Classification of Different Types of Plants by their Chlorophyll...

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University of Africa Journal of Sciences

Classification of Different Types of Plants by their

Chlorophyll Fluorescence Spectra

Using Nitrogen Laser

I. M. K. Medani1, A. K. Sabir Ali2 , S. T. Kafic and A. M. Ahmed3

1 Physics Department, Faculty of Pure and Applied Sciences, International University of Africa, Khartoum, Sudan. 2 School of Life Sciences, Faculty of Science and Technology, Al Neelain University, Khartoum, Sudan. 3 School of Applied Physics, Faculty of Science and Technology, Al Neelain University, Khartoum, Sudan.

Abstract

This work was aimed to differentiate between six major plant

types.These include hardwood dicots, herbaceous dicots, hardwood

monocots, herbaceous monocots, gymnosperms, and algae. Each of

these plant types exhibited a characteristic (LIF) laser induced

chlorophyll a fluorescence spectra when excited by a pulsed N2 laser

emitting at 337.8 nm. All the plant types studied here agree in having

a green fluorescence, and a red fluorescence emitted from their leaves.

But the gymnosperms and the hardwood dicots were the only plant

types that had a measurable far red fluorescence. The gymnosperms

can be differentiated from the hardwood dicots by the additional

fluorescence at 715 nm. Herbaceous dicots and herbaceous monocots

possess common fluorescence maxima at 522, 680 and 715nm, they

could be differentiated from each other by using the average of the

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peak intensity ratio, and the average of the area ratio between these

different three peaks. In all cases the values of the herbaceous

monocots are approximately three times that of the herbaceous dicots.

The hardwood monocots and the algae were differentiated from the

other groups by the absence of the far red fluorescence together with

the peak of 715nm fluorescence and these can be differentiated from

each other by the position of the green fluorescence. So the potential

use of the LIF technique for discrimination between different species

appears to be suitable.

Keywords: Discrimination; Dicots, monocots, gymnosperms and

algae; Fluorescence; Chlorophyll a; Nitrogen laser 337.8nm.

Introduction

Classification of the different types of plants by means of laser-

induced chlorophyll fluorescence emission techniques, is a developing

tool in remote sensing studies. It plays a great role in the ability to

distinguish between plant types with specific identification, and can

place every plant in special category. The study of the chlorophyll

spectral measurements is used to deal only with the vegetation

changes in the different areas (Denison and Russotti 1997; Tejada et al

2005), however, if the discrimination between the difference types of

plants is available, one can correlate any difference in the spectral

measurement to the environmental stress effect (Schächtl 2005;

Subhash 2004). The objective of this study is to classify the plant

types according to their differences (variability) and physiological

status. The most important plant constituent is chlorophyll, which

3


plays an important role in photosynthesis process. Beside chlorophyll

there are many other pigments which emit fluorescence when excited

with a proper exciting wavelength. Of course, the genetic differences

of the plant include the differences in their pigments, so it is

reasonable to find the different spectral shapes of the different plant

types. We present here the results of six different groups (hardwood

dicots, herbaceous dicots, hardwood monocots, herbaceous monocots,

gymnosperms, and algae), and see whether these groups can be

identified on the basis of their different fluorescence spectral shapes.

Materials and methods

The hardwood dicots studied here were Diospyrus mispiliformis,

Tectonia, Zygophyllum, Olea europoea, and Eucalyptus. The

herbaceous dicots contained of Alternanthera, Cilinus lotoides,

Euphorbia heterophylla, Solanum nigrum L, and Amaranthera viridis

L. The hardwood monocots included Lamintania cantaroides,

Oreodoxa regia, Phoenix sp, Washintonia sp, and Phoenix dactylifera.

The herbaceous monocots were Citichnizia cemmelinaceae, Aspargus

sprangeri, Aspargus plumosus, Caralluma, and Cymbopogan citrates.

The gymnosperms chosen were Thuja orientalis, and Cycas revileota.

And the algae sample was Spirogyra. The leaves samples for all these

groups, except the algae group, investigated in this study were

obtained from the National Botanical Garden in Khartoum (Sudan).

