Structural investigations of trichomes and essential oilcomposition of Salvia verticillata

Lana Krstic1 Djordje Malencic2 and Goran Anackov1

1 University of Novi Sad Faculty of Sciences Department of Biology and EcologyTrg D Obradovica 2 Novi Sad Serbia e-mail lanaibnsacyu

2 University of Novi Sad Faculty of Agriculture Novi Sad Serbia

Manuscript accepted 6 September 2006

Abstract

Krstic L Malencic Dj and Anackov G 2006 Structural investigations of trichomesand the essential oil composition of Salvia verticillata BotHelv 116 159 ndash 168

The morphology of glandular and non-glandular trichomes and the essential oilcomposition ofLamiaceae play an important role in the ecology of these species as wellas for their industrial use they may also serve as taxonomic criteria We studied thetrichome anatomy and essential oil composition of three wild-growing populations ofSalvia verticillata in Serbia to determine how strongly these traits vary among andwithin Salvia species and to evaluate their potential taxonomic or economicsignificance Microscopic investigations of leaf and calyx indumentum revealed thatS verticillata has the same types of peltate and capitate trichomes as other Salviaspecies In combination however the trichome characteristics of S verticillata differedfrom previously examined Salvia species and might therefore help in speciesidentification The density of glandular trichomes differed among the three populationsand was lowest in a dry steppe habitat but trichome density also varied substantiallyamong individual leaves within each population The essential oil content (determinedgravimetrically in n-hexane) ranged from 040 to 042 of dry mass In total 39different oil components were identified using GC-MS Oil composition variedconsiderably among the three populations Only three of the 39 compounds were wellrepresented (gt01 of oil) in all three populations The main component wasgermacrene D in two populations (480 and 246 of oil respectively) but (E)-caryophyllene (102) in the third Low oil content and high variability in oilcomposition may restrict the industrial use of wild-growing S verticillata plants

Key words Epidermis anatomy glandular trichomes intraspecific morphologicalvariation non-glandular trichomes secondary compounds secretory glands

BotHelv 116 (2006) 159 ndash 1680253-145306020159-10DOI 101007s00035-006-0767-6BirkhAuser Verlag Basel 2006 Botanica Helvetica

Introduction

Glandular trichomes are primary secretory organs of the Lamiaceae (Duke 1994Marin 1996 Gersbach et al 2001) They are distributed on vegetative and reproductiveorgans and produce bioactive lipophilic secondary products (essential oils) whichmayfor example contribute to the plantCs defence against herbivores and pathogens Someof these products are of interest to the pharmaceutical food and pesticide industry Thetype and distribution of glandular trichomes can also be used for the differentiation ofclosely related taxa for example within the genus Salvia (Chakalova et al 1993) orStachys (Demissew and Harley 1992) This is particularly valuable when small plantparts (eg leaves) need to be identified (Chakalova et al 1993) The anatomy ofglandular trichomes and their essential oil composition were also useful in solvingtaxonomic problems within the Satureja complex (Bosabalidis 1990)

Two main types of glandular trichomes are found in the Lamiaceae ndash capitate andpeltate (Metcalfe andChalk 1950Marin 1996) Capitate glandular trichomes consist ofa basal cell of epidermal origin a unicellular or multicellular stalk and a round to pear-shaped uni- or multicellular secretory head Peltate glandular trichomes consist of abasal cell embedded in the epidermis a short stalk cell and awide headmade of severalsecretory cells covered with a common cuticlewhich can be arranged in one or twocircles and with varyingmorphology Capitate hairs are active on younger leaves whilepeltate hairs start with their secretory function once the secretion process of capitatehairs is finished The number of trichomes per unit plant surface varies among andwithin species (Uphof and Hummel 1962 Bosabalidis and Kokkini 1997) Hightrichome density tends to be associated with greater heat tolerance light protectionreduced transpiration and reduced predation by insects and herbivores (Dickison2000)

Essential oils serve as a barrier against various external factors includingherbivores pathogens UV-B radiation extreme temperatures and drought and theymay reduce transpiration or attract pollinators (Uphof and Hummel 1962 Corsi andBottega 1999 Valkama et al 2004) The secretion of essential oils varies among andwithin species and also depends on the density of glandular trichomes (Krstic et al2001) Intraspecific variation can be considerable and may be related to growthconditions or to genetic differentiation For example Krstic et al (2001) found a loweressential oil content in Salvia glutinosa plants from a shaded forest edge (0095) thanin plants cultivated in full sun (0152) even lower contents were reported from SouthItaly (003ndash005) by Senatore and de Fusco (1997) Such variations may be relevantto the commercial value of a species

ALamiaceae genus of particular economic relevance is the genus Salvia In the floraof Serbia 15 species of Salvia have been described some of which are well known fortheir medicinal use and their aromatic and antioxidant properties (Diklic 1974) Salviaspecies used in folkmedicine include S aegyptiaca S aethiopis S divinorum S plebeiaS sclarea and S verbenaca but the most important ones are S officinalis S triloba andS miltiorrhizaAccording to the German Pharmacopeia (Deutsches Arzneibuch DAB10) only S officinalis and S triloba are widely used inEurope (Liu et al 1995Malencic2001) Accordingly most other wild-growing Salvia species have hardly beeninvestigated phytochemically or pharmacologically One of these poorly investigatedspecies is Salvia verticillata L a close relative of medicinally important Salvia speciesand therefore a species of potential pharmacological and commercial value

160 Lana Krstic Djordje Malencic and Goran Anackov

S verticillata (lilac whorled sage) is a herbaceous perennial native to central andeastern Europe and western Asia where it grows under semi-arid continental climaticconditions Stems are up to 80 cm heigh erect and eglandular-hairy (Hedge 1972)Leaves are mostly simple ovate-triangular with 1ndash2 pairs of small lateral segmentscordate to truncate at the base with an acute tip Inflorescences consist of (8ndash)15ndash30lilac-blue flowers Sefidkon and Khajavi (1999) analysed the chemical composition ofthe essential oil of this species in Iran but no chemotaxonomic data are available fromEurope

We investigated the glandular and non-glandular trichomes of S verticillata leavesand calyces to determine their anatomical structure and the chemical composition oftheir products Our aims were to determine (1) whether trichome characteristics of Sverticillata differ from those of other Salvia species and (2) how strongly trichomedensity essential oil content and essential oil composition vary among three Sverticillata populations from contrasting habitats

Materials and Methods

Plant material was collected during flowering from three wild-growing populationsrepresenting different types of habitats where S verticillata occurs Population 1 wascollected from Vrdnik (North Serbia 400 m altitude) in a mesic meadow allianceArrhenatherion elatioris Population 2 was collected from Rimski Sanac (Pannonianpart of North Serbia 80 m above sea level) in a xeric steppe habitat alliance Festucionrupicolae and population 3 fromTaramountain (Southwest Serbia altitude 1000m) ina mesic meadow alliance Arrhenatherion elatioris Voucher specimens were depositedin the Herbarium of the Department of Biology and Ecology University of Novi Sad(BUNS)

Anatomical and morphological investigations were carried out on ten plants perpopulation using leaves from the middle part of the plants For light microscopy crosssections of fresh leaves weremade at the region of themain vein and at 14 of leaf widthusing Leica CM 1850 cryostat at temperature 18 to 20 8C with cutting intervals of25 mm Sections were investigated using the Image Analyzing System Motic 2000 Thenumber of glandular trichomes was counted and the diameter of peltate trichomesmeasured on fifty leaves per population The significance of differences betweenpopulations was determined with DuncanGs test using STATISTICA version 70 Forscanning electron microscopy (SEM) small pieces of dry leaves and calyces of tenplants were sputter coated with gold for 180 seconds 30mA (BAL-TEC SCD 005) andviewed with a JEOL JSM-6460LVelectron microscope at an acceleration voltage of 20kV

Essential oils were isolated from dry leaves and flowering tops in full blossom of 50plants per population by the Europaean Pharmacopeia hydrodestillation methodsusing n-hexane as collecting solvent The extracts were dried over anhydrous sodiumsulphate and decanted Hexane was evaporated under reduced pressure to measure oilyields A suitable dilution of each oil in hexane (10 mgml) was then analyzed by GC-FID and GC-MS The GC conditions used were column HP-5 fused silica capillarycolumn 30 mI025 mm film thickness 025 mm column temperature 50 8C for 5 minthen heated to 250 8C at a rate of 3 8Cmin on a Perkin-Elmer 8500 gas chromatographcarrier gas helium constant flow (1mlmin) injector 280 8C FIDdetector 280 8CMassspectra were obtained from aHewlett-Packard 5973-6890GC-MS system operating on

Botanica Helvetica 116 2006 161

EI mode at 70 eV equipped with HP-5 MS capillary column (30 mI025 mm filmthickness 025 mm) The initial temperature of the column was 60 8C and then it washeated to 280 8C at a rate of 3 8Cmin The identification of individual compounds wasmade by comparison of their retention times andmass spectra with those obtained fromauthentic samples andor the NISTNBS Wiley libraries spectra as well as withliterature data

Results

The indumentum of S verticillata leaves was made of non-glandular and glandulartrichomes which were present on both lamina surfaces Non-glandular trichomes werelong uniseriate multicellular (2ndash6 cells) with acute apical cells On their surface smallpapillae were noticed A ring of raised epidermal cells surrounded larger trichomesNon-glandular trichomes densely covered the whole leaf surface but were moreabundant on the abaxial epidermis and along the veins

Both peltate and capitate glandular trichomes were observed Peltate trichomeswere present on the abaxial epidermis only in epidermal depressions (Fig 1ab 2a)They consisted of a basal cell a short stalk cell and a broad round head composed offour secretory cells Above the secretory cells the thick cuticle elevated to form a largesubcuticular space for an accumulation of secreted material Pores or cracks were notrecorded on cuticle using SEM so it could be supposed that secreted material wasreleased after the cuticle had been broken The number of peltate hairs per mm2 of leafsurface and their diameter were lower on the leaves of population 2 than on those ofpopulations 1 and 3 (Tab 1) The number of peltate trichomes also varied stronglywithin populations whereas their diameter showed low variability (cf variationcoefficients in Tab 1)

Capitate glandular trichomeswere present onboth leaf surfaces butmore numerouson the adaxial epidermis and along the veins (Fig 1cd 2bcd) Three types could bedistinguished The hairs of type I had a short stalk cell and a bicellular head Type II hada short unicellular stalk and a unicellular secretory head The hairs of type III were notvery common and their density was always low They were larger with a long stalkcomposed of one elongated stalk cell and one short narrower neck cell and had a pear-

Tab 1 Number of glandular trichomes per mm2 of leaf surface area and diameter of peltatetrichomes Data are means standard errors and coefficients of variation () of 50 leaves perpopulation Letters in superscript point to significance of differences between the populations(Duncan test) means without common letter differ significantly (plt^005)

Population 1 Population 2 Population 3

Capitate trichomes Nr on adaxial epidermis 6102a(248)

4302b(280)

5903a(237)

Nr on abaxial epidermis 3902a(285)

