquatic system, mainly due to low mineral content and disturbing the
entire soil chemistry.
• Acid rain is the common name for acid deposition, such as rain, snow, sleet, hail and other
forms of polluted precipitation.
• Acid deposition is a worldwide problem for all natural things including bodies of water,
forests and other things.
• Acid precipitation has been moving more around the world.

3. • The problem of acid rain has become a concern for agriculture.
• Leaf is the most sensitive and reliable part than any other parts of plant like stem, root,
flower, fruit and seed; it may act as a persistent absorber and exploiter in polluted
environment.
• Acidic rain solutions enter the leaf tissue through the cuticle and produce toxic effects
on plants.
• Researches conducted using SAR have shown that it decreases the plant productivity.
• Among the plant metabolites, plant pigments are very sensitive to air pollutants and
identified as indicator of the physiological states of plants influenced by acid rain.
• The decrease in growth occurs when the acidity is due to sulfuric acid or together with
nitric acid.
• Scientists have reported that the impact of acid rain on plant biomass (fresh and dry
weight) differs with cultivars.
• Histological examination of plant tissues has been a useful tool for diagnosing the
sensitivity of plants to pollutants and acid rain.

4. Figure: 1 Formation of Acid Rain from Air Pollutant
Gases
Certain chemical compounds such as nitrogen oxides and sulfur
dioxide mix with the moisture in the air and are formed into clouds
and then produce acid precipitation.

5. OBJECTIVES
• The major objectives of the proposed study were to:
• Evaluate the effect of simulated acid rain on plant growth
behavior particularly roots, shoots and leaves.
• Study the effect of simulated acid rain on flowering behavior
and pollen germination.
• Investigate the effect of simulated acid rain on pH and
conductivity of leaf cell sap.
• Study the effect of simulated acid rain on leaf
photosynthetic pigment contents.
• Record the effect of simulated acid rain on seed yield.

6. MATERIAL
• The proposed study has been conducted with sunflower
(Helianthus annuus L.) variety ‘Morden’ was used as test
plant.
• The present study was carried out in the experimental plot
available at Oilseed Farm, C.S.A.U.A.T., Kanpur.
• The experiments works were conducted in Zaid season (April
to June) to find out the effect of simulated acid rain on
sunflower (Helianthus annuus L.).

7. Figure: 2 SUNFLOWER

8. METHODS
• Sowing of sunflower seeds was done on 12 April 2006 and 2007
• 60 cm inter-row and 30 cm inter-plant spacing (within the row) was
finally maintained.
• The field experiment was conducted in a randomized block design.
• The pH of the acid rain solution was adjusted to different pH by
mixing 1N HNO3 and 1N H2SO4 at 1:2 ratio in all acidic solutions.
• Three concentrations of SAR i.e. pH 3.0, 4.5, 5.7 and control (pH 7.0)
were applied in their respective plots.
• These plots were then irrigated regularly with normal deionized water.
• All treatments of SAR and control plants were treated with 30 ml
solution/plant of different pH, starting from two leaves stage till
initiation of flower buds at weekly intervals with the help of hand
sprayer.
• Acid rain sprayings were given in the early morning.
• Data were recorded at peak growth and maturity stages.
• Data were collected on nine replications for each parameter using
randomly selected from plants in each treatment.

9. • The observations on plants treated with various SAR levels were
recorded for twelve different parameters.
• Biomass (fresh weight and dry weight) and length of root, shoot
and leaf at peak growth and maturity stage.
• Leaf area at peak growth stage.
• Budding behavior and flowering behavior
• Leaf abscission behavior
• pH and conductivity of leaf cell sap
• In vitro pollen germination and pollen tube measurement.
• Photosynthetic pigment contents of leaf at peak growth stage in
terms of total Chl, chlorophyll a, chlorophyll b, carotenoids,
chlorophyll a: b ratio, total chlorophyll: carotenoid ratio.
• Seed yield
• Percent phytotoxicity

10. RESULTS
• The results are described under following heads.
• 1. Analyses of variance (ANOVA)
• 2. Biomass Studies
• 3. Leaf area at peak growth
• 4. Study of budding, flowering and leaf abscission
behavior
• 5. pH and conductivity of leaf cell sap
• 6. In vitro pollen germination and pollen tube growth
• 7. Photosynthetic pigment contents of leaf
• 8. Seed yield
• 9. Percent phytotoxicity

11. ANOVA
• The analyses of variance (ANOVA) for various
variables of the experiments are presented in
Tables.
• These values showed that means have wide
variations for all the characters under study.