The algae sample was obtained from the biological laboratory at

Elneelain University. The samples were taken from plants in the

similar stages of physiological development. The measurements were

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taken from different points of the fully outermost leaves. The spectra

of ten leaves from each plant were measured then the average was

taken. The algae sample was exposed to laser excitation in liquid

culture in a Pyrex test tube, repeated for five times. A constant area of

π(2)2 (mm)2 of the uppermost fully developed surface was exposed to

the Homebuilding UV pulsed nitrogen laser which emits at 337.8 nm

at a distance of 1 m. This laser was run with a pulse output power of

0.4MW. The leaf was fixed to a nonfluorescent stainless steel plate. A

compact software controlled spectrophotometer of the type USB2000

from Ocean optics company, Dunedin, USA/Northampton, USA) was

used for recording the fluorescence signal emitted from plants intact

leaf. The resolution of the USB2000 spectrometer used was 1.34nm

FWHM (Ocean optics 2005), and its detector covers the range from

350nm to 1100nm. The whole setup is coupled to a laptop computer

for mobile use, and field measurement. Data recorded were analyzed

using the software ORIGIN 6.1. The software uses (Marquardt-

Levenberg) algorithm for iterative non-linear curve fitting with a

combination of Gaussian spectral functions to analyze the spectra.

Results and Discussion

The spectra of the five hardwood dicots, five herbaceous dicots, five

hardwood monocots, five herbaceous monocots, two gymnosperms

and one algae on the same fluorescence intensity scale were shown

from Fig. 1 up to Fig. 6 Also the spectra of the different groups on the

same fluorescence intensity scale are shown in Fig. 7. Example of

each group is shown from Fig. 8 up to Fig. 13. Each spectrum is the

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average of 10 different leaves of the same plant. The evaluation of the

standard errors for the wavelength at maximum peak (λmax), and the

fluorescence intensity peak amplitude (IF), show that the

determination of the peaks was acceptable with minimum standard

error (Marquard (1963). The determination of the band area (A)

became easier with limited standard error when a Gaussian fitting was

made on them (Anderson et al 2004). Thus the Gaussian fitting was

made on each spectrum , (the Figs. from (8 to13). So the smoothing

and the averaging of the randomness on the profile of the curve were

done as well as getting separate curves for area ratio evaluation. In

addition full width at half maximum (FWHM) Δλ was determined.

Fig.1. The spectra of hardwood dicots

1 Diospyrus mispiliformis

2 Tectonia grandis

3 Zygophyllum sp.

4 Olea europoea

5 Eucalyptus sp.

Fig.2. The spectra of herbaceous dicots

1 Alternanthera sp.

2 Cilinus lotoides

3 Euphorbia heterophylla

4 Solanum nigrum L

5 Amaranthera viridis L

350 400 450 500 550 600 650 700 750 800 850 900 9500

330

660

990

1320

1650

1980

2310

2640

2970

3300

5

4

3

2

1

I F(a.u

.)

(nm)350 400 450 500 550 600 650 700 750 800 850 900 950

0

360

720

1080

1440

1800

2160

2520

2880

3240

3600

5

4

3

2

1

I F(a.u

.)

(nm)

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Fig..3. The spectra of hardwood monocots

Fig.4. The spectra of herbaceous monocots Fig.5. The spectra of gymnosperms 1 Thuja orientalis

2 Cycas revileota

1 Lamintania cantaroides 2 Oreodoxa regia 3 Phoenix sp

4 Washintonia sp 5 Phoenix dactylifera

1 Citichnizia cemmelinaceae 2 Aspargus sprangeri 3 Aspargus plumosus 4 Caralluma 5 Cynbopogon citrates

350 400 450 500 550 600 650 700 750 800 850 900 9500

300

600

900

1200

1500

1800

2100

2400

2700

3000

5

43

2

1

I F(a.u

.) (nm )

350 400 450 500 550 600 650 700 750 800 850 900 9500

350

700

1050

1400

1750

2100

2450

2800

3150

3500

543 21

I F(a.u

.)

(nm)350 400 450 500 550 600 650 700 750 800 850 900 950

0

350

700

1050

1400

1750

2100

2450

2800

3150

3500

21

I F(a.u

.)

(nm)

7


Fig.6.. The spectra of alga (Spirogyra)

Fig.7. The spectra of different plant types on the same IF scale

1 Algae 2 Gymnosperms 3 Hardwood Dicots

4 Herbaceous Dicots 5 Hardwood Monocots 6 Herbaceous Monocots

300 350 400 450 500 550 600 650 700 750 800 850 900 9500

45

90

135

180

225

270

315

360

405

450

I F(a.u

.)