3703a(410)

3503a(426)

Peltate trichomes Nr on abaxial epidermis 2102a(810)

0901b(698)

1702a(643)

Diameter (mm) 48707ab(90)

46807b(75)

50709a(93)

162 Lana Krstic Djordje Malencic and Goran Anackov

shaped unicellular secretory head The total number of capitate hairs did not differamong the examined populations but their number on the adaxial epidermis was sig-nificantly lower in population 2 than in the two other populations The number ofcapitate trichomes varied strongly within populations (Tab 1) though less than thenumber of peltate trichomes

The calyx indumentum was composed of the same types of glandular and non-glandular trichomes but compared to leaves they were more abundant (Fig 3) Thenon-glandular trichomes had larger papillae on their surfaces andweremore numerousbetween the calyx ribs

The essential oil content in dry herb (leaves and flowers) of S verticillata rangedfrom 040 to 042 In total 39 different oil components were identified but only 23to 27 components in individual populations (Tab 2) Oil composition varied conside-rably among the three populations only three of the 39 compounds were well re-presented (gt01of oil) in all three populations (Tab 2) For populations 1 and 2 onlyeight compounds were identified in an amount higher than 01 compared to 27compounds in population 3 The dominant components in populations 1 and 2 weregermacrene D (480 and 246 respectively) and (E)-caryophyllene (134 and190 respectively) whereas the dominant components in population 3 were (E)-caryophyllene (102) b-cubebene (86) and eicosane (85) Using GC-MC only

Fig 1 Salvia verticillata scanning electron micrographs A peltate hair four secretory cellsvisible B peltate hair C capitate hair type II D capitate hair type III Magnification scalebar AndashC 10 mm D 50 mm

Botanica Helvetica 116 2006 163

75 of volatile compounds could be identified in population 3 suggesting that theseplants were also rich in other non-identified volatile carbohydrates besides hy-drocarbons and sesquiterpenes

Fig 2 Salvia verticillata light micrographs population 1 A peltate hair B capitate hair typeI C capitate hair type II D capitate hair type III Magnification scale bar A D 20 mmB C10 mm

Fig 3 Salvia verticillata scanning electron micrographs population 1 A calyx B calyx non-glandular hairs-detail Magnification scale bar A 500 mm B 10 mm

164 Lana Krstic Djordje Malencic and Goran Anackov

Tab 2 Composition of the essential oil of three populations of Salvia verticillata expressed asrelative percentage of each compound as obtained from peak areas (t = trace lt 01) Con-stituents are sorted according to retention time in GC

Constituents Pop 1 Pop 2 Pop 3

a-pinene t t 05sabinene t t ndashb-pinene t t ndashmyrcene t t ndashb-phellandrene t 86 17camphene ndash ndash 06(Z)- b-ocimene ndash 6 ndash(E)- b-ocimene t 75 ndashlinalool t t ndashterpinen-4-ol t t ndasha-terpineol t t ndasha-cubebene t t 05a-copaene t t ndashb-bourbonene t t 04b-elemene t t 11(E)-caryophyllene 134 19 102b-cubebene ndash ndash 86a-elemene ndash ndash 11a-humulene 72 102 48a-muurolene ndash ndash 10germacrene D 48 246 ndashbicyclogermacrene 53 167 ndashnaphthalene 1 2 3 4 4a 5 6 8a-octahydroxyl ndash ndash 40g-cadinene t t 13d-cadinene 60 t 37naphthalene 1 2 3 4 4a 7-hexahydroxyl ndash ndash 13spathulenol 35 72 65caryophyllene oxide t t 29g-gurjunene ndash ndash 09a-cadinol 104 t 37eudesma-4(15)7-dien-1-beta-ol 60 t ndashcaryophyllenol-11 ndash ndash 06aromadendrenepoxide ndash ndash 21octacosane ndash ndash 05nonahexacontanoic acid ndash ndash 05cyclopentane ndash ndash 45eicosane ndash ndash 852-pentadecanone 6 10 14-trimethyl ndash ndash 2512-benzenedicarboxylic acid ndash ndash 25

Total of identified compounds 998 998 754

Botanica Helvetica 116 2006 165

Discussion

Non-glandular and glandular trichomes of S verticillata leaves were similar to thetrichome types described previously for Lamiaceae (Tab 3) The number of head cellsin peltate trichomes varied from four to 16 in different Salvia species (Tab 3) Accor-ding to our findings peltate trichomes of S verticillata leaves had a four-celled secretoryhead Although the structure of trichomes should be of taxonomic value and constantfor the species (Uphof and Hummel 1962) this disagreed with the findings of Remenyi(1997a) who reported 6- 8- and 12-celled heads of sessile glandular trichomes Four-celled head had also been recorded for S blepharophylla and S glutinosa (Tab 3)Capitate glandular trichomes of three typeswere recorded for S verticillataTypes I andII with a unicellular stalk and a bi- and unicellular secretory head respectively cor-responded to trichomes described for S blepharophylla S tomentosa and S officinalis(Tab 3)Glandular trichomes of type III of S verticillatawere similar to those describedby Corsi and Bottega (1999) for S officinalis type III and IV Despite the overallsimilarity of trichome structures the combination of a four-celled head on peltatetrichomes together with the occurrence of types I II and III for capitate trichomes hasnot yet been described for another Salvia species suggesting that it might be a criterionfor the identification of S verticillata However information about trichomes is stillmissing for the majority of the wild-growing Serbian Salvia species so that we cannotexclude that other species have the same trichome characteristics as S verticillataAnother taxonomically important finding is that trichome morphology did not differbetween leaves and calyces

Capitate glandular trichomes were more numerous than peltate trichomes Com-pared with S glutinosa S tomentosa and S officinalis (Tab 3) S verticillata seems tohave a lower total number of glandular trichomes on the abaxial epidermis and a highernumber on the adaxial epidermis of leaves However the number of glandular tri-chomes is generally a very variable character which is strongly influenced by externalfactors such as temperature and light intensity (Uphof and Hummel 1962) so thatinterspecific comparisons based on a small number of populations must be consideredwith caution

The yield of the essential oil (040ndash042) permits the assignment of this species tooil-poor representatives of Lamiaceae as the widely accepted borderline between oil-poor and oil-rich representatives of Salvia is 1 (Mathe et al 1992 Senatore and deFusco 1997 Malencic et al 2004) Sesquiterpenes were present in all populationsconsistent with previous reports that oil-poor species of Lamiaceae possess oil rich insesquiterpene compounds (Malencic et al 2004) There were striking differences in theessential oil composition of the three populations Monoterpenes were well reACHTUNGTRENNUNGpresent-ACHTUNGTRENNUNGed in populations 2 and 3 but only present in traces in population 1Hydrocarbonsweremissing in populations 1 and 2 from the North of Serbia while population 3 fromsouthwest mountainous regions was rich in them The number of compounds found inindividual populations (23ndash27) was similar to the number of compounds found in awild-growing population in Iran (Sefidkon and Khajavi 1999) Two dominant compo-nents in Iran (E)-caryophyllene (247) and a-humulene (78) were also dominantcomponents in all three Serbian populations suggesting that these are generally presentin S verticillata Of the two other components that were present in significant amountsin the Iranian population a-muurolene (228) and limonene (89) only a-muu-rolene was recorded in Serbia and only in population 3

166 Lana Krstic Djordje Malencic and Goran Anackov

Both environmental factors (Mathe et al 1992) and genetic differences (Werker etal 1985) have been found to cause variation in oil composition in other species ofLamiaceae The number of populations investigated in this study does not yet allowconclusions about factors determining the essential oil composition of S verticillataHowever our results suggest that low oil content and high intraspecific variability in oilcomposition may restrict the commercial use of wild-growing S verticillata plants

This work was financially supported by theMinistry of Science andEnvironmental Protection of Serbia andMontenegro Grant No 143037 We would like to thank Mr Milos Bokorov University Center for ElectronMicroscopy Novi Sad for his technical assistance and SEM micrographs

References

Bisio A Corallo A Gastaldo P Romussi G Ciarallo G Fontana N de Tommasi N andProfumoP 1999Glandular hairs and secretedmaterial in Salvia blepharophyllaBrandegeeex Epling grown in Italy Ann Bot 83 441 ndash 452

Bosabalidis AM and Kokkini S 1997 Infraspecific variation of leaf anatomy in Origanumvulgare grown wild in Greece Bot J Linn Soc 123 353 ndash 362

Bosabalidis AM 1990 Glandular trichomes in Satureja thymbra leaves Ann Bot 65 71 ndash 78

Tab 3 Non-glandular and glandular trichome characteristics of various Salvia species (adeadaxial epidermis abe abaxial epidermis ndash not determined)

Species Non-glandulartrichomesnr of cells

Peltatetrichomesnr of headcells

Capitatetrichome type

Meannumber ofglandulartrichomesmm2

References

ade abe

S verticillata 3ndash6 4 I II and III 57 53 This studyS verticillata ndash 6 8 and 12 ndash ndash ndash Remenyi (1997a)S aethiops 3 or more 16 ndash ndash ndash Remenyi (1997b)S aurea ndash 6ndash8 I ndash ndash Serrato-Valenti et

al (1997)S blepharo-

phylla1 4ndash5 or more 4 I and II ndash ndash Bisio et al (1999)

S glutinosa 2ndash7 4 I 80 245 Krstic et al (2001)S officinalis 3ndash4 12 I and II 30 63 Venkatachalam et

al (1984)Corsi and Bottega(1999)

2ndash4 III and IV Chakalova et al(1993)

S officinalis faurea

2ndash4 12 48 87 Chakalova et al(1993)

S tomentosa 2ndash4 12 I and II 066 106 Chakalova et al(1993)

plus trichomes with multicellular stalk and unicellular head

Botanica Helvetica 116 2006 167

Chakalova ES Genova EM and Thuy NT 1993 Comparative anatomical investigations onleaves of Salvia officinalis L and S tomentosa Mill Fitologija 46 3 ndash 11

Corsi G and Bottega S 1999 Glandular hairs of Salvia officinalis new data on morphologylocalization and histochemistry in relation to function Ann Bot 84 657 ndash 664

Demissev S and Harley MM 1992 Trichome seed surface and pollen characters in Stachys(Lamioidae Labiatae) in tropical Africa In Harley R M and Reynolds T (eds) Ad-vances in Labiate Science Royal Botanic Gardens Kew 149 ndash 166

Dickison WC 2000 Integrative Plant Anatomy Academic Press San Diego 297 ndash 307Diklic N 1974 Rod Salvia L In Josifovic M (ed) Flora SR Srbije VI SANU Beograd 432 ndash

453Duke SO 1994 Glandular trichomes ndash focal point of chemical and structural interactions Int

J Plant Sci 155 617 ndash 620Gersbach PV Wyllie SG and Sarafis V 2001 A new histochemical method for localization of

the site of monoterpene phenol accumulation in plant secretory structures Ann Bot 88521 ndash 525

Hedge IC 1972 Genus Salvia L In Tutin et al (eds) Flora Europaea Vol 3 CambridgeUniversity Press London p 192

