Acta Botanica Hungarica 50(1–2), pp. 81–91, 2008DOI: 10.1556/ABot.50.2008.1–2.5

ESTIMATION OF LEAF AREAIN SELECTED HYPERICUM SPECIES

C. ÇÝRAK1, M. S. ODABAŞ1, A. K. AYAN2, B. SAÐLAM1 and K. KEVSEROÐLU1

1Department of Agronomy, Faculty of Agriculture, University of Ondokuz Mayýs55139 Kurupelit, Samsun, Turkey; E-mail: cuneytc@omu.edu.tr; msodabas@omu.edu.tr;

birsensaglam@mynet.com; kudretk@omu.edu.tr2The High School of Profession of Bafra, University of Ondokuz Mayýs, Samsun, Turkey

E-mail: akayan@omu.edu.tr

(Received 2 June, 2006; Accepted 30 October, 2006)

The genus Hypericum has attracted scientific interest for recent years, since it is a source of avariety of compounds including hypericin. The present study was conducted out to developleaf area prediction models for some Hypericum species containing hypericin, namely H.pruinatum, H. perfoliatum, H. aviculariifolium subsp. depilatum var. depilatum (endemic), H. mon-tanum, H. montbretii, H. linarioides, H. triquetrifolium, H. bithynicum and H. perforatum growingwild in Northern Turkey. Lamina width, length and leaf area were measured without de-stroying to develop the models. The actual leaf areas of the plants were measured by Placomdigital planimeter, and multiple regression analysis with Excel 7.0 computer package pro-gram was performed for the plants separately. The produced leaf area prediction models inthe present study were formulised as LA = (a) + (b1 × L) + [b2 × (L × W)] + (b3 × L2) + (b4 × W2)+ [b5 × (L × W2)] + [b6 × (L2 × W)] + [b7 × (L2 + W2)] where LA is leaf area, Wis leaf width, L isleaf length and a, b1, b2, b3, b4, b5, b6, and b7 are coefficients. R2 values varied with species from0.80 in H. aviculariifolium subsp. depilatum var. depilatum to 0.97 in H. pruinatum. All R2 valuesand standard errors were found to be significant at the p < 0.001 level.

Key words: dark glands, hypericin, Hypericum species, leaf area, modelling

INTRODUCTION

Hypericum species are of great interest for mankind throughout manycenturies and have been used as traditional medicinal plants due to theirwound-healing (Yazaki and Okuda 1990), bactericide (Ishiguro et al. 1998),anti-inflammatory (Dias et al. 1998), diuretic and sedative properties (Holz andOstrowiski 1987) for last two hundred years. The Hypericum genus of Gutti-

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ferae is represented in Turkey by 89 species of which 43 are endemic. This ge-nus is widespread in Turkey. The most abundant and well-known species is H.perforatum L. (Davis 1988). Use of the crude extract of this plant as especially anantidepressant is very popular today (Biffignandi and Bilia 2000).

The biological activities of the genus Hypericum derived from mainly theirhypericin contents (Sakar and Tamer 1990, Diwu 1995, Kubin et al. 1996, Gree-son et al. 2001, Agostinis et al. 2002) and total hypericin content of products hasbeen used for standardisation purposes within the botanical industry (Nahr-stedt and Butterweck 1997). Hypericin is found in dark glands of leaves themost intensively (Ciccarelli et al. 2001). That is why; leaves are the most impor-tant organs of these plants. Of all Hypericum, only a few have been reported tocontain hypericin to date (Gerassim and Kitanov 2001, Ferraz et al. 2002). AllHypericum species evaluated in the present study namely, H. pruinatum Boiss.et Bal., H. perfoliatum L., H. aviculariifolium Jaup. et Spach subsp. depilatum (Freynet Bornm.) Robson var. depilatum (endemic), H. montanum L., H. montbretiiSpach, H. linarioides Bosse, H. triquetrifolium Turra, H. bithynicum Boiss. and H.perforatum L. contain hypericin in various levels (Kitanov 2001, Ayan et al.2004).