(nm)

350 400 450 500 550 600 650 700 750 800 850 900 9500

350

700

1050

1400

1750

2100

2450

2800

3150

3500

6

5

4 32

1

I F(a.u

.)

(nm)

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Fig.8. Diospyrus mispiliformis Fig.9 Alternanthera (herbaceous dicots)

(hardwood dicots)

Fig.10. Lamintania Cantaroides Fig.11. Citichnizia

(hardwood monocots)

cemmelinacea(herbaceousmonocots)

300 350 400 450 500 550 600 650 700 750 800 850 900 950

35

70

105

140

175

210

245

280

315

350

733680

528

I F(a.u

.)

(nm)350 400 450 500 550 600 650 700 750 800 850 900 950

0

350

700

1050

1400

1750

2100

2450

2800

3150

3500

715

680

522

I F(a.u

.)

(nm)

300 350 400 450 500 550 600 650 700 750 800 850 900 9500

208

416

624

832

1040

1248

1456

1664

1872

2080

680

530

I F(a.u

.)

(nm)350 400 450 500 550 600 650 700 750 800 850 900 950

0

348

696

1044

1392

1740

2088

2436

2784

3132

3480

715

680

522

I F(a.u

.)

(nm)

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Fig.12. Cycas revileota (gymnosperm) Fig.13. Spirogyra (Algae)

Green plants under ultra violet excitation emit a red fluorescence, a far

red fluorescence, both from chlorophyll, and a blue-green

fluorescence coming mainly from hydroxycinnamic acids (Apostol

2005). All the plant types studied here have a green fluorescence, and

a red fluorescence emitted from their leaves. The gymnosperms and

the hardwood dicots were the only plant types that had a measurable

far red fluorescence emitted from their leaves. The gymnosperms

differed from the hardwood dicots in that they had an additional

fluorescence at 715 nm. The herbaceous dicots and monocots also

have a fluorescence at 715 nm. The absence of the blue fluorescence

in all the plant types does not mean there is no hydroxycinnamic

acids. Also the absence of the far red fluorescence in algae, hardwood

monocots, and herbaceous dicots and monocots does not mean the

absence of chlorophyll in photosystem I. The reason in the first case

may be due to the sink of the transfer excitation energy from the blue

350 400 450 500 550 600 650 700 750 800 850 900 950

350

700

1050

1400

1750

2100

2450

2800

3150

3500

736715

680

500

I F(a.u

.)

(nm)300 350 400 450 500 550 600 650 700 750 800 850 900 950

45

90

135

180

225

270

315

360

405

450

680500

I F(a.u

.)

(nm)

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Soret band to the green Soret band and then to the lower energy Q

bands in the photosynthetic cycle, so there is no efficient energy to

dissipate as fluorescence at the blue region (Chappelle et al 1985). But

in the other case, the reason may be due to the high intensity of

photosystem II so there is no sufficient energy to dissipate as

fluorescence at photosystem I (Subhash et al 1995). From the Figs.1 to

6, it was shown that the wavelengths at maximum peaks, peak centers

(λmax), were identical in the same group. But from Fig.7, which

shows the spectra of all the different plant types on the same

fluorescence intensity scale, it was shown that the wavelengths at

maximum peaks differed from one group to other.

Having observed these spectral differences among the different

studied groups, we can differentiate between them. The absence of the

maximum at 715 nm in hardwood dicots Fig.8, distinguishes them

from gymnosperms Fig.12. The presence of the maximum at 715 nm

in herbaceous dicots Fig.9 and herbaceous monocots Fig.11,

differentiates them from the hardwood monocots Fig.10, and algae

Fig.13. Hardwood monocots and algae can be differentiated from each

other by the position of the maximum wavelength peak at the green

fluorescence. Herbaceous monocots and herbaceous dicots exhibit the

same spectral shape, and the positions of their maximum wavelength

peaks are the same. But the intensity of the Soret band maximum in

the herbaceous monocots is considerably higher than that of the

herbaceous dicots. So to distinguish between them, we use the peak

intensity ratio (P.I.R.) and the area ratio (A.R.) between the Soret band

and photosystem II. Also between the Soret band and the chlorophyll

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fluorescence at 715 nm. See table13. Thus, on the basis of the

presence or absence of certain fluorescence maxima, positions of the

maximum wavelength peaks, and by using (P.I.R.) and (A.R.), six

plant groups can be identified. Such classification of the different

types of plants using a pulse N2 laser was done by (Chappelle et al

(1985). He distinguished between five major plant types, and there is

no contradiction between the findings of his study and the present

study. If the scale of the fluorescence is decreased, the spectra of the

peaks of all this groups will appear more elaborated and more clearly,

but because the peak at photosystem II has a very large intensity, it

will not appear completely.