Krstic L Malencic Dj Merkulov Lj Popovic M Adamovic D and Boza P 2001 A con-tribution to themorpho-anatomical study ofwild-growing and cultivated Salvia glutinosaLMed Plant Rep 8 (8) 38 ndash 47 (in Serbian English summary)

Liu J Zapp J and Becker H 1995 Comparative phytochemical investigation of Salvia mil-tiorrhiza and Salvia triloba Planta Med 61 453 ndash 455

Malencic Dj 2001 Biochemical investigations of selected wild-growing Salvia species fromVojvodina province PhD Thesis University of Novi Sad Faculty of Science (In SerbianEnglish summary)

Malencic Dj Couladis M Mimica-Dukic N Popovic M and Boza P 2004 Essential oils ofthree Salvia species from the Pannonian part of Serbia Flavor Fragr J 19 225 ndash 228

Marin P 1996 Orasice i trihome u familiji Lamiaceae Bioloski fakultet BelgradeMathe JrI Olah L Mathe A Miklossy V Bernath J Blunden G Patel A and Mathe I

1992 Changes in the essential oil production of Salvia officinalis under climatic conditionsof the temperate belt Planta Med 58 680 ndash 686

Metcalfe CR and Chalk L 1950 Anatomy of the dicotyledons Vol II Clarendon PressOxford

PharmacopM EuropMenne vol 1 1983 Maisonneuve SA Sainte-RuffineRemMnyi ML 1997a Research on epidermal appendices of Salvia aethiops L Magyar Nccedil-

vMnyanatOmiai SzimpOzium Abstracts Szeged 89RemMnyi ML 1997b Variations in tissue structures on the epidermis of Salvia verticillata L

Magyar NccedilvMnyanatOmiai SzimpOzium Abstracts Szeged 91Sefidkon F and Khajavi MS 1999 Chemical composition of the essential oils of two Salvia

species from Iran Salvia verticillata L and Salvia santolinifolia Boiss Flavor Fragr J 1477 ndash 78

Senatore F and de Fusco R 1997 Essential oils from Salvia spp (Lamiaceae) I Chemicalcomposition of the essential oils from Salvia glutinosa L growing wild in Southern Italy JEssent Oil Res 9 151 ndash 157

Serrato-Valenti G Bisio A Cornara L and Ciarallo G 1997 Structural and histochemicalinvestigation of the glandular trichomes of Salvia aurea L leaves and chemical analysis ofthe essential oil Ann Bot 79 329 ndash 336

Uphof JCT and Hummel K 1962 Plant Hairs Borntraeger BerlinValkama E Salminen J-P Koricheva J and Pihlaja K 2004 Changes in leaf trichomes and

epicuticular flavonoids during leaf development in three birch taxaAnnBot 94 233 ndash 242Venkatachalam KV Kjonaas R and Crotean R 1984 Development and essential oil content

of secretory glands of sage (Salvia officinalis) Plant Physiol 76 148 ndash 150Werker E Putievsky E and Rivid V 1985 The essential oils and glandular hairs in different

chemotypes of Origanum vulgare L Ann Bot 55 793 ndash 801

168 Lana Krstic Djordje Malencic and Goran Anackov

S verticillata (lilac whorled sage) is a herbaceous perennial native to central andeastern Europe and western Asia where it grows under semi-arid continental climaticconditions Stems are up to 80 cm heigh erect and eglandular-hairy (Hedge 1972)Leaves are mostly simple ovate-triangular with 1ndash2 pairs of small lateral segmentscordate to truncate at the base with an acute tip Inflorescences consist of (8ndash)15ndash30lilac-blue flowers Sefidkon and Khajavi (1999) analysed the chemical composition ofthe essential oil of this species in Iran but no chemotaxonomic data are available fromEurope

We investigated the glandular and non-glandular trichomes of S verticillata leavesand calyces to determine their anatomical structure and the chemical composition oftheir products Our aims were to determine (1) whether trichome characteristics of Sverticillata differ from those of other Salvia species and (2) how strongly trichomedensity essential oil content and essential oil composition vary among three Sverticillata populations from contrasting habitats

Materials and Methods

Plant material was collected during flowering from three wild-growing populationsrepresenting different types of habitats where S verticillata occurs Population 1 wascollected from Vrdnik (North Serbia 400 m altitude) in a mesic meadow allianceArrhenatherion elatioris Population 2 was collected from Rimski Sanac (Pannonianpart of North Serbia 80 m above sea level) in a xeric steppe habitat alliance Festucionrupicolae and population 3 fromTaramountain (Southwest Serbia altitude 1000m) ina mesic meadow alliance Arrhenatherion elatioris Voucher specimens were depositedin the Herbarium of the Department of Biology and Ecology University of Novi Sad(BUNS)

Anatomical and morphological investigations were carried out on ten plants perpopulation using leaves from the middle part of the plants For light microscopy crosssections of fresh leaves weremade at the region of themain vein and at 14 of leaf widthusing Leica CM 1850 cryostat at temperature 18 to 20 8C with cutting intervals of25 mm Sections were investigated using the Image Analyzing System Motic 2000 Thenumber of glandular trichomes was counted and the diameter of peltate trichomesmeasured on fifty leaves per population The significance of differences betweenpopulations was determined with DuncanGs test using STATISTICA version 70 Forscanning electron microscopy (SEM) small pieces of dry leaves and calyces of tenplants were sputter coated with gold for 180 seconds 30mA (BAL-TEC SCD 005) andviewed with a JEOL JSM-6460LVelectron microscope at an acceleration voltage of 20kV

Essential oils were isolated from dry leaves and flowering tops in full blossom of 50plants per population by the Europaean Pharmacopeia hydrodestillation methodsusing n-hexane as collecting solvent The extracts were dried over anhydrous sodiumsulphate and decanted Hexane was evaporated under reduced pressure to measure oilyields A suitable dilution of each oil in hexane (10 mgml) was then analyzed by GC-FID and GC-MS The GC conditions used were column HP-5 fused silica capillarycolumn 30 mI025 mm film thickness 025 mm column temperature 50 8C for 5 minthen heated to 250 8C at a rate of 3 8Cmin on a Perkin-Elmer 8500 gas chromatographcarrier gas helium constant flow (1mlmin) injector 280 8C FIDdetector 280 8CMassspectra were obtained from aHewlett-Packard 5973-6890GC-MS system operating on

Botanica Helvetica 116 2006 161

EI mode at 70 eV equipped with HP-5 MS capillary column (30 mI025 mm filmthickness 025 mm) The initial temperature of the column was 60 8C and then it washeated to 280 8C at a rate of 3 8Cmin The identification of individual compounds wasmade by comparison of their retention times andmass spectra with those obtained fromauthentic samples andor the NISTNBS Wiley libraries spectra as well as withliterature data

Results

The indumentum of S verticillata leaves was made of non-glandular and glandulartrichomes which were present on both lamina surfaces Non-glandular trichomes werelong uniseriate multicellular (2ndash6 cells) with acute apical cells On their surface smallpapillae were noticed A ring of raised epidermal cells surrounded larger trichomesNon-glandular trichomes densely covered the whole leaf surface but were moreabundant on the abaxial epidermis and along the veins

Both peltate and capitate glandular trichomes were observed Peltate trichomeswere present on the abaxial epidermis only in epidermal depressions (Fig 1ab 2a)They consisted of a basal cell a short stalk cell and a broad round head composed offour secretory cells Above the secretory cells the thick cuticle elevated to form a largesubcuticular space for an accumulation of secreted material Pores or cracks were notrecorded on cuticle using SEM so it could be supposed that secreted material wasreleased after the cuticle had been broken The number of peltate hairs per mm2 of leafsurface and their diameter were lower on the leaves of population 2 than on those ofpopulations 1 and 3 (Tab 1) The number of peltate trichomes also varied stronglywithin populations whereas their diameter showed low variability (cf variationcoefficients in Tab 1)

Capitate glandular trichomeswere present onboth leaf surfaces butmore numerouson the adaxial epidermis and along the veins (Fig 1cd 2bcd) Three types could bedistinguished The hairs of type I had a short stalk cell and a bicellular head Type II hada short unicellular stalk and a unicellular secretory head The hairs of type III were notvery common and their density was always low They were larger with a long stalkcomposed of one elongated stalk cell and one short narrower neck cell and had a pear-

Tab 1 Number of glandular trichomes per mm2 of leaf surface area and diameter of peltatetrichomes Data are means standard errors and coefficients of variation () of 50 leaves perpopulation Letters in superscript point to significance of differences between the populations(Duncan test) means without common letter differ significantly (plt^005)

Population 1 Population 2 Population 3

Capitate trichomes Nr on adaxial epidermis 6102a(248)

4302b(280)

5903a(237)

Nr on abaxial epidermis 3902a(285)

3703a(410)

3503a(426)

Peltate trichomes Nr on abaxial epidermis 2102a(810)

0901b(698)

1702a(643)

Diameter (mm) 48707ab(90)

46807b(75)

50709a(93)

162 Lana Krstic Djordje Malencic and Goran Anackov

shaped unicellular secretory head The total number of capitate hairs did not differamong the examined populations but their number on the adaxial epidermis was sig-nificantly lower in population 2 than in the two other populations The number ofcapitate trichomes varied strongly within populations (Tab 1) though less than thenumber of peltate trichomes

The calyx indumentum was composed of the same types of glandular and non-glandular trichomes but compared to leaves they were more abundant (Fig 3) Thenon-glandular trichomes had larger papillae on their surfaces andweremore numerousbetween the calyx ribs

The essential oil content in dry herb (leaves and flowers) of S verticillata rangedfrom 040 to 042 In total 39 different oil components were identified but only 23to 27 components in individual populations (Tab 2) Oil composition varied conside-rably among the three populations only three of the 39 compounds were well re-presented (gt01of oil) in all three populations (Tab 2) For populations 1 and 2 onlyeight compounds were identified in an amount higher than 01 compared to 27compounds in population 3 The dominant components in populations 1 and 2 weregermacrene D (480 and 246 respectively) and (E)-caryophyllene (134 and190 respectively) whereas the dominant components in population 3 were (E)-caryophyllene (102) b-cubebene (86) and eicosane (85) Using GC-MC only

Fig 1 Salvia verticillata scanning electron micrographs A peltate hair four secretory cellsvisible B peltate hair C capitate hair type II D capitate hair type III Magnification scalebar AndashC 10 mm D 50 mm

Botanica Helvetica 116 2006 163

75 of volatile compounds could be identified in population 3 suggesting that theseplants were also rich in other non-identified volatile carbohydrates besides hy-drocarbons and sesquiterpenes

Fig 2 Salvia verticillata light micrographs population 1 A peltate hair B capitate hair typeI C capitate hair type II D capitate hair type III Magnification scale bar A D 20 mmB C10 mm

Fig 3 Salvia verticillata scanning electron micrographs population 1 A calyx B calyx non-glandular hairs-detail Magnification scale bar A 500 mm B 10 mm