12. TABLE: ANALYSIS OF VARIANCE
Root Biomass (fresh weight and dry weight) and Length Shoot Biomass (fresh weight and dry weight) and Length Leaf Biomass (fresh weight and dry weight) and Length
Source of D.F. FW DW Length FW DW Length FW DW Length
variation
PG MS PG MS PG MS PG MS PG MS PG MS PG MS PG MS PG MS
Replication 8 0.6775 0.6183 0.1395 0.2474 0.5924 0.1149 24.312 32.687 5.626 2.487 5.3402 6.415 0.0816 0.0094 0.000436 0.000565 0.0958 0.032113
Treatment 3 83.866** 288.296** 19.567** 22.479** 29.676** 75.613** 12188.66** 13130.91** 4144.47** 4595.36** 2096.88** 2100.33** 5.4505** 9.6283** 0.369456** 0.220137** 18.7499** 20.28373**
Error 24 1.0859 0.5315 0.3269 0.3231 0.2909 0.4343 21.645 26.479 7.904 3.17 8.701 5.823 0.0837 0.0092 0.000656 0.001708 0.5125 0.044575
Leaf Area Bud initiation Flowering Leaf cell sap In vitro Pollen Photosynthesis pigment
Total Chl:
Source of Degree of
Carotenoid
variation freedom
PG First Duration First Period Size pH Conductivity germination tube length Total Chl Chl a Chl b Carotenoid Chl a: b ratio ratio Seed yield
Replication 8 11.22603 0.548611 0.548611 0.375 0.465278 0.0867 0.014624 0.000003 5.125 0.000507 0.015928 0.114 0.000475 0.00105 0.04254 0.010181 3.395277
Treatment 3 4620.1814** 57.36111** 29.74074** 68.259** 20.472** 23.75** 15.3179** 1.02784** 3457.361** 3.01208** 0.41918** 0.2766** 0.0176** 0.05309** 0.9216 0.00062 1097.4195**
Error 24 9.552294 0.548611 1.344907 0.717593 0.659722 0.08396 0.011483 0.000005 3.319444 0.000647 0.002925 0.001765 0.000525 0.00039 0.04901 0.004806 2.994536

13. • The ANOVA showed highly significant differences of SAR
treatments for the characters like root biomass (fresh weight
and dry weight) and length at peak growth and maturity stage,
shoot (biomass and length) at peak growth and maturity stage
and leaf (biomass and length) at peak growth and maturity stage
in Table.
• Leaf area at peak growth stage, behavior of budding, flowering,
pH and conductivity of leaf cell sap, in vitro pollen germination
and pollen tube growth, total chlorophyll, chlorophyll a,
chlorophyll b, carotenoids and seed yield.
• Non-significant differences of simulated acid rain treatments
were observed for the characters like chlorophyll a: b ratio and
total chlorophyll: carotenoid ratio.

14. Biomass Studies
• The effects of simulated acid rain (SAR) treatment
on biomass (fresh weight and dry weight) and
length of root, shoot and leaf at peak growth and
maturity stages of sunflower (Helianthus annuus L.)
plants are summarized in Figure 3 to 11.

15. Figure: 3 Effect of SAR on Root Fresh Weight at
peak growth and maturity stage
Peak growth Maturity stage
30
25
20
Root Fresh Weight (g)
15
10
5
0
7 5.7 Treatments (SAR pH) 4.5 3

16. Figure: 4 Effect of SAR on Root Dry Weight at
peak growth and maturity stage
Peak growth Maturity stage
9
8
7
6
Root Dry Weight (g)
5
4
3
2
1
0
7 5.7 4.5 3
Treatments (SAR pH)

17. Figure: 5 Effect of SAR on Root Length at peak
growth and maturity stage

18. Figure: 6 Effect of SAR on Shoot Fresh Weight at
peak growth and maturity stage
Peak growth Maturity stage
200
180
160
140
Shoot Fresh Weight (g)
120
100
80
60
40
20
0
7 5.7
Treatments (SAR pH) 4.5 3

19. Figure: 7 Effect of SAR on Shoot Dry Weight at
peak growth and maturity stage
Peak growth Maturity stage
90
80
Shoot Dry Weight (g)
70
60
50
40
30
20
10
0
7 5.7 4.5 3
Treatments (SAR pH)

20. Figure: 8 Effect of SAR on Shoot Length at peak
growth and maturity stage
Peak growth Maturity stage
100
90
80
70
Shoot Length (cm)
60
50
40
30
20
10
0
7 5.7Treatments (SAR pH) 4.5 3

21. Figure: 9 Effect of SAR on Leaf Fresh Weight at
peak growth and maturity stage

22. Figure: 10 Effect of SAR on Leaf Dry Weight at
peak growth and maturity stage

23. Figure: 11 Effect of SAR on Leaf Length at peak
growth and maturity stage

24. Leaf Area
• Leaf Area showed significant difference with
control. Comparison of SAR treatments showed
that the leaf area decreased from control (167.18
cm²) to pH 3.0 (113.61 cm²).