Table1. Parameters obtainen from the Gaussian fitting curves for the hardwood

dicots

Table1.a Diospyrus mispiliformis

Table1.b Tectonia

Diospyrusmispilifor

mis λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

P3

733.29±0.37

680.10±0.83

528.08±0.27

49.739±0.052

12.237±0.941

104.165±0.149

5869.80±3.72

5319.89±8.61

2761.64±6.50

97.46±0.87

345.36±0.43

20.78±0.43

Tectonia λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

P3

733.16±0.38

680.98±0.29

528.22±0.79

61.793±0.292

11.824±0.836

96.085±0.764

1929.39±8.64

21047.22±2.73

2529.68±5.28

63.03±0.37

3211.07±0.28

71.38±0.19

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Table1.c Zygophyllum

Table1.d Olea europo

Table 1.e Eucalyptus

Table2. Parameters obtained from the Gaussian fitting curves for the herbaceous

dicots

Table 2.a Alternanthera

Zygophyllum λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

P3

733.26±0.27

680.38±0.19

528.62±0.46

58.387±0.923

11.248±0.567

92.876±0.645

1829.76±4.43

23158.23±5.38

2629.69±3.29

28.54±0.46

1429.32±0.27

32.26±0.38

Olea europoea λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

P3

733.25±0.19

680.12±0.28

528.48±0.37

49.837±0.564

12.268±0.298

136.815±0365

2860.17±4.91

2297.78±9.82

1617.82±2.73

46.21±0.29

159.32±0.18

10.16±0.93

Eucalyptus λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

P3

733.71±0.38

680.67±0.63

528.92±0.27

48.166±0.876

11.470±0.923

92.550±0.487

5121.44±5.28

29509.18±6.45

5988.38±3.74

90.13±0.48

1997.15±0.94

65.03±0.34

P1 : PSI P2 : PSII P3 : Soret band

λmax : Peak center Δλ: FWHM (full width half maximum) A : Gaussian area

IF : Fluorescence Intensity

Alternantheraternata λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

P3

715.39±0.83

680.14±0.27

522.15±0.16

18.153±0.384

12.234±0.576

90.309±0.786

1145.51±2.92

51754.00±8.74

9269.88±5.32

67.88±0.23

3502.99±0.76

94.84±0.45

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Table 2.b Cilinus lotoides

Table 2.c Euphorbia heterophylla

Table 2.d Solanum nigrum L

Table 2.e Amaranthera viridis L

P1 : Chlorophyll Fluorescence at 715 P2 : PSII P3 : Soret band

Cilinus lotoides λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

P3

715.00±0.35

680.95±0.75

522.79±0.32

16.073±0.967

11.982±0.342

98.136±0.645

646.07±4.52

48686.11±9.74

10741.16±6.49

38.59±0.92

3302.82±0.83

97.89±0.74

Euphorbiaheterophylla λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

P3

715.02±0.78

680.87±0.37

522.02±0.47

13.936±0.976

11.784±0.546

96.306±0.476

567.73±3.45

46583.99±2.65

5239.36±8.23

37.42±0.98

3161.67±0.56

53.85±0.67

SolanumnigrumL λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

P3

715.45±0.95

680.15±0.37

522.68±0.96

18.086±0.435

12.229±0.567

100.928±0.436

797.23±4.38

35419.84±9.74

7676.57±6.65

46.55±0.25

2383.57±0.87

65.03±0.64

AmarantheraviridisL λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

P3

715.91±0.65

680.13±0.45

522.81±0.43

17.247±0.354

12.157±0.867

109.797±0.476

814.37±6.67

51160.42±8.23

3728.71±5.36

49.91±0.85

3476.04±0.46

40.93±0.64

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Table3. Parameters obtained from the Gaussian fitting curvesfor the hardwood