164 Lana Krstic Djordje Malencic and Goran Anackov

Tab 2 Composition of the essential oil of three populations of Salvia verticillata expressed asrelative percentage of each compound as obtained from peak areas (t = trace lt 01) Con-stituents are sorted according to retention time in GC

Constituents Pop 1 Pop 2 Pop 3

a-pinene t t 05sabinene t t ndashb-pinene t t ndashmyrcene t t ndashb-phellandrene t 86 17camphene ndash ndash 06(Z)- b-ocimene ndash 6 ndash(E)- b-ocimene t 75 ndashlinalool t t ndashterpinen-4-ol t t ndasha-terpineol t t ndasha-cubebene t t 05a-copaene t t ndashb-bourbonene t t 04b-elemene t t 11(E)-caryophyllene 134 19 102b-cubebene ndash ndash 86a-elemene ndash ndash 11a-humulene 72 102 48a-muurolene ndash ndash 10germacrene D 48 246 ndashbicyclogermacrene 53 167 ndashnaphthalene 1 2 3 4 4a 5 6 8a-octahydroxyl ndash ndash 40g-cadinene t t 13d-cadinene 60 t 37naphthalene 1 2 3 4 4a 7-hexahydroxyl ndash ndash 13spathulenol 35 72 65caryophyllene oxide t t 29g-gurjunene ndash ndash 09a-cadinol 104 t 37eudesma-4(15)7-dien-1-beta-ol 60 t ndashcaryophyllenol-11 ndash ndash 06aromadendrenepoxide ndash ndash 21octacosane ndash ndash 05nonahexacontanoic acid ndash ndash 05cyclopentane ndash ndash 45eicosane ndash ndash 852-pentadecanone 6 10 14-trimethyl ndash ndash 2512-benzenedicarboxylic acid ndash ndash 25

Total of identified compounds 998 998 754

Botanica Helvetica 116 2006 165

Discussion

Non-glandular and glandular trichomes of S verticillata leaves were similar to thetrichome types described previously for Lamiaceae (Tab 3) The number of head cellsin peltate trichomes varied from four to 16 in different Salvia species (Tab 3) Accor-ding to our findings peltate trichomes of S verticillata leaves had a four-celled secretoryhead Although the structure of trichomes should be of taxonomic value and constantfor the species (Uphof and Hummel 1962) this disagreed with the findings of Remenyi(1997a) who reported 6- 8- and 12-celled heads of sessile glandular trichomes Four-celled head had also been recorded for S blepharophylla and S glutinosa (Tab 3)Capitate glandular trichomes of three typeswere recorded for S verticillataTypes I andII with a unicellular stalk and a bi- and unicellular secretory head respectively cor-responded to trichomes described for S blepharophylla S tomentosa and S officinalis(Tab 3)Glandular trichomes of type III of S verticillatawere similar to those describedby Corsi and Bottega (1999) for S officinalis type III and IV Despite the overallsimilarity of trichome structures the combination of a four-celled head on peltatetrichomes together with the occurrence of types I II and III for capitate trichomes hasnot yet been described for another Salvia species suggesting that it might be a criterionfor the identification of S verticillata However information about trichomes is stillmissing for the majority of the wild-growing Serbian Salvia species so that we cannotexclude that other species have the same trichome characteristics as S verticillataAnother taxonomically important finding is that trichome morphology did not differbetween leaves and calyces

Capitate glandular trichomes were more numerous than peltate trichomes Com-pared with S glutinosa S tomentosa and S officinalis (Tab 3) S verticillata seems tohave a lower total number of glandular trichomes on the abaxial epidermis and a highernumber on the adaxial epidermis of leaves However the number of glandular tri-chomes is generally a very variable character which is strongly influenced by externalfactors such as temperature and light intensity (Uphof and Hummel 1962) so thatinterspecific comparisons based on a small number of populations must be consideredwith caution

The yield of the essential oil (040ndash042) permits the assignment of this species tooil-poor representatives of Lamiaceae as the widely accepted borderline between oil-poor and oil-rich representatives of Salvia is 1 (Mathe et al 1992 Senatore and deFusco 1997 Malencic et al 2004) Sesquiterpenes were present in all populationsconsistent with previous reports that oil-poor species of Lamiaceae possess oil rich insesquiterpene compounds (Malencic et al 2004) There were striking differences in theessential oil composition of the three populations Monoterpenes were well reACHTUNGTRENNUNGpresent-ACHTUNGTRENNUNGed in populations 2 and 3 but only present in traces in population 1Hydrocarbonsweremissing in populations 1 and 2 from the North of Serbia while population 3 fromsouthwest mountainous regions was rich in them The number of compounds found inindividual populations (23ndash27) was similar to the number of compounds found in awild-growing population in Iran (Sefidkon and Khajavi 1999) Two dominant compo-nents in Iran (E)-caryophyllene (247) and a-humulene (78) were also dominantcomponents in all three Serbian populations suggesting that these are generally presentin S verticillata Of the two other components that were present in significant amountsin the Iranian population a-muurolene (228) and limonene (89) only a-muu-rolene was recorded in Serbia and only in population 3

166 Lana Krstic Djordje Malencic and Goran Anackov

Both environmental factors (Mathe et al 1992) and genetic differences (Werker etal 1985) have been found to cause variation in oil composition in other species ofLamiaceae The number of populations investigated in this study does not yet allowconclusions about factors determining the essential oil composition of S verticillataHowever our results suggest that low oil content and high intraspecific variability in oilcomposition may restrict the commercial use of wild-growing S verticillata plants

This work was financially supported by theMinistry of Science andEnvironmental Protection of Serbia andMontenegro Grant No 143037 We would like to thank Mr Milos Bokorov University Center for ElectronMicroscopy Novi Sad for his technical assistance and SEM micrographs

References

Bisio A Corallo A Gastaldo P Romussi G Ciarallo G Fontana N de Tommasi N andProfumoP 1999Glandular hairs and secretedmaterial in Salvia blepharophyllaBrandegeeex Epling grown in Italy Ann Bot 83 441 ndash 452

Bosabalidis AM and Kokkini S 1997 Infraspecific variation of leaf anatomy in Origanumvulgare grown wild in Greece Bot J Linn Soc 123 353 ndash 362

Bosabalidis AM 1990 Glandular trichomes in Satureja thymbra leaves Ann Bot 65 71 ndash 78

Tab 3 Non-glandular and glandular trichome characteristics of various Salvia species (adeadaxial epidermis abe abaxial epidermis ndash not determined)

Species Non-glandulartrichomesnr of cells

Peltatetrichomesnr of headcells

Capitatetrichome type

Meannumber ofglandulartrichomesmm2

References

ade abe

S verticillata 3ndash6 4 I II and III 57 53 This studyS verticillata ndash 6 8 and 12 ndash ndash ndash Remenyi (1997a)S aethiops 3 or more 16 ndash ndash ndash Remenyi (1997b)S aurea ndash 6ndash8 I ndash ndash Serrato-Valenti et

al (1997)S blepharo-

phylla1 4ndash5 or more 4 I and II ndash ndash Bisio et al (1999)

S glutinosa 2ndash7 4 I 80 245 Krstic et al (2001)S officinalis 3ndash4 12 I and II 30 63 Venkatachalam et

al (1984)Corsi and Bottega(1999)

2ndash4 III and IV Chakalova et al(1993)

S officinalis faurea

2ndash4 12 48 87 Chakalova et al(1993)

S tomentosa 2ndash4 12 I and II 066 106 Chakalova et al(1993)

plus trichomes with multicellular stalk and unicellular head

Botanica Helvetica 116 2006 167

Chakalova ES Genova EM and Thuy NT 1993 Comparative anatomical investigations onleaves of Salvia officinalis L and S tomentosa Mill Fitologija 46 3 ndash 11

Corsi G and Bottega S 1999 Glandular hairs of Salvia officinalis new data on morphologylocalization and histochemistry in relation to function Ann Bot 84 657 ndash 664

Demissev S and Harley MM 1992 Trichome seed surface and pollen characters in Stachys(Lamioidae Labiatae) in tropical Africa In Harley R M and Reynolds T (eds) Ad-vances in Labiate Science Royal Botanic Gardens Kew 149 ndash 166

Dickison WC 2000 Integrative Plant Anatomy Academic Press San Diego 297 ndash 307Diklic N 1974 Rod Salvia L In Josifovic M (ed) Flora SR Srbije VI SANU Beograd 432 ndash

453Duke SO 1994 Glandular trichomes ndash focal point of chemical and structural interactions Int

J Plant Sci 155 617 ndash 620Gersbach PV Wyllie SG and Sarafis V 2001 A new histochemical method for localization of

the site of monoterpene phenol accumulation in plant secretory structures Ann Bot 88521 ndash 525

Hedge IC 1972 Genus Salvia L In Tutin et al (eds) Flora Europaea Vol 3 CambridgeUniversity Press London p 192

Krstic L Malencic Dj Merkulov Lj Popovic M Adamovic D and Boza P 2001 A con-tribution to themorpho-anatomical study ofwild-growing and cultivated Salvia glutinosaLMed Plant Rep 8 (8) 38 ndash 47 (in Serbian English summary)

Liu J Zapp J and Becker H 1995 Comparative phytochemical investigation of Salvia mil-tiorrhiza and Salvia triloba Planta Med 61 453 ndash 455

Malencic Dj 2001 Biochemical investigations of selected wild-growing Salvia species fromVojvodina province PhD Thesis University of Novi Sad Faculty of Science (In SerbianEnglish summary)

Malencic Dj Couladis M Mimica-Dukic N Popovic M and Boza P 2004 Essential oils ofthree Salvia species from the Pannonian part of Serbia Flavor Fragr J 19 225 ndash 228

Marin P 1996 Orasice i trihome u familiji Lamiaceae Bioloski fakultet BelgradeMathe JrI Olah L Mathe A Miklossy V Bernath J Blunden G Patel A and Mathe I

1992 Changes in the essential oil production of Salvia officinalis under climatic conditionsof the temperate belt Planta Med 58 680 ndash 686

Metcalfe CR and Chalk L 1950 Anatomy of the dicotyledons Vol II Clarendon PressOxford

PharmacopM EuropMenne vol 1 1983 Maisonneuve SA Sainte-RuffineRemMnyi ML 1997a Research on epidermal appendices of Salvia aethiops L Magyar Nccedil-

vMnyanatOmiai SzimpOzium Abstracts Szeged 89RemMnyi ML 1997b Variations in tissue structures on the epidermis of Salvia verticillata L

Magyar NccedilvMnyanatOmiai SzimpOzium Abstracts Szeged 91Sefidkon F and Khajavi MS 1999 Chemical composition of the essential oils of two Salvia

species from Iran Salvia verticillata L and Salvia santolinifolia Boiss Flavor Fragr J 1477 ndash 78

Senatore F and de Fusco R 1997 Essential oils from Salvia spp (Lamiaceae) I Chemicalcomposition of the essential oils from Salvia glutinosa L growing wild in Southern Italy JEssent Oil Res 9 151 ndash 157