25. Figure: 12 Effect of SAR on Leaf Area at peak
growth stage

26. BUDDING BEHAVIOR
• It was observed that bud initiation took minimum
days in control which got increased with
decreasing pH level of 5.7, 4.5 and 3.0 to
53.11, 54.11 and 57.88 days, respectively. The
maximum difference of first bud initiation was
recorded between pH 4.5 and pH 3.0.
• Budding period decreased with increasing level of
acidity. The minimum difference was recorded
between control to pH 5.7 i.e. 1.11 (4.28 %) and
maximum difference between pH 4.5 and pH 3.0
i.e. 1.78 (7.59 %).

27. Figure: 13 Effect of SAR on Budding behavior
and Duration of bud initiation(days)
Days taken to First bud initiation Duration of bud initiation
70
60
Budding Behavior (days)
50
40
30
20
10
0
7 5.7 4.5 3
Treatments (SAR pH)

28. Figure: 14 Effect of SAR on First flower
opening, Duration of Flowering(days) and Average
flower size(cm)

29. FLOWERING BEHAVIOR
• The acidity was increased days taken to first flower
opening increased accordingly. It was 57.88 in pH
4.5 followed by pH 3.0 with 62.11 days (maximum).
• An increasing acidity showed decreasing trend on
duration of flowering (days) in the plants. The pH
5.7, 4.5 and pH 3.0 showered plants, period of
flowering were showed 27.22, 25.88 and 24.77
days, respectively.
• The decrease over the control in head diameter was
recorded by 3.87 cm or 33.87 % in pH 3.0, 2.74 cm
or 17.08 % in pH 4.5 and 1.79 cm or 11.15 % in pH
5.7.

30. Figure: 15 Comparison Showing Effect of
SAR on Delay in first flower opening

31. FLOWER
• An observation of Figure 15 clearly reveals that
the days taken to first flower opening was
significantly affected by different level of pH.

32. Figure: 16 Comparison Showing Effect of
SAR on head Diameter (cm)

33. LEAF SYMPTOMS
• When young leaves were exposed to acid rai n at
pH 3.0 during the early development stage, they
became severely necrotic, crinkled and wrinkled.
However, mature leaves were only slightly injured
(Figure 17).
• Leaves abscissions was maximum at pH 3.0 (15.10
%) as compared to control.

34. Figure: 17a Mature Leaves of Sunflower

35. Figure: 17b Effect of SAR on Young Leaves

36. Figure: 18 Leaf Abscission Percentage (%)

37. CONDUCTIVITY
• The comparative study of entire treated plants
showed that the pH 4.5 and 3.0 was
maximum difference of reduced pH of leaf cell
sap observed as compared to control.
• The maximum difference was observed between
pH 4.5 and pH 3.0 (26.43 %). The values also
showed significant increases in conductivity of leaf
cell sap as compared to entire treated plants.
•

38. Figure: 19 Effect of SAR on pH of leaf cell
sap at peak growth stage

39. Figure: 20 Effect of SAR on Conductivity of
leaf cell sap at peak growth stage

40. IN VITRO POLLEN
• Comparison of SAR treatment showed that the
pollen germination decreased from control (86.88
%) to pH 3.0 (43.22 %). Pollen exposed to SAR at
pH 3.0 was minimum germinated among all
treated pollens.
• It is clear from the Figure 22 that measurement of
pollen tube was decreased significantly with as
increase in the level of acidity.