Leaf area has been measured in experiments concerning some physiolog-ical phenomenon such as light, photosynthesis, respiration, plant water con-sumption and transpiration (Rieger and Duemmel 1992, Horsley and Gott-schalk 1993, Gottschalk 1994, Kersteins and Hawes 1994, Picchioni and Wein-baum 1995, Uzun 1996, Centritto et al. 2000). In addition, leaf number and areaof a plant have an important role in some cultural practices such as training,pruning, irrigation, fertilisation, etc. The leaf area estimation models aiming topredict leaf area non-destructively can provide researches with many advan-tages in agricultural experiments. Moreover, these kinds of models enable re-searchers to carry out leaf area measurements on the same plants over thecourse of a study, resulting in reduced experimental variability (Gamiely et al.1991, NeSmith 1991, 1992). Leaf area can be measured by using expensive in-struments and/or predictive models. Recently, new instruments, tools andmachines such as hand scanners and laser optical apparatuses have been de-veloped for leaf and fruit measurements. These are very expensive and com-plex devices for both basic and simple studies. Furthermore, non-destructiveestimation of leaf area saves time as compared with geometrical measure-ments (Robbins and Pharr 1987). For this reason, several leaf area predictionmodels were produced for some plant species in previous studies. But, to theauthors’ knowledge, there are no published reports concerning leaf area pre-diction model for Hypericum plants. Due to the lack of such information, in thepresent study, we aimed to develop reliable equations that allow for the non-

84 ÇÝRAK, C., ODABAÞ, M. S., AYAN, A. K., SAÐLAM, B. AND KEVSEROÐLU, K.

Acta Bot. Hung. 50, 2008

destructive estimation of leaf area through linear measurements of the afore-said species of Hypericum.

MATERIALS AND METHODS

Brief description of plant materials

H. pruinatum: Stems 15–35 cm, erect or ascending from a rooting and branch-ing base, pruinose. Leaves on main stems 10–35 mm, oblong to elliptic,pruinose. H. perfoliatum: Stems 15–80 cm, erect or decumbent. Leaves 13–60mm, ovate to triangular-lanceolate or rarely linear, the upper most sometimesblack-glandular-ciliate, usually densely pellucid-dotted, with obscure reticu-late venation. H. aviculariifolium subsp. depilatum var. depilatum: Stems 5–60cm, erect or prostrate, branching and sometimes rooting at the base. Leaves5–35 mm, oblong or linear to elliptic or obovate, with intramarginal blackglands, glabrous or shortly pubescent. H. montanum: Stems 20–80 cm, erect,glabrous. Leaves 20–70 mm, ovate to lanceolate or oblong-elliptic, scabrid be-neath or rarely wholly glabrous. H. montbretii: Stems 15–60 cm, erect or decum-bent, rarely rooting. Leaves 15–55 mm, ovate to oblong or triangular-lanceo-late, the upper most sometimes black-glandular-denticulate. H. linarioides:Stems 5–33 cm, erect or ascending from a rooting and branching base, gla-brous. Leaves 5–30 mm, oblong to elliptic to linear, often revolute and gla-brous. H. triquetrifolium: Stems 15–55 cm, erect or decumbent, with branchesspreading usually forming a pyramid. Leaves 3–20 mm, triangular-lanceolateor rarely narrowly ovate to linear-oblong, amplexicaul, undulate, sometimeswith medium to small pellucid dots. H. bithynicum: Stems 10–60 cm, erect orascending, rooting. Leaves 10–55 mm, ovate or ovate-oblong to suborbicular,entire without pellucid dots or with a few in the upper most pairs, sometimeswith superficial black dots. H. perforatum: Stems 10–110 cm, erect, sometimesrooting. Leaves 5–30 mm, narrowly ovate or lanceolate to elliptic oblong, al-ways with large pellucid dots (Davis 1988).

All plants described above grow wild in Black Sea region of Turkey andthey were identified by Dr Hasan Korkmaz, Department of Biology, Univer-sity of 19 Mayis, Samsun, Turkey.

Experimental procedures

Leaf samples were randomly taken from wild populations of the Hyperi-cum plants tested between March and August 2004 at a five time intervals. Inthis period, 60 leaves were collected for each plant within the first three day of

ESTIMATION OF LEAF AREA IN SELECTED HYPERICUM SPECIES 85

Acta Bot. Hung. 50, 2008

April, May, June, July and August to catch the different phases of leaf morpho-genesis. Thus, 300 leaf samples for each species and a total of 2,700 leaves foreight species were processed at the same day as they were collected in the fol-lowing manner. First, they were placed on the photocopier desktop by holdingflat and secure and copied on A3 sheet (at 1:1 ratio). Second, a Placom digitalplanimeter (Sokkisha Planimeter Inc., Model KP-90) was used to measure ac-tual leaf area of the copy. Selection of leaf dimensions for measurement wasgoverned by variation in leaf characteristics (e.g. size, shape, and symmetry)and practical constraints (e.g. ease and accuracy of measurements under fieldconditions). Given these concerns, maximum leaf width (W) and length (L)were chosen to correlate with leaf area. Leaf width (cm) was measured fromtip to tip at the widest part of the lamina and leaf length (cm) was measuredfrom lamina tip to the point of petiole intersection along the midrib (Fig. 1).The leaf positions were selected with regard to points that could be easily iden-tified and used to facilitate the measurement of leaf length and width.