Serrato-Valenti G Bisio A Cornara L and Ciarallo G 1997 Structural and histochemicalinvestigation of the glandular trichomes of Salvia aurea L leaves and chemical analysis ofthe essential oil Ann Bot 79 329 ndash 336

Uphof JCT and Hummel K 1962 Plant Hairs Borntraeger BerlinValkama E Salminen J-P Koricheva J and Pihlaja K 2004 Changes in leaf trichomes and

epicuticular flavonoids during leaf development in three birch taxaAnnBot 94 233 ndash 242Venkatachalam KV Kjonaas R and Crotean R 1984 Development and essential oil content

of secretory glands of sage (Salvia officinalis) Plant Physiol 76 148 ndash 150Werker E Putievsky E and Rivid V 1985 The essential oils and glandular hairs in different

chemotypes of Origanum vulgare L Ann Bot 55 793 ndash 801

168 Lana Krstic Djordje Malencic and Goran Anackov

EI mode at 70 eV equipped with HP-5 MS capillary column (30 mI025 mm filmthickness 025 mm) The initial temperature of the column was 60 8C and then it washeated to 280 8C at a rate of 3 8Cmin The identification of individual compounds wasmade by comparison of their retention times andmass spectra with those obtained fromauthentic samples andor the NISTNBS Wiley libraries spectra as well as withliterature data

Results

The indumentum of S verticillata leaves was made of non-glandular and glandulartrichomes which were present on both lamina surfaces Non-glandular trichomes werelong uniseriate multicellular (2ndash6 cells) with acute apical cells On their surface smallpapillae were noticed A ring of raised epidermal cells surrounded larger trichomesNon-glandular trichomes densely covered the whole leaf surface but were moreabundant on the abaxial epidermis and along the veins

Both peltate and capitate glandular trichomes were observed Peltate trichomeswere present on the abaxial epidermis only in epidermal depressions (Fig 1ab 2a)They consisted of a basal cell a short stalk cell and a broad round head composed offour secretory cells Above the secretory cells the thick cuticle elevated to form a largesubcuticular space for an accumulation of secreted material Pores or cracks were notrecorded on cuticle using SEM so it could be supposed that secreted material wasreleased after the cuticle had been broken The number of peltate hairs per mm2 of leafsurface and their diameter were lower on the leaves of population 2 than on those ofpopulations 1 and 3 (Tab 1) The number of peltate trichomes also varied stronglywithin populations whereas their diameter showed low variability (cf variationcoefficients in Tab 1)

Capitate glandular trichomeswere present onboth leaf surfaces butmore numerouson the adaxial epidermis and along the veins (Fig 1cd 2bcd) Three types could bedistinguished The hairs of type I had a short stalk cell and a bicellular head Type II hada short unicellular stalk and a unicellular secretory head The hairs of type III were notvery common and their density was always low They were larger with a long stalkcomposed of one elongated stalk cell and one short narrower neck cell and had a pear-

Tab 1 Number of glandular trichomes per mm2 of leaf surface area and diameter of peltatetrichomes Data are means standard errors and coefficients of variation () of 50 leaves perpopulation Letters in superscript point to significance of differences between the populations(Duncan test) means without common letter differ significantly (plt^005)

Population 1 Population 2 Population 3

Capitate trichomes Nr on adaxial epidermis 6102a(248)

4302b(280)

5903a(237)

Nr on abaxial epidermis 3902a(285)

3703a(410)

3503a(426)

Peltate trichomes Nr on abaxial epidermis 2102a(810)

0901b(698)

1702a(643)

Diameter (mm) 48707ab(90)

46807b(75)

50709a(93)

162 Lana Krstic Djordje Malencic and Goran Anackov

shaped unicellular secretory head The total number of capitate hairs did not differamong the examined populations but their number on the adaxial epidermis was sig-nificantly lower in population 2 than in the two other populations The number ofcapitate trichomes varied strongly within populations (Tab 1) though less than thenumber of peltate trichomes

The calyx indumentum was composed of the same types of glandular and non-glandular trichomes but compared to leaves they were more abundant (Fig 3) Thenon-glandular trichomes had larger papillae on their surfaces andweremore numerousbetween the calyx ribs

The essential oil content in dry herb (leaves and flowers) of S verticillata rangedfrom 040 to 042 In total 39 different oil components were identified but only 23to 27 components in individual populations (Tab 2) Oil composition varied conside-rably among the three populations only three of the 39 compounds were well re-presented (gt01of oil) in all three populations (Tab 2) For populations 1 and 2 onlyeight compounds were identified in an amount higher than 01 compared to 27compounds in population 3 The dominant components in populations 1 and 2 weregermacrene D (480 and 246 respectively) and (E)-caryophyllene (134 and190 respectively) whereas the dominant components in population 3 were (E)-caryophyllene (102) b-cubebene (86) and eicosane (85) Using GC-MC only

Fig 1 Salvia verticillata scanning electron micrographs A peltate hair four secretory cellsvisible B peltate hair C capitate hair type II D capitate hair type III Magnification scalebar AndashC 10 mm D 50 mm

Botanica Helvetica 116 2006 163

75 of volatile compounds could be identified in population 3 suggesting that theseplants were also rich in other non-identified volatile carbohydrates besides hy-drocarbons and sesquiterpenes

Fig 2 Salvia verticillata light micrographs population 1 A peltate hair B capitate hair typeI C capitate hair type II D capitate hair type III Magnification scale bar A D 20 mmB C10 mm

Fig 3 Salvia verticillata scanning electron micrographs population 1 A calyx B calyx non-glandular hairs-detail Magnification scale bar A 500 mm B 10 mm

164 Lana Krstic Djordje Malencic and Goran Anackov

Tab 2 Composition of the essential oil of three populations of Salvia verticillata expressed asrelative percentage of each compound as obtained from peak areas (t = trace lt 01) Con-stituents are sorted according to retention time in GC

Constituents Pop 1 Pop 2 Pop 3

a-pinene t t 05sabinene t t ndashb-pinene t t ndashmyrcene t t ndashb-phellandrene t 86 17camphene ndash ndash 06(Z)- b-ocimene ndash 6 ndash(E)- b-ocimene t 75 ndashlinalool t t ndashterpinen-4-ol t t ndasha-terpineol t t ndasha-cubebene t t 05a-copaene t t ndashb-bourbonene t t 04b-elemene t t 11(E)-caryophyllene 134 19 102b-cubebene ndash ndash 86a-elemene ndash ndash 11a-humulene 72 102 48a-muurolene ndash ndash 10germacrene D 48 246 ndashbicyclogermacrene 53 167 ndashnaphthalene 1 2 3 4 4a 5 6 8a-octahydroxyl ndash ndash 40g-cadinene t t 13d-cadinene 60 t 37naphthalene 1 2 3 4 4a 7-hexahydroxyl ndash ndash 13spathulenol 35 72 65caryophyllene oxide t t 29g-gurjunene ndash ndash 09a-cadinol 104 t 37eudesma-4(15)7-dien-1-beta-ol 60 t ndashcaryophyllenol-11 ndash ndash 06aromadendrenepoxide ndash ndash 21octacosane ndash ndash 05nonahexacontanoic acid ndash ndash 05cyclopentane ndash ndash 45eicosane ndash ndash 852-pentadecanone 6 10 14-trimethyl ndash ndash 2512-benzenedicarboxylic acid ndash ndash 25

Total of identified compounds 998 998 754

Botanica Helvetica 116 2006 165

Discussion

Non-glandular and glandular trichomes of S verticillata leaves were similar to thetrichome types described previously for Lamiaceae (Tab 3) The number of head cellsin peltate trichomes varied from four to 16 in different Salvia species (Tab 3) Accor-ding to our findings peltate trichomes of S verticillata leaves had a four-celled secretoryhead Although the structure of trichomes should be of taxonomic value and constantfor the species (Uphof and Hummel 1962) this disagreed with the findings of Remenyi(1997a) who reported 6- 8- and 12-celled heads of sessile glandular trichomes Four-celled head had also been recorded for S blepharophylla and S glutinosa (Tab 3)Capitate glandular trichomes of three typeswere recorded for S verticillataTypes I andII with a unicellular stalk and a bi- and unicellular secretory head respectively cor-responded to trichomes described for S blepharophylla S tomentosa and S officinalis(Tab 3)Glandular trichomes of type III of S verticillatawere similar to those describedby Corsi and Bottega (1999) for S officinalis type III and IV Despite the overallsimilarity of trichome structures the combination of a four-celled head on peltatetrichomes together with the occurrence of types I II and III for capitate trichomes hasnot yet been described for another Salvia species suggesting that it might be a criterionfor the identification of S verticillata However information about trichomes is stillmissing for the majority of the wild-growing Serbian Salvia species so that we cannotexclude that other species have the same trichome characteristics as S verticillataAnother taxonomically important finding is that trichome morphology did not differbetween leaves and calyces

Capitate glandular trichomes were more numerous than peltate trichomes Com-pared with S glutinosa S tomentosa and S officinalis (Tab 3) S verticillata seems tohave a lower total number of glandular trichomes on the abaxial epidermis and a highernumber on the adaxial epidermis of leaves However the number of glandular tri-chomes is generally a very variable character which is strongly influenced by externalfactors such as temperature and light intensity (Uphof and Hummel 1962) so thatinterspecific comparisons based on a small number of populations must be consideredwith caution

The yield of the essential oil (040ndash042) permits the assignment of this species tooil-poor representatives of Lamiaceae as the widely accepted borderline between oil-poor and oil-rich representatives of Salvia is 1 (Mathe et al 1992 Senatore and deFusco 1997 Malencic et al 2004) Sesquiterpenes were present in all populationsconsistent with previous reports that oil-poor species of Lamiaceae possess oil rich insesquiterpene compounds (Malencic et al 2004) There were striking differences in theessential oil composition of the three populations Monoterpenes were well reACHTUNGTRENNUNGpresent-ACHTUNGTRENNUNGed in populations 2 and 3 but only present in traces in population 1Hydrocarbonsweremissing in populations 1 and 2 from the North of Serbia while population 3 fromsouthwest mountainous regions was rich in them The number of compounds found inindividual populations (23ndash27) was similar to the number of compounds found in awild-growing population in Iran (Sefidkon and Khajavi 1999) Two dominant compo-nents in Iran (E)-caryophyllene (247) and a-humulene (78) were also dominantcomponents in all three Serbian populations suggesting that these are generally presentin S verticillata Of the two other components that were present in significant amountsin the Iranian population a-muurolene (228) and limonene (89) only a-muu-rolene was recorded in Serbia and only in population 3

166 Lana Krstic Djordje Malencic and Goran Anackov

Both environmental factors (Mathe et al 1992) and genetic differences (Werker etal 1985) have been found to cause variation in oil composition in other species ofLamiaceae The number of populations investigated in this study does not yet allowconclusions about factors determining the essential oil composition of S verticillataHowever our results suggest that low oil content and high intraspecific variability in oilcomposition may restrict the commercial use of wild-growing S verticillata plants