41. Figure: 21 Effect of SAR on in vitro Pollen
Germination
100
90
80
70
Pollen Germination (%)
60
50
40
30
20
10
0
7 5.7 4.5 3
Treatments (SAR pH)

42. Figure: 22 Effect of SAR on in vitro Pollen Tube
Length
3
2.5
Pollen Tube Length (μm)
2
1.5
1
0.5
0
7 5.7 4.5 3
Treatments (SAR pH)

43. Photosynthetic pigment contents
• SAR application showed decreasing trend in
chlorophyll content.
• Chl a and Chl b contents followed similar trend in
change as was observed in case of total chlorophyll.
• The carotenoids content showed highest difference
between pH 4.5 and pH 3.0 i.e. (0.084). It has lowest
difference between pH 5.7 and pH 4.5 i.e. (0.023).
• The values were statistically at par in case of
chlorophyll a: b ratio at peak growth (Figure 23b).
• These values showed unaffected total chlorophyll to
carotenoid ratio from SAR.

44. Figure: 23a Effect of SAR on Photosynthetic
pigment contents at peak growth stage
Total Chl Chl a Chl b
2.5
Photosynthetic Pigment Contents (mg g-1 fw)
2
1.5
1
0.5
0
7 5.7 4.5 3
Treatments (SAR pH)

45. Figure: 23b Effect of SAR on Carotenoids, Chl
a:b ratio and Total Chl : carotenoid ratio
Carotenoid Chl a: b ratio Total Chl: Carotenoid ratio
4.5
Photosynthetic Pigments Contents (mg g -1 fw)
4
3.5
3
2.5
2
1.5
1
0.5
0
7 5.7 4.5 3
Treatments (SAR pH)

46. Figure: 24 Effect of SAR on Seed Yield
50
45
40
35
30
Seed Yield (g)
25
20
15
10
5
0
7 5.7 4.5 3
Treatments (SAR pH)

47. SEED YIELD
• The comparison of different SAR treatments
showed that the seed yield per head decreased
highest at pH 3.0 from control.
• Average seed weight and size were decreased with
increasing level of acidity (Figure 25).

48. Figure: 25 Comparison Showing Effect of
SAR on variation of the Seeds size
(a) pH 7.0 (b) pH 5.7
-
(c) pH 4.5 (d) pH 3.0

49. PHYTOTOXICITY
• The effects of simulated acid rain (SAR) treatment
on percent phytotoxicity in shoot and root tissues
at peak growth and maturity stage was measured
keeping pH 7.0 as control and results are
summarized below.

50. Figure: 26 Effect of SAR on Percent Shoot phytotoxicity
at peak growth and maturity stage

51. PERCENT SHOOT PHYTOTOXICITY
• Percent phytotoxicity in shoot tissues at peak
growth stage was measured keeping pH 7.0 as
control. SAR application caused increase in
percent shoots phytotoxicity and was 27.30, 35.81
and 38.90 at pH 5.7, 4.5 and 3.0, respectively.

52. Figure: 27 Effect of SAR on Percent Root phytotoxicity
at peak growth and maturity stage

53. PERCENT ROOT PHYTOTOXICITY
• Comparative evaluation of SAR treated plants
showed that the percent root phytotoxicity was
highest at pH 3.0. SAR effect on roots showed
significantly increasing trend of percent
phytotoxicity from pH 4.5 (34.01) to 3.0 (46.73).

54. Shoot and Root Percent Phytotoxicity
• Comparison between shoot and root percent
phytotoxicity of sunflower variety Morden exposed
to simulated acid rain revealed that percent shoot
phytotoxicity and percent root phytotoxicity could
serve as good biological parameters for evaluating
relative sensitivity.

55. CONCLUSION
• The study reveals that acid rain decreased length and biomass
accumulation of root, shoot and leaf.
• The adverse effects of simulated acid rain reported here and elsewhere
demonstrate the potential for acute effects of atmospheric acidic
depositions on growth parameters, photosynthetic pigment content in
terms of Total Chl, Chl a, Chl b, carotenoids, Chl a: b ratio, Total Chl:
carotenoid ratio, seed yield and yield contributing characters like
pollen germination and pollen tube length in treated plants.
• The percent phytotoxicity determination in plant roots and shoot
provides indicators for biomonitoring the sensitivity to acid rain in the
sunflower plant.

56. • The results further suggest that problems related to acid rain are
likely to arise in future in view of rapid and uncontrolled
industrialization in the all parts of the world, particularly
developed countries as acid rain is harmful for normal
survival, growth and yield in major crops. However, this clue needs
to be examined widely before making generalizations.
• There is a need to identify suitable variety to be grown in acid rain
affected zones.
• The concentration of SAR may increase to an extent causing an
acidification of cytoplasm to decrease intracellular pH.
• The capacity of acidic buffering and the mechanism(s) involved are
still unclear and require further in depth investigation.

57. • THANK YOU VERY MUCH