86 ÇÝRAK, C., ODABAÞ, M. S., AYAN, A. K., SAÐLAM, B. AND KEVSEROÐLU, K.

Acta Bot. Hung. 50, 2008

Fig. 1. Leaf diagram of H. pruinatum (1), H. perfoliatum (2), H. aviculariifolium subsp. depilatumvar. depilatum (3), H. montanum (4), H. montbretii (5), H. linarioides (6), H. triquetrifolium (7), H.bithynicum (8) and H. perforatum (9) showing the position of leaf length (L) and leaf width

(W). Bars seen at the lower right corner of each figure represent 1 cm

ESTIMATION OF LEAF AREA IN SELECTED HYPERICUM SPECIES 87

Acta Bot. Hung. 50, 2008

Tabl

e1

The

equa

tion

ofle

afar

eaLA

=(a

)+(b

1×

L)+

[b2

×(L

×W

)]+

(b3

×L2 )+

(b4

×W

2 )+[b

5×

(L×

W2 )]

+[b

6×

(L2

×W

)]+

[b7

×(L

2+

W2 )]

for

the

Hyp

eric

umsp

ecie

ste

sted

Spec

ies

Ab 1

b 2b 3

b 4b 5

b 6b 7

b 8R

2

Hyp

eric

umpr

uina

tum

LA0.

1092

68–0

.013

060.

7549

170.

97SE

0.04

4***

1.8

***

0.01

06**

*H

yper

icum

perf

olia

tum

LA–1

.113

421.

596

–0.3

690.

373

0.32

1–0

.04

0.94

SE0.

161*

**0.

0927

***

0.01

***

0.02

2***

0.00

89**

*0.

002*

**H

yper

icum

avic

ular

iifol

ium

subs

p.de

pila

tum

var.

depi

latu

m

LA0.

229

0.71

6–0

.066

0.80

SE0.

05**

*0.

034*

**0.

035*

**H

yper

icum

mon

tanu

mLA

3.65

–2.2

9–8

222.

326.

93–1

.83

–0.4

90.

84SE

0.58

7***

0.31

6***

1.33

***

0.56

6***

0.82

4***

0.28

5***

0.09

6***

Hyp

eric

umm

ontb

retii

LA1.

70–8

.074

10.9

22.

94–6

.18

0.88

0.87

SE0.

634*

**1.

877*

**1.

545*

**0.

406*

**0.

805*

**0.

121*

**H

yper

icum

linar

ioid

esLA

0.59

2–4

.198

2.86

30.

202

–0.5

080.

88SE

0.06

8***

0.22

4***

0.15

4***

0.02

1***

0.04

8***

Hyp

eric

umtr

ique

trifo

lium

LA0.

0397

0.86

20.

51–0

.639

0.91

SE0.

0235

***

0.10

8***

0.03

18**

*0.

059*

**H

yper

icum

bith

ynic

umLA

0.90

7–1

.743

1.04

790.

80SE

0.05

2***

0.40

4***

0.17

3***

Hyp

eric

umpe

rfor

atum

LA–0

.171

41.

0417

2–0

.192

040.

92SE

0.06

6***

0.02

5***

0.00

6***

LA=

leaf

area

,L=

leaf

leng

th,W

=le

afw

idth

,SE

=st

anda

rder

ror.

***

R2

and

allS

Eva

lues

are

sign

ific

anta

tp<

0.00

1

88 ÇÝRAK, C., ODABAÞ, M. S., AYAN, A. K., SAÐLAM, B. AND KEVSEROÐLU, K.