This work was financially supported by theMinistry of Science andEnvironmental Protection of Serbia andMontenegro Grant No 143037 We would like to thank Mr Milos Bokorov University Center for ElectronMicroscopy Novi Sad for his technical assistance and SEM micrographs

References

Bisio A Corallo A Gastaldo P Romussi G Ciarallo G Fontana N de Tommasi N andProfumoP 1999Glandular hairs and secretedmaterial in Salvia blepharophyllaBrandegeeex Epling grown in Italy Ann Bot 83 441 ndash 452

Bosabalidis AM and Kokkini S 1997 Infraspecific variation of leaf anatomy in Origanumvulgare grown wild in Greece Bot J Linn Soc 123 353 ndash 362

Bosabalidis AM 1990 Glandular trichomes in Satureja thymbra leaves Ann Bot 65 71 ndash 78

Tab 3 Non-glandular and glandular trichome characteristics of various Salvia species (adeadaxial epidermis abe abaxial epidermis ndash not determined)

Species Non-glandulartrichomesnr of cells

Peltatetrichomesnr of headcells

Capitatetrichome type

Meannumber ofglandulartrichomesmm2

References

ade abe

S verticillata 3ndash6 4 I II and III 57 53 This studyS verticillata ndash 6 8 and 12 ndash ndash ndash Remenyi (1997a)S aethiops 3 or more 16 ndash ndash ndash Remenyi (1997b)S aurea ndash 6ndash8 I ndash ndash Serrato-Valenti et

al (1997)S blepharo-

phylla1 4ndash5 or more 4 I and II ndash ndash Bisio et al (1999)

S glutinosa 2ndash7 4 I 80 245 Krstic et al (2001)S officinalis 3ndash4 12 I and II 30 63 Venkatachalam et

al (1984)Corsi and Bottega(1999)

2ndash4 III and IV Chakalova et al(1993)

S officinalis faurea

2ndash4 12 48 87 Chakalova et al(1993)

S tomentosa 2ndash4 12 I and II 066 106 Chakalova et al(1993)

plus trichomes with multicellular stalk and unicellular head

Botanica Helvetica 116 2006 167

Chakalova ES Genova EM and Thuy NT 1993 Comparative anatomical investigations onleaves of Salvia officinalis L and S tomentosa Mill Fitologija 46 3 ndash 11

Corsi G and Bottega S 1999 Glandular hairs of Salvia officinalis new data on morphologylocalization and histochemistry in relation to function Ann Bot 84 657 ndash 664

Demissev S and Harley MM 1992 Trichome seed surface and pollen characters in Stachys(Lamioidae Labiatae) in tropical Africa In Harley R M and Reynolds T (eds) Ad-vances in Labiate Science Royal Botanic Gardens Kew 149 ndash 166

Dickison WC 2000 Integrative Plant Anatomy Academic Press San Diego 297 ndash 307Diklic N 1974 Rod Salvia L In Josifovic M (ed) Flora SR Srbije VI SANU Beograd 432 ndash

453Duke SO 1994 Glandular trichomes ndash focal point of chemical and structural interactions Int

J Plant Sci 155 617 ndash 620Gersbach PV Wyllie SG and Sarafis V 2001 A new histochemical method for localization of

the site of monoterpene phenol accumulation in plant secretory structures Ann Bot 88521 ndash 525

Hedge IC 1972 Genus Salvia L In Tutin et al (eds) Flora Europaea Vol 3 CambridgeUniversity Press London p 192

Krstic L Malencic Dj Merkulov Lj Popovic M Adamovic D and Boza P 2001 A con-tribution to themorpho-anatomical study ofwild-growing and cultivated Salvia glutinosaLMed Plant Rep 8 (8) 38 ndash 47 (in Serbian English summary)

Liu J Zapp J and Becker H 1995 Comparative phytochemical investigation of Salvia mil-tiorrhiza and Salvia triloba Planta Med 61 453 ndash 455

Malencic Dj 2001 Biochemical investigations of selected wild-growing Salvia species fromVojvodina province PhD Thesis University of Novi Sad Faculty of Science (In SerbianEnglish summary)

Malencic Dj Couladis M Mimica-Dukic N Popovic M and Boza P 2004 Essential oils ofthree Salvia species from the Pannonian part of Serbia Flavor Fragr J 19 225 ndash 228

Marin P 1996 Orasice i trihome u familiji Lamiaceae Bioloski fakultet BelgradeMathe JrI Olah L Mathe A Miklossy V Bernath J Blunden G Patel A and Mathe I

1992 Changes in the essential oil production of Salvia officinalis under climatic conditionsof the temperate belt Planta Med 58 680 ndash 686

Metcalfe CR and Chalk L 1950 Anatomy of the dicotyledons Vol II Clarendon PressOxford

PharmacopM EuropMenne vol 1 1983 Maisonneuve SA Sainte-RuffineRemMnyi ML 1997a Research on epidermal appendices of Salvia aethiops L Magyar Nccedil-

vMnyanatOmiai SzimpOzium Abstracts Szeged 89RemMnyi ML 1997b Variations in tissue structures on the epidermis of Salvia verticillata L

Magyar NccedilvMnyanatOmiai SzimpOzium Abstracts Szeged 91Sefidkon F and Khajavi MS 1999 Chemical composition of the essential oils of two Salvia

species from Iran Salvia verticillata L and Salvia santolinifolia Boiss Flavor Fragr J 1477 ndash 78

Senatore F and de Fusco R 1997 Essential oils from Salvia spp (Lamiaceae) I Chemicalcomposition of the essential oils from Salvia glutinosa L growing wild in Southern Italy JEssent Oil Res 9 151 ndash 157

Serrato-Valenti G Bisio A Cornara L and Ciarallo G 1997 Structural and histochemicalinvestigation of the glandular trichomes of Salvia aurea L leaves and chemical analysis ofthe essential oil Ann Bot 79 329 ndash 336

Uphof JCT and Hummel K 1962 Plant Hairs Borntraeger BerlinValkama E Salminen J-P Koricheva J and Pihlaja K 2004 Changes in leaf trichomes and

epicuticular flavonoids during leaf development in three birch taxaAnnBot 94 233 ndash 242Venkatachalam KV Kjonaas R and Crotean R 1984 Development and essential oil content

of secretory glands of sage (Salvia officinalis) Plant Physiol 76 148 ndash 150Werker E Putievsky E and Rivid V 1985 The essential oils and glandular hairs in different

chemotypes of Origanum vulgare L Ann Bot 55 793 ndash 801

168 Lana Krstic Djordje Malencic and Goran Anackov

shaped unicellular secretory head The total number of capitate hairs did not differamong the examined populations but their number on the adaxial epidermis was sig-nificantly lower in population 2 than in the two other populations The number ofcapitate trichomes varied strongly within populations (Tab 1) though less than thenumber of peltate trichomes

The calyx indumentum was composed of the same types of glandular and non-glandular trichomes but compared to leaves they were more abundant (Fig 3) Thenon-glandular trichomes had larger papillae on their surfaces andweremore numerousbetween the calyx ribs

The essential oil content in dry herb (leaves and flowers) of S verticillata rangedfrom 040 to 042 In total 39 different oil components were identified but only 23to 27 components in individual populations (Tab 2) Oil composition varied conside-rably among the three populations only three of the 39 compounds were well re-presented (gt01of oil) in all three populations (Tab 2) For populations 1 and 2 onlyeight compounds were identified in an amount higher than 01 compared to 27compounds in population 3 The dominant components in populations 1 and 2 weregermacrene D (480 and 246 respectively) and (E)-caryophyllene (134 and190 respectively) whereas the dominant components in population 3 were (E)-caryophyllene (102) b-cubebene (86) and eicosane (85) Using GC-MC only

Fig 1 Salvia verticillata scanning electron micrographs A peltate hair four secretory cellsvisible B peltate hair C capitate hair type II D capitate hair type III Magnification scalebar AndashC 10 mm D 50 mm

Botanica Helvetica 116 2006 163

75 of volatile compounds could be identified in population 3 suggesting that theseplants were also rich in other non-identified volatile carbohydrates besides hy-drocarbons and sesquiterpenes

Fig 2 Salvia verticillata light micrographs population 1 A peltate hair B capitate hair typeI C capitate hair type II D capitate hair type III Magnification scale bar A D 20 mmB C10 mm

Fig 3 Salvia verticillata scanning electron micrographs population 1 A calyx B calyx non-glandular hairs-detail Magnification scale bar A 500 mm B 10 mm

164 Lana Krstic Djordje Malencic and Goran Anackov

Tab 2 Composition of the essential oil of three populations of Salvia verticillata expressed asrelative percentage of each compound as obtained from peak areas (t = trace lt 01) Con-stituents are sorted according to retention time in GC

Constituents Pop 1 Pop 2 Pop 3

a-pinene t t 05sabinene t t ndashb-pinene t t ndashmyrcene t t ndashb-phellandrene t 86 17camphene ndash ndash 06(Z)- b-ocimene ndash 6 ndash(E)- b-ocimene t 75 ndashlinalool t t ndashterpinen-4-ol t t ndasha-terpineol t t ndasha-cubebene t t 05a-copaene t t ndashb-bourbonene t t 04b-elemene t t 11(E)-caryophyllene 134 19 102b-cubebene ndash ndash 86a-elemene ndash ndash 11a-humulene 72 102 48a-muurolene ndash ndash 10germacrene D 48 246 ndashbicyclogermacrene 53 167 ndashnaphthalene 1 2 3 4 4a 5 6 8a-octahydroxyl ndash ndash 40g-cadinene t t 13d-cadinene 60 t 37naphthalene 1 2 3 4 4a 7-hexahydroxyl ndash ndash 13spathulenol 35 72 65caryophyllene oxide t t 29g-gurjunene ndash ndash 09a-cadinol 104 t 37eudesma-4(15)7-dien-1-beta-ol 60 t ndashcaryophyllenol-11 ndash ndash 06aromadendrenepoxide ndash ndash 21octacosane ndash ndash 05nonahexacontanoic acid ndash ndash 05cyclopentane ndash ndash 45eicosane ndash ndash 852-pentadecanone 6 10 14-trimethyl ndash ndash 2512-benzenedicarboxylic acid ndash ndash 25

Total of identified compounds 998 998 754

Botanica Helvetica 116 2006 165

Discussion

Non-glandular and glandular trichomes of S verticillata leaves were similar to thetrichome types described previously for Lamiaceae (Tab 3) The number of head cellsin peltate trichomes varied from four to 16 in different Salvia species (Tab 3) Accor-ding to our findings peltate trichomes of S verticillata leaves had a four-celled secretoryhead Although the structure of trichomes should be of taxonomic value and constantfor the species (Uphof and Hummel 1962) this disagreed with the findings of Remenyi(1997a) who reported 6- 8- and 12-celled heads of sessile glandular trichomes Four-celled head had also been recorded for S blepharophylla and S glutinosa (Tab 3)Capitate glandular trichomes of three typeswere recorded for S verticillataTypes I andII with a unicellular stalk and a bi- and unicellular secretory head respectively cor-responded to trichomes described for S blepharophylla S tomentosa and S officinalis(Tab 3)Glandular trichomes of type III of S verticillatawere similar to those describedby Corsi and Bottega (1999) for S officinalis type III and IV Despite the overallsimilarity of trichome structures the combination of a four-celled head on peltatetrichomes together with the occurrence of types I II and III for capitate trichomes hasnot yet been described for another Salvia species suggesting that it might be a criterionfor the identification of S verticillata However information about trichomes is stillmissing for the majority of the wild-growing Serbian Salvia species so that we cannotexclude that other species have the same trichome characteristics as S verticillataAnother taxonomically important finding is that trichome morphology did not differbetween leaves and calyces