monocots

Table 3.a Lamintania cantaroider

Table 3.b Oreodoxa regia

Table 3.c Phoenix sp

Table 3.d Washintonia sp

Table 3.e Phoenix dactylifera

P1 : PSII P2 : Soret band

Lamintaniacantaroider λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

680.66±0.67

530.65±0.46

11.338±0.735

97.981±0.587

29939.33±9.54

6405.76±7.87

2087.12±0.85

56.36±0.76

Oreodoxa regia λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

680.48±0.58

530.39±0.73

11.216±0.576

113.769±0.843

13253.06±6.87

4074.16±7.95

909.70±0.24

30.30±0.75

Phoenix sp λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

680.49±0.67

530.63±0.94

11.191±0.387

83.439±0.487

16477.21±6.75

3284.58±2.38

1124.42±0.78

34.11±0.74

Washintonia sp λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

680.49±0.45

530.55±0.95

11.188±0.756

83.636±0.978

21412.06±9.83

4177.51±7.73

1494.01±0.65

44.92±0.48

Phoenixdactylifera λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

680.66±0.96

530.19±0.45

11.336±0.967

98.293±0.438

41303.00±9.29

8824.93±7.45

2842.93±0.85

86.02±0.63

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Table4. Parameters obtained from the Gaussian fitting curvesfor the herbaceous

monocots

Table 4.a Citichnizia cemmelinaceae

Table 4.b Aspargus sprangeri

Table 4.c Asparagus plumosus

Table 4.d Caralluma

Table 4.e Cynbopogon citrates

P1 : Chlorophyll Fluorescence at 715 P2 : PSII P3 : Soret band

Citichniziacemmeli

naceae λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

P3

715.45±0.47

680.06±0.39

522.81±0.94

17.487±0.374

12.186±0.856

95.451±0.756

1002.20±5.96

51008.70±8.54

29727.25±3.67

67.50±0.64

3447.24±0.34

254.10±0.26

Aspargussprangeri λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

P3

715.53±0.47

680.69±0.32

522.32±0.56

13.791±0.478

11.679±0.376

91.594±0.264

496.13±5.83

36558.13±7.58

17102.60±3.45

29.24±0.78

2476.47±0.54

150.46±0.35

Asparagusplumosus λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

P3

715.03±0.67

680.69±0.54

522.13±0.87

18.646±0.534

11.661±0.362

90.237±0.936

879.82±5.94

48633.82±7.28

22090.70±6.74

39.18±0.85

3309.87±0.34

201.61±0.27

Caralluma λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

P3

715.33±0.46

680.06±0.54

522.44±0.37

12.197±0.374

12.184±0.645

96.644±0.786

649.07±4.48

39231.32±7.29

22936.78±6.02

44.59±0.24

2638.66±0.76

200.30±0.67

Cynbopogoncitrats λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

P3

715.04±0.85

680.95±0.97

522.42±0.58

13.216±0.476

12.001±0.598

93.919±0.467

787.74±3.75

49074.94±9.49

10334.27±8.82

30.58±0.74

3318.47±0.93

99.37±0.54

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Table5. Parameters obtained from the Gaussian fitting curves for the gymnosperms

Table 5.a Cycas revileota

Table 5.b Thuja orientalis

P1 : PSI P2 : Chlorophyll Fluorescence at 715


Table6. Parameters obtained from the Gaussian fitting curves for the algae


Table 7. Peak intensity ratio (P.I.R.) and area ratio (A.R.)

GF: Green fluorescence PSII : Photosystem II PSI : Photosystem I

Cycas revileota λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

P3

P4

736.15±0.34

715.75±0.92

680.01±0.36

500.46±0.84

41.092±0.271

30.404±0.592

12.264±0.502

69.992±0.435

9530.94±5.83

6858.99±3.71

48110.92±8.73

32181.88±6.95

188.36±0.82

197.47±0.59

3262.75±0.38

375.29±0.37

Thujaorientalis λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

P3

P4

736.09±0.36

715.59±0.34

680.15±0.81

500.23±0.83

41.791±0.644

28.509±0.276

12.329±0.175

83.613±0.276

7489.32±2.38

4602.27±7.72

51458.29±3.34

19267.58±4.95

148.75±0.92

139.76±0.56

3432.12±0.67

202.65±0.34

Algae λmax(nm) Δλ (nm) A(cm2) IF (a.u.)

P1

P2

680.51±0.32

500.48±0.84

11.098±0.643

85.473±0.362

6208.19±6.47

4551.52±7.50

445.55±0.27

43.05±0.29

Hardwood

Dicots

P.I.R.