Acta Bot. Hung. 50, 2008

R2=

0.7

761

0

0.51

1.52

2.5

00.5

11.5

22.5

R2=

0.9

375

012345678

01

23

45

67

8

R2=

0.9

70

1

0123456789

02

46

81

0

R2=

0.8

76

0

0.51

1.52

2.53

3.5

00

.51

1.5

22

.53

3.5

R2=

0.8

40

8

0

0.51

1.52

2.53

3.5

00

.51

1.5

22

.5

R2=

0.8

748

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

01

23

45

R2

=0

.92

08

00

.511

.52

2.53

3.54

4.55

12

34

5

R2=

0.8

0

0.51

1.52

2.5

00.5

11.5

22.5

R2

=0

.91

29

0

0.2

0.4

0.6

0.81

1.2

1.4

1.6

00.2

0.4

0.6

0.8

11.2

1.4

1.6

Actu

alle

af

are

a(c

m)

2

Predictedleafarea(cm)2

Predictedleafarea(cm)2

Predictedleafarea(cm)2

Actu

alle

af

are

a(c

m)

2A

ctu

alle

af

are

a(c

m)

2

12

3

45

6 98

7

Fig.

2.R

elat

ions

hip

betw

een

actu

alle

afar

ea(c

m2 )a

ndpr

edic

ted

leaf

area

(cm

2 )for

H.p

ruin

atum

(1),

H.p

erfo

liatu

m(2

),H

.avi

cula

riifo

lium

(3),

H.m

onta

num

(4),

H.m

ontb

retii

(5),

H.l

inar

ioid

es(6

),H

.tri

quet

rifo

lium

(7),

H.b

ithyn

icum

(8)a

ndH

.per

fora

tum

(9)

Model constructions

Multiple regression analysis of the data was performed for each plantseparately. A search for the best model for predicting leaf area was conductedwith various subsets of the independent variables, namely, length (L), (L × W),width (W), (L2), (W2), (L × W2) ( L2 × W) and (L2 × W2). The best estimatingequations for the leaf area (LA) of the plants tested were determined with theExcel 7.0 and formulised as LA = (a) + (b1 × L) + [b2 × (L × W)] + (b3 × L2) + (b4 ×W2) + [b5 × (L × W2)] + [b6 × (L2 × W)] + [b7 × (L2 + W2)] where LAis leaf area, Wisleaf width, L is leaf length and a, b1, b2, b3, b4, b5, b6 and b7 are coefficients of theproduced equation. Multiple regression analysis was carried out until the leastsum of square was obtained.

RESULTS

Multiple regression analysis used for determination of the best fittingequation for estimation of leaf area in the medicinal plants evaluated hereshowed that most of the variation in leaf area values was explained by the se-lected parameters (length and width). The variation explained by the parame-ters was 97% for H. pruinatum, 94% for H. perfoliatum, 80% for H. aviculariifo-lium subsp. depilatum var. depilatum, 84% for H. montanum, 87% for H. mont-bretii, 88% for H. linarioides, 91% for H. triquetrifolium, 80% for H. bithynicumand 92% for H. perforatum. The produced leaf area prediction models in thepresent study were LA = 0.109268 + (–0.01306 × W2) + [0.754917 × (L × W)] forH. pruinatum, LA = –1.11342 + (1.596 × L) + (–0.369 × L2) + (0.373 × W2) + [0.321× (L2 × W)] + [–0.04 × (L2 × W2)] for H. perfoliatum, LA = 0.229 + [0.716 × (L × W)]+ [–0.066 × (L × W2)] for H. aviculariifolium, LA = 3.65 + (–2.29 × L) + (–8.22 × W)+ (2.32 × W2) + [6.93 × (L × W)] + [–0.49 × (L2 × W)] + [–1.83 × (L × W2)] for H.montanum, LA = 1.70 + (–8.074 × W) + (10.92 × W2) + [2.94 × (L × W)] + [–6.18 ×(L × W2)] + [0.88 × (L2 × w2)] for H. montbretii, LA = (0.592) + (–4.198 × W2) +[2.863 × (L × W2)] + [–0.508 × (L2 × W2)] + [0.202 × (L2 × W)] for H. linarioides, LA= 0.0397 + [0.51 × (L2 ×] + [0.862 × (L × W2)] + [–0.639 × (L2 × W2)] for H.triquetrifolium, LA = 0.907 + (–1.743 × W2) + [1.0479 × (L × W2)] for H.bithynicum and LA = –0.1714 + [1.04172 × (L × W)] + [–0.19204 × (W2 × L)] for H.perforatum (Table 1).