Capitate glandular trichomes were more numerous than peltate trichomes Com-pared with S glutinosa S tomentosa and S officinalis (Tab 3) S verticillata seems tohave a lower total number of glandular trichomes on the abaxial epidermis and a highernumber on the adaxial epidermis of leaves However the number of glandular tri-chomes is generally a very variable character which is strongly influenced by externalfactors such as temperature and light intensity (Uphof and Hummel 1962) so thatinterspecific comparisons based on a small number of populations must be consideredwith caution

The yield of the essential oil (040ndash042) permits the assignment of this species tooil-poor representatives of Lamiaceae as the widely accepted borderline between oil-poor and oil-rich representatives of Salvia is 1 (Mathe et al 1992 Senatore and deFusco 1997 Malencic et al 2004) Sesquiterpenes were present in all populationsconsistent with previous reports that oil-poor species of Lamiaceae possess oil rich insesquiterpene compounds (Malencic et al 2004) There were striking differences in theessential oil composition of the three populations Monoterpenes were well reACHTUNGTRENNUNGpresent-ACHTUNGTRENNUNGed in populations 2 and 3 but only present in traces in population 1Hydrocarbonsweremissing in populations 1 and 2 from the North of Serbia while population 3 fromsouthwest mountainous regions was rich in them The number of compounds found inindividual populations (23ndash27) was similar to the number of compounds found in awild-growing population in Iran (Sefidkon and Khajavi 1999) Two dominant compo-nents in Iran (E)-caryophyllene (247) and a-humulene (78) were also dominantcomponents in all three Serbian populations suggesting that these are generally presentin S verticillata Of the two other components that were present in significant amountsin the Iranian population a-muurolene (228) and limonene (89) only a-muu-rolene was recorded in Serbia and only in population 3

166 Lana Krstic Djordje Malencic and Goran Anackov

Both environmental factors (Mathe et al 1992) and genetic differences (Werker etal 1985) have been found to cause variation in oil composition in other species ofLamiaceae The number of populations investigated in this study does not yet allowconclusions about factors determining the essential oil composition of S verticillataHowever our results suggest that low oil content and high intraspecific variability in oilcomposition may restrict the commercial use of wild-growing S verticillata plants

This work was financially supported by theMinistry of Science andEnvironmental Protection of Serbia andMontenegro Grant No 143037 We would like to thank Mr Milos Bokorov University Center for ElectronMicroscopy Novi Sad for his technical assistance and SEM micrographs

References

Bisio A Corallo A Gastaldo P Romussi G Ciarallo G Fontana N de Tommasi N andProfumoP 1999Glandular hairs and secretedmaterial in Salvia blepharophyllaBrandegeeex Epling grown in Italy Ann Bot 83 441 ndash 452

Bosabalidis AM and Kokkini S 1997 Infraspecific variation of leaf anatomy in Origanumvulgare grown wild in Greece Bot J Linn Soc 123 353 ndash 362

Bosabalidis AM 1990 Glandular trichomes in Satureja thymbra leaves Ann Bot 65 71 ndash 78

Tab 3 Non-glandular and glandular trichome characteristics of various Salvia species (adeadaxial epidermis abe abaxial epidermis ndash not determined)

Species Non-glandulartrichomesnr of cells

Peltatetrichomesnr of headcells

Capitatetrichome type

Meannumber ofglandulartrichomesmm2

References

ade abe

S verticillata 3ndash6 4 I II and III 57 53 This studyS verticillata ndash 6 8 and 12 ndash ndash ndash Remenyi (1997a)S aethiops 3 or more 16 ndash ndash ndash Remenyi (1997b)S aurea ndash 6ndash8 I ndash ndash Serrato-Valenti et

al (1997)S blepharo-

phylla1 4ndash5 or more 4 I and II ndash ndash Bisio et al (1999)

S glutinosa 2ndash7 4 I 80 245 Krstic et al (2001)S officinalis 3ndash4 12 I and II 30 63 Venkatachalam et

al (1984)Corsi and Bottega(1999)

2ndash4 III and IV Chakalova et al(1993)

S officinalis faurea

2ndash4 12 48 87 Chakalova et al(1993)

S tomentosa 2ndash4 12 I and II 066 106 Chakalova et al(1993)

plus trichomes with multicellular stalk and unicellular head

Botanica Helvetica 116 2006 167

Chakalova ES Genova EM and Thuy NT 1993 Comparative anatomical investigations onleaves of Salvia officinalis L and S tomentosa Mill Fitologija 46 3 ndash 11

Corsi G and Bottega S 1999 Glandular hairs of Salvia officinalis new data on morphologylocalization and histochemistry in relation to function Ann Bot 84 657 ndash 664

Demissev S and Harley MM 1992 Trichome seed surface and pollen characters in Stachys(Lamioidae Labiatae) in tropical Africa In Harley R M and Reynolds T (eds) Ad-vances in Labiate Science Royal Botanic Gardens Kew 149 ndash 166

Dickison WC 2000 Integrative Plant Anatomy Academic Press San Diego 297 ndash 307Diklic N 1974 Rod Salvia L In Josifovic M (ed) Flora SR Srbije VI SANU Beograd 432 ndash

453Duke SO 1994 Glandular trichomes ndash focal point of chemical and structural interactions Int

J Plant Sci 155 617 ndash 620Gersbach PV Wyllie SG and Sarafis V 2001 A new histochemical method for localization of

the site of monoterpene phenol accumulation in plant secretory structures Ann Bot 88521 ndash 525

Hedge IC 1972 Genus Salvia L In Tutin et al (eds) Flora Europaea Vol 3 CambridgeUniversity Press London p 192

Krstic L Malencic Dj Merkulov Lj Popovic M Adamovic D and Boza P 2001 A con-tribution to themorpho-anatomical study ofwild-growing and cultivated Salvia glutinosaLMed Plant Rep 8 (8) 38 ndash 47 (in Serbian English summary)

Liu J Zapp J and Becker H 1995 Comparative phytochemical investigation of Salvia mil-tiorrhiza and Salvia triloba Planta Med 61 453 ndash 455

Malencic Dj 2001 Biochemical investigations of selected wild-growing Salvia species fromVojvodina province PhD Thesis University of Novi Sad Faculty of Science (In SerbianEnglish summary)

Malencic Dj Couladis M Mimica-Dukic N Popovic M and Boza P 2004 Essential oils ofthree Salvia species from the Pannonian part of Serbia Flavor Fragr J 19 225 ndash 228

Marin P 1996 Orasice i trihome u familiji Lamiaceae Bioloski fakultet BelgradeMathe JrI Olah L Mathe A Miklossy V Bernath J Blunden G Patel A and Mathe I

1992 Changes in the essential oil production of Salvia officinalis under climatic conditionsof the temperate belt Planta Med 58 680 ndash 686

Metcalfe CR and Chalk L 1950 Anatomy of the dicotyledons Vol II Clarendon PressOxford

PharmacopM EuropMenne vol 1 1983 Maisonneuve SA Sainte-RuffineRemMnyi ML 1997a Research on epidermal appendices of Salvia aethiops L Magyar Nccedil-

vMnyanatOmiai SzimpOzium Abstracts Szeged 89RemMnyi ML 1997b Variations in tissue structures on the epidermis of Salvia verticillata L

Magyar NccedilvMnyanatOmiai SzimpOzium Abstracts Szeged 91Sefidkon F and Khajavi MS 1999 Chemical composition of the essential oils of two Salvia

species from Iran Salvia verticillata L and Salvia santolinifolia Boiss Flavor Fragr J 1477 ndash 78

Senatore F and de Fusco R 1997 Essential oils from Salvia spp (Lamiaceae) I Chemicalcomposition of the essential oils from Salvia glutinosa L growing wild in Southern Italy JEssent Oil Res 9 151 ndash 157

Serrato-Valenti G Bisio A Cornara L and Ciarallo G 1997 Structural and histochemicalinvestigation of the glandular trichomes of Salvia aurea L leaves and chemical analysis ofthe essential oil Ann Bot 79 329 ndash 336

Uphof JCT and Hummel K 1962 Plant Hairs Borntraeger BerlinValkama E Salminen J-P Koricheva J and Pihlaja K 2004 Changes in leaf trichomes and

epicuticular flavonoids during leaf development in three birch taxaAnnBot 94 233 ndash 242Venkatachalam KV Kjonaas R and Crotean R 1984 Development and essential oil content

of secretory glands of sage (Salvia officinalis) Plant Physiol 76 148 ndash 150Werker E Putievsky E and Rivid V 1985 The essential oils and glandular hairs in different

chemotypes of Origanum vulgare L Ann Bot 55 793 ndash 801

168 Lana Krstic Djordje Malencic and Goran Anackov

75 of volatile compounds could be identified in population 3 suggesting that theseplants were also rich in other non-identified volatile carbohydrates besides hy-drocarbons and sesquiterpenes

Fig 2 Salvia verticillata light micrographs population 1 A peltate hair B capitate hair typeI C capitate hair type II D capitate hair type III Magnification scale bar A D 20 mmB C10 mm

Fig 3 Salvia verticillata scanning electron micrographs population 1 A calyx B calyx non-glandular hairs-detail Magnification scale bar A 500 mm B 10 mm

164 Lana Krstic Djordje Malencic and Goran Anackov

Tab 2 Composition of the essential oil of three populations of Salvia verticillata expressed asrelative percentage of each compound as obtained from peak areas (t = trace lt 01) Con-stituents are sorted according to retention time in GC

Constituents Pop 1 Pop 2 Pop 3

a-pinene t t 05sabinene t t ndashb-pinene t t ndashmyrcene t t ndashb-phellandrene t 86 17camphene ndash ndash 06(Z)- b-ocimene ndash 6 ndash(E)- b-ocimene t 75 ndashlinalool t t ndashterpinen-4-ol t t ndasha-terpineol t t ndasha-cubebene t t 05a-copaene t t ndashb-bourbonene t t 04b-elemene t t 11(E)-caryophyllene 134 19 102b-cubebene ndash ndash 86a-elemene ndash ndash 11a-humulene 72 102 48a-muurolene ndash ndash 10germacrene D 48 246 ndashbicyclogermacrene 53 167 ndashnaphthalene 1 2 3 4 4a 5 6 8a-octahydroxyl ndash ndash 40g-cadinene t t 13d-cadinene 60 t 37naphthalene 1 2 3 4 4a 7-hexahydroxyl ndash ndash 13spathulenol 35 72 65caryophyllene oxide t t 29g-gurjunene ndash ndash 09a-cadinol 104 t 37eudesma-4(15)7-dien-1-beta-ol 60 t ndashcaryophyllenol-11 ndash ndash 06aromadendrenepoxide ndash ndash 21octacosane ndash ndash 05nonahexacontanoic acid ndash ndash 05cyclopentane ndash ndash 45eicosane ndash ndash 852-pentadecanone 6 10 14-trimethyl ndash ndash 2512-benzenedicarboxylic acid ndash ndash 25