GF/PSII

A.R.

GF/PSII

P.I.R.

GF/PSI

A.R.

GF/PSI

P.I.R.

PSII/PSI

A.R.

PSII/PSI

Diospyrusmispiliformis

Tectonia grandis

Zygophyllum

Olea europoea

Eucalyptus

0.060

0.022

0.023

0.064

0.033

0.519

0.120

0.114

0.704

0.203

0.213

1.132

1.130

0.220

0.722

0.470

1.311

1.437

0.566

1.169

3.544

50.945

50.081

3.448

22.159

0.906

10.909

12.656

0.803

5.762

17



GF : Green fluorescence PSII : Photosystem II Chl. F : Chlorophyll

fluorescence



Herbaceous

Dicots

P.I.R.

GF/PSII

A.R.

GF/PSII

P.I.R.

GF/Chl.F

A.R.

GF/Chl.F

P.I.R.

PSII/Chl.F

A.R.

PSII/Chl.F

Alternanthera

Cilinus lotoides

Euphorbiaheterophylla

Solanum nigrum L

Amaranthera viridis L

0.027

0.030

0.017

0.027

0.012

0.179

0.221

0.112

0.217

0.073

1.397

2.537

0.958

1.397

0.820

8.092

16.625

9.229

9.629

4.579

51.606

85.587

84.491

51.205

69.646

45.180

75.357

82.053

44.429

62.822

Hardwood monocots P.I.R.GF/PSII A.R.GF/PSII

Lamintania cantaroides

Oreodoxa regia

Phoenix sp

Washintonia sp

Phoenix dactylifera

0.031

0.033

0.030

0.030

0.030

0.214

0.307

0.199

0.195

0.214

Herbaceous monocots P.I.R.

GF/PSII

A.R.

GF/PSII

P.I.R.

GF/Chl.F

A.R.

GF/Chl

.F

P.I.R.

PSII/Ch

l.F

A.R.

PSII/Ch

l.F

Citichniziacemmelinaceae

Aspargus sprangeri

Aspargus plumosus

Caralluma

Cynbopogon citrates

0.074

0.061

0.061

0.076

0.030

0.583

0.468

0.454

0.585

0.211

3.764

5.146

5.146

4.492

3.250

29.662

34.472

25.108

35.338

13.119

51.070

84.695

84.479

59.176

108.518

50.897

73.687

55.277

60.442

62.298

18




Table13. Comparison between herbaceous monocots and herbaceous dicots at

different average intensity fluorescence, different average Gaussian area , different

average peak intensity ratio, and different average area ratio

A.IF : Average of fluorescence intensity A.A : Average of Gaussian area

A.P.I.R. : Average of peak intensity ratio A.A.R. : Average of area ratio

Gymnosperms P.I.R.

GF/PSII

A.R

GF/PSII

P.I.R.

GF/Chl.F

A.R.

GF/Chl.F

P.I.R.

GF/PSI

A.R.

GF/PSI

Cycasrevileota

Thujaorientalis

0.115

0.059

0.669

0.374

1.900

1.450

4.692

4.187

1.992

1.362

3.377

2.573

Gymnosperms P.I.R.

PSII/Chl.F

A.R.

PSII/Chl.F

P.I.R.

PSII/PSI

A.R.

PSII/PSI

P.I.R.

Chl.F/PSI

A.R.

Chl.F/PSI

Cycasrevileota

Thujaorientalis

16.523

24.557

7.014

11.181

17.322

23.073

5.048

6.871

1.048

0.940

0.720

0.615

Algae P.I.R. GF/PSII A.R. GF/PSII

Spirogyra 0.070 0.733

Plant

types

A.IF (a.u.)

GF

A.A(cm2)

GF

A.IF (a.u.)

PSII

A.A(cm2)

PSII

A.IF (a.u.)

Chl.F

A.A(cm2)

Chl.F

Monocots

Dicots

181.168

70.508

20438.32

7331.136

3038.142

3165.418

44901.382

46720.872

42.218

48.07

762.992

794.182

Plant

types

A.P.I.R.

GF/PSII

A.A.R.

GF/PSII

A.P.I.R.

GF/Chl.F

A.A.R.

GF/Chl.F

A.P.I.R.

PSII/Chl.F

A.A.R.