ESTIMATION OF LEAF AREA IN SELECTED HYPERICUM SPECIES 89

Acta Bot. Hung. 50, 2008

DISCUSSION

Hypericum plants are characterised mainly by the presence of three typesof secretory structures, light glands, dark glands and secretory canals (Cicca-relli et al. 2001). The dark glands on the vegetative organs of many species ofHypericum have been used by botanists as an important distinguishing markfor the classification of this genus (Kitanov 2001). In addition, there is a markedevidence and general agreement about localisation of hypericins in the darkglands (Lu et al. 2001). The presence of dark glands in Hypericum species is ex-pressed as its density in leaves for which it is necessary to calculate the leafarea. Therefore, leaf area has been routinely measured in morphological andtaxonomic studies on Hypericum genus (Walker et al. 2001). For example, in aprevious study we determined a positive and significant relationship betweendark gland density of leaves and both leaf and whole plant contents of hype-ricin in H. perforatum, H. pruinatum and H. aviculariifolium subsp. depilatum var.depilatum. For this purpose, we measured the total area of 50 leaves for eachspecies by using of Placom digital planimeter and it was very time-consumingprocess (Çýrak et al. 2006). Thus, we developed very useful, simple and accu-rate models for estimation leaf area for some hypericin-contained species ofHypericum in the present study.

Similarly, a number of studies have been carried out to estimate leaf areaby linear measurements of leaf width and length in the following plants: soy-bean (Lieth et al. 1986), cucumbers (Robbins and Pharr 1987), orange (Arias etal. 1989, Ramkhelawan and Brathwaite 1992), French bean (Rai et al. 1990), co-conut (Mathes et al. 1990), banana (Potdar and Pawar 1995), grape (Uzun andÇelik 1999), Miscanthus (Vargas et al. 2002), broad bean (Odabaş 2003), cherry(Demirsoy and Demirsoy 2003, Pinto et al. 2004), strawberry (Demirsoy et al.2005), peach (Demirsoy et al. 2004), summer snowflake (Çýrak et al. 2005a), net-tle and lemon balm (Çýrak et al. 2005b). The same authors found that there wereclose relationship between leaf area value, leaf length and leaf width for theseplants (R2 = 0.94 for soybean, R2 = 0.76 to 0.99 for cucumber, R2 = 0.89 to 0.93 fororange, R2 = 0.99 for French bean, R2 = 0.95 to 0.98 for coconut, R2 = 0.96 for ba-nana, R2 = 0.98 for grapes, R2 = 0.91 for Miscanthus, R2 = 0.99 for broad bean,R2 = 0.95 and 0.99 for cherry, R2 = 0.99 for strawberry, R2 = 0.99 for peach, 0.97for summer snowflake, R2 = 0.97 for nettle and R2 = 0.98 for lemon balm). Wefound that there was very close relationship between actual and predicted leafarea for all Hypericum species tested (Fig. 2).

Our results were consistent with those of other studies mentioned abovethat used linear measurements of leaves from different plants for estimatingleaf area. Although coefficients of determination were generally high (R2 >0.85) in both the current and previous studies, it is interesting to note that R2

90 ÇÝRAK, C., ODABAÞ, M. S., AYAN, A. K., SAÐLAM, B. AND KEVSEROÐLU, K.

Acta Bot. Hung. 50, 2008

values for Hypericum species tested varied with species greatly from 0.80 in H.aviculariifolium subsp. depilatum var. depilatum to 0.97 in H. pruinatum. How-ever, the difference is not surprising when the evident difference in size andshape of leaves among the species is taken into consideration (Fig. 1). Apartfrom different species, regression coefficients of leaf area estimation can evenbe different between the cultivars of the same species. For example, using thegrapevine cultivars ‘Niagara’ and ‘DeChaunac’, Williams and Martinson (2003)found that the product of maximum leaf length and width was most highlycorrelated with leaf area, but R2 values of cultivars were different (0.99 for Ni-agara and 0.96 for Dechaunac).

CONCLUSION

Here, we developed leaf area prediction models for nine Hypericum spe-cies, each has potential using fields in medicinal treatments and botanical in-dustry with their well-documented hypericin contents. As the understandingof plant growth and development has been increasing, such mathematicalmodels as this shown in Table 1 will be very useful tools for prediction of leafarea for many plants without using of expensive devices. Also, considering theimportance of expressing the presence of dark glands in vegetative parts ofHypericum plants, prediction of leaf area easily by simple equations instead ofusing expensive and time-consuming devices during the course of experimentis an also important topic for morphological and taxonomic studies on the ge-nus Hypericum. Therefore, the models produced in the present study can beused safely by Hypericum researchers for the species used in this research. Onthe other hand, different models can be developed by researches studying onHypericum species different from those used in the present study.

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