Total of identified compounds 998 998 754

Botanica Helvetica 116 2006 165

Discussion

Non-glandular and glandular trichomes of S verticillata leaves were similar to thetrichome types described previously for Lamiaceae (Tab 3) The number of head cellsin peltate trichomes varied from four to 16 in different Salvia species (Tab 3) Accor-ding to our findings peltate trichomes of S verticillata leaves had a four-celled secretoryhead Although the structure of trichomes should be of taxonomic value and constantfor the species (Uphof and Hummel 1962) this disagreed with the findings of Remenyi(1997a) who reported 6- 8- and 12-celled heads of sessile glandular trichomes Four-celled head had also been recorded for S blepharophylla and S glutinosa (Tab 3)Capitate glandular trichomes of three typeswere recorded for S verticillataTypes I andII with a unicellular stalk and a bi- and unicellular secretory head respectively cor-responded to trichomes described for S blepharophylla S tomentosa and S officinalis(Tab 3)Glandular trichomes of type III of S verticillatawere similar to those describedby Corsi and Bottega (1999) for S officinalis type III and IV Despite the overallsimilarity of trichome structures the combination of a four-celled head on peltatetrichomes together with the occurrence of types I II and III for capitate trichomes hasnot yet been described for another Salvia species suggesting that it might be a criterionfor the identification of S verticillata However information about trichomes is stillmissing for the majority of the wild-growing Serbian Salvia species so that we cannotexclude that other species have the same trichome characteristics as S verticillataAnother taxonomically important finding is that trichome morphology did not differbetween leaves and calyces

Capitate glandular trichomes were more numerous than peltate trichomes Com-pared with S glutinosa S tomentosa and S officinalis (Tab 3) S verticillata seems tohave a lower total number of glandular trichomes on the abaxial epidermis and a highernumber on the adaxial epidermis of leaves However the number of glandular tri-chomes is generally a very variable character which is strongly influenced by externalfactors such as temperature and light intensity (Uphof and Hummel 1962) so thatinterspecific comparisons based on a small number of populations must be consideredwith caution

The yield of the essential oil (040ndash042) permits the assignment of this species tooil-poor representatives of Lamiaceae as the widely accepted borderline between oil-poor and oil-rich representatives of Salvia is 1 (Mathe et al 1992 Senatore and deFusco 1997 Malencic et al 2004) Sesquiterpenes were present in all populationsconsistent with previous reports that oil-poor species of Lamiaceae possess oil rich insesquiterpene compounds (Malencic et al 2004) There were striking differences in theessential oil composition of the three populations Monoterpenes were well reACHTUNGTRENNUNGpresent-ACHTUNGTRENNUNGed in populations 2 and 3 but only present in traces in population 1Hydrocarbonsweremissing in populations 1 and 2 from the North of Serbia while population 3 fromsouthwest mountainous regions was rich in them The number of compounds found inindividual populations (23ndash27) was similar to the number of compounds found in awild-growing population in Iran (Sefidkon and Khajavi 1999) Two dominant compo-nents in Iran (E)-caryophyllene (247) and a-humulene (78) were also dominantcomponents in all three Serbian populations suggesting that these are generally presentin S verticillata Of the two other components that were present in significant amountsin the Iranian population a-muurolene (228) and limonene (89) only a-muu-rolene was recorded in Serbia and only in population 3

166 Lana Krstic Djordje Malencic and Goran Anackov

Both environmental factors (Mathe et al 1992) and genetic differences (Werker etal 1985) have been found to cause variation in oil composition in other species ofLamiaceae The number of populations investigated in this study does not yet allowconclusions about factors determining the essential oil composition of S verticillataHowever our results suggest that low oil content and high intraspecific variability in oilcomposition may restrict the commercial use of wild-growing S verticillata plants

This work was financially supported by theMinistry of Science andEnvironmental Protection of Serbia andMontenegro Grant No 143037 We would like to thank Mr Milos Bokorov University Center for ElectronMicroscopy Novi Sad for his technical assistance and SEM micrographs

References

Bisio A Corallo A Gastaldo P Romussi G Ciarallo G Fontana N de Tommasi N andProfumoP 1999Glandular hairs and secretedmaterial in Salvia blepharophyllaBrandegeeex Epling grown in Italy Ann Bot 83 441 ndash 452

Bosabalidis AM and Kokkini S 1997 Infraspecific variation of leaf anatomy in Origanumvulgare grown wild in Greece Bot J Linn Soc 123 353 ndash 362

Bosabalidis AM 1990 Glandular trichomes in Satureja thymbra leaves Ann Bot 65 71 ndash 78

Tab 3 Non-glandular and glandular trichome characteristics of various Salvia species (adeadaxial epidermis abe abaxial epidermis ndash not determined)

Species Non-glandulartrichomesnr of cells

Peltatetrichomesnr of headcells

Capitatetrichome type

Meannumber ofglandulartrichomesmm2

References

ade abe

S verticillata 3ndash6 4 I II and III 57 53 This studyS verticillata ndash 6 8 and 12 ndash ndash ndash Remenyi (1997a)S aethiops 3 or more 16 ndash ndash ndash Remenyi (1997b)S aurea ndash 6ndash8 I ndash ndash Serrato-Valenti et

al (1997)S blepharo-

phylla1 4ndash5 or more 4 I and II ndash ndash Bisio et al (1999)

S glutinosa 2ndash7 4 I 80 245 Krstic et al (2001)S officinalis 3ndash4 12 I and II 30 63 Venkatachalam et

al (1984)Corsi and Bottega(1999)

2ndash4 III and IV Chakalova et al(1993)

S officinalis faurea

2ndash4 12 48 87 Chakalova et al(1993)

S tomentosa 2ndash4 12 I and II 066 106 Chakalova et al(1993)

plus trichomes with multicellular stalk and unicellular head

Botanica Helvetica 116 2006 167

Chakalova ES Genova EM and Thuy NT 1993 Comparative anatomical investigations onleaves of Salvia officinalis L and S tomentosa Mill Fitologija 46 3 ndash 11

Corsi G and Bottega S 1999 Glandular hairs of Salvia officinalis new data on morphologylocalization and histochemistry in relation to function Ann Bot 84 657 ndash 664

Demissev S and Harley MM 1992 Trichome seed surface and pollen characters in Stachys(Lamioidae Labiatae) in tropical Africa In Harley R M and Reynolds T (eds) Ad-vances in Labiate Science Royal Botanic Gardens Kew 149 ndash 166

Dickison WC 2000 Integrative Plant Anatomy Academic Press San Diego 297 ndash 307Diklic N 1974 Rod Salvia L In Josifovic M (ed) Flora SR Srbije VI SANU Beograd 432 ndash

453Duke SO 1994 Glandular trichomes ndash focal point of chemical and structural interactions Int

J Plant Sci 155 617 ndash 620Gersbach PV Wyllie SG and Sarafis V 2001 A new histochemical method for localization of

the site of monoterpene phenol accumulation in plant secretory structures Ann Bot 88521 ndash 525

Hedge IC 1972 Genus Salvia L In Tutin et al (eds) Flora Europaea Vol 3 CambridgeUniversity Press London p 192

Krstic L Malencic Dj Merkulov Lj Popovic M Adamovic D and Boza P 2001 A con-tribution to themorpho-anatomical study ofwild-growing and cultivated Salvia glutinosaLMed Plant Rep 8 (8) 38 ndash 47 (in Serbian English summary)

Liu J Zapp J and Becker H 1995 Comparative phytochemical investigation of Salvia mil-tiorrhiza and Salvia triloba Planta Med 61 453 ndash 455

Malencic Dj 2001 Biochemical investigations of selected wild-growing Salvia species fromVojvodina province PhD Thesis University of Novi Sad Faculty of Science (In SerbianEnglish summary)

Malencic Dj Couladis M Mimica-Dukic N Popovic M and Boza P 2004 Essential oils ofthree Salvia species from the Pannonian part of Serbia Flavor Fragr J 19 225 ndash 228

Marin P 1996 Orasice i trihome u familiji Lamiaceae Bioloski fakultet BelgradeMathe JrI Olah L Mathe A Miklossy V Bernath J Blunden G Patel A and Mathe I

1992 Changes in the essential oil production of Salvia officinalis under climatic conditionsof the temperate belt Planta Med 58 680 ndash 686

Metcalfe CR and Chalk L 1950 Anatomy of the dicotyledons Vol II Clarendon PressOxford

PharmacopM EuropMenne vol 1 1983 Maisonneuve SA Sainte-RuffineRemMnyi ML 1997a Research on epidermal appendices of Salvia aethiops L Magyar Nccedil-

vMnyanatOmiai SzimpOzium Abstracts Szeged 89RemMnyi ML 1997b Variations in tissue structures on the epidermis of Salvia verticillata L

Magyar NccedilvMnyanatOmiai SzimpOzium Abstracts Szeged 91Sefidkon F and Khajavi MS 1999 Chemical composition of the essential oils of two Salvia

species from Iran Salvia verticillata L and Salvia santolinifolia Boiss Flavor Fragr J 1477 ndash 78

Senatore F and de Fusco R 1997 Essential oils from Salvia spp (Lamiaceae) I Chemicalcomposition of the essential oils from Salvia glutinosa L growing wild in Southern Italy JEssent Oil Res 9 151 ndash 157

Serrato-Valenti G Bisio A Cornara L and Ciarallo G 1997 Structural and histochemicalinvestigation of the glandular trichomes of Salvia aurea L leaves and chemical analysis ofthe essential oil Ann Bot 79 329 ndash 336

Uphof JCT and Hummel K 1962 Plant Hairs Borntraeger BerlinValkama E Salminen J-P Koricheva J and Pihlaja K 2004 Changes in leaf trichomes and

epicuticular flavonoids during leaf development in three birch taxaAnnBot 94 233 ndash 242Venkatachalam KV Kjonaas R and Crotean R 1984 Development and essential oil content

of secretory glands of sage (Salvia officinalis) Plant Physiol 76 148 ndash 150Werker E Putievsky E and Rivid V 1985 The essential oils and glandular hairs in different

chemotypes of Origanum vulgare L Ann Bot 55 793 ndash 801

168 Lana Krstic Djordje Malencic and Goran Anackov

Tab 2 Composition of the essential oil of three populations of Salvia verticillata expressed asrelative percentage of each compound as obtained from peak areas (t = trace lt 01) Con-stituents are sorted according