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.

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.

• 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.

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.

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.

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.).

Figure: 2 SUNFLOWER

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.

• 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

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

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.

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 variation

D.F. FW DW Length FW DW Length FW DW Length

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

Source of variation

Degree of freedom

PG First Duration First Period Size pH Conductivity germinationtube length

Total Chl Chl a Chl b Carotenoid Chl a: b ratio

Total Chl: Carotenoid

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

• 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.

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.

Figure: 3 Effect of SAR on Root Fresh Weight at peak growth and maturity stage

7 5.7 4.5 30

5

10

15

20

25

30

Peak growth Maturity stage

Treatments (SAR pH)

Root

Fre

sh W

eigh

t (g)

Figure: 4 Effect of SAR on Root Dry Weight at peak growth and maturity stage

7 5.7 4.5 30

1

2

3

4

5

6

7

8

9

Peak growth Maturity stage

Treatments (SAR pH)

Root

Dry

Wei

ght (

g)

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

Figure: 6 Effect of SAR on Shoot Fresh Weight at peak growth and maturity stage

7 5.7 4.5 30

20

40

60

80

100

120

140

160

180

200

Peak growth Maturity stage

Treatments (SAR pH)

Shoo

t Fre

sh W

eigh

t (g)

Figure: 7 Effect of SAR on Shoot Dry Weight at peak growth and maturity stage

7 5.7 4.5 30

10

20

30

40

50

60

70

80

90

Peak growth Maturity stage

Treatments (SAR pH)

Shoo

t Dry

Wei

ght (

g)

Figure: 8 Effect of SAR on Shoot Length at peak growth and maturity stage

7 5.7 4.5 30

10

20

30

40

50

60

70

80

90

100

Peak growth Maturity stage

Treatments (SAR pH)

Shoo

t Len

gth

(cm

)

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

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

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

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).

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

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 %).

Figure: 13 Effect of SAR on Budding behavior and Duration of bud initiation(days)

7 5.7 4.5 30

10

20

30

40

50

60

70

Days taken to First bud initiation Duration of bud initiation

Treatments (SAR pH)

Budd

ing

Beha

vior

(day

s)

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

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.

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

FLOWER

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

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

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.

Figure: 17a Mature Leaves of Sunflower

Figure: 17b Effect of SAR on Young Leaves

Figure: 18 Leaf Abscission Percentage (%)

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. 

•

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

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

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.

Figure: 21 Effect of SAR on in vitro Pollen Germination

7 5.7 4.5 30

10

20

30

40

50

60

70

80

90

100

Treatments (SAR pH)

Polle

n Ge

rmin

ation

(%)

Figure: 22 Effect of SAR on in vitro Pollen Tube Length

7 5.7 4.5 30

0.5

1

1.5

2

2.5

3

Treatments (SAR pH)

Polle

n Tu

be Le

ngth

(μm

)

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.

Figure: 23a Effect of SAR on Photosynthetic pigment contents at peak growth stage

7 5.7 4.5 30

0.5

1

1.5

2

2.5

Total Chl Chl a Chl b

Treatments (SAR pH)

Phot

osyn

theti

c Pig

men

t Con

tent

s (m

g g-

1 fw

)

7 5.7 4.5 30

0.5

1

1.5

2

2.5

3

3.5

4

4.5

Carotenoid Chl a: b ratio Total Chl: Carotenoid ratio

Treatments (SAR pH)

Phot

osyn

theti

c Pig

men

ts C

onte

nts (

mg

g -1

fw)

Figure: 23b Effect of SAR on Carotenoids, Chl a:b ratio and Total Chl : carotenoid ratio

7 5.7 4.5 30

5

10

15

20

25

30

35

40

45

Treatments (SAR pH)

Seed

Yie

ld (g

)Figure: 24 Effect of SAR on Seed Yield

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).

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

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.

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

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.

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

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).

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.

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.

  

• 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.

• THANK YOU VERY MUCH