PSII/Chl.F

Monocots

Dicots

0.060

0.022

0.455

0.157

4.291

1.467

26.787

9.231

71.963

65.850

58.849

58.829

19


From the results obtained from table1.a to1.e for the different

members of the hardwood dicot group, it could be observed that each

member has special characteristic properties, or special fingerprint.

This is the same for the other results that obtained from table2.a to

table6. These results belong to the different members of herbaceous

dicot, hardwood monocot, herbaceous monocot, gymnosperm, and

algae groups respectively.

The absolute emission signal of leaves can vary from leaf to leaf of

the same sample due to small differences such as the excitation and

sensing angles of the fluorescence, and the roughness and scattering

properties of the leaf surface (Ndao et al 2005). The intensity of the

emitted fluorescence declined with the distance from the irradiated

surface (Vogelmann and Evans 2002). Thus, the absolute fluorescence

usually varies to a large degree than the fluorescence ratio. The

fluorescence ratio, in turn exhibit much lower variation from leaf to

leaf, and represents reliable means for studying the changes in the

fluorescence characteristics of a certain samples. The peak intensity

ratio (P.I.R.), and the area ratio (A.R.) for all the different peaks

maxima of the different spectra, calculated from the Gaussian fitting

curves parameters, are seen in table7 to table12. The (P.I.R.) and the

(A.R.) of the different spectra, give information about the chlorophyll

pigment, thickness of sample, light scattering properties, geometrical

and other factors are related to the wavelength of the exciting light

(Agati 1998). The (P.I.R.) and the (A.R) changes or differences in the

values between the different members and groups are due to plant

genetic variability, age, geographical location, and environmental

20


factors such as climatic conditions, soil fertility and composition,

moisture content and light conditions (Buschmann 2007).

In table13, the average of the fluorescence intensity (A.IF.), average

of the Gaussian area (A.A.), average of the peak intensity ratio

(A.P.I.R.), and average of the area ratio (A.A.R.), for the different

peaks maxima of all the different spectra of the herbaceous dicots and

the herbaceous monocots were calculated. They help in the

discrimination between the herbaceous dicots and the herbaceous

monocots. As was observed the value of the (A.IF.), and the value of

the (A.A.), at the Soret band (GF) of the herbaceous monocots are

considerably greater than the values at the (GF) of the herbaceous

dicots. Also the (A.P.I.R.) between the Soret band and photosystem II

(GF/PSII) and between the Soret band and the chlorophyll

fluorescence at 715 nm (GF/Chl.F), in the case of the herbaceous

monocots are approximately more than two and half times than that of

the herbaceous dicots. The same with the (A.A.R.) between (GF/PSII)

and (GF/Chl.F), the values of the herbaceous monocots are nearly

three times that of the herbaceous dicots. See Fig.14.

Comparisons between the herbaceous monocots and the

herbaceous dicots according to their (A.P.I.R.) and (A.A.R.), between

the Soret band (GF) and photosystem II (GF/PSII) also between the

Soret band and chlorophyll fluorescence at 715 nm (GF/Chl.F)

respectively, were seen in Fig.14.

21


(a) (b)

Fig.14 (a) The A.P.I.R. of GF/PSII and GF/Chl.F for the herbaceous

monocots and the herbaceous dicots, (b) the A.A.R. of GF/PSII and

GF/Chl.F for the herbaceous monocots and the herbaceous dicots.

Conclusions

In this study, six plant groups were identified according to their

different fluorescence spectral shapes. These groups were genetically

different, that means their constituent pigments were also different so

their fluorescence spectra were expected to be different. Each plant of

these different groups had a special characteristic properties or a

special fingerprint, which appeared from the P.I.R. and the A.R.

calculations, after the Gaussian fitting of the fluorescence spectra.

Although these observations were obtained from a limited number of

plant samples, the LIF techniques using a pulsed nitrogen source

provide considerable acceptable results which could be used for plant

type identification.

GF/PSII GF/Chl. F0.00

0.45

0.90

1.35

1.80

2.25

2.70

3.15

3.60

4.05

4.50

21

2

1

A.P.

I.R.

Herbaceous Varieties

1 Dicots 2 Monocots

GF/PSII GF/Chl. F0.00

2.74

5.48

8.22

10.96

13.70

16.44

19.18

21.92

24.66

27.40

21

2

1

A.A.

R.

Herbaceous Varieties

1 Dicots 2 Monocots

22


Acknowledgments

The authors extend their gratitude to the Ministry of Higher Education

and Research of Sudan for financial support of this work.

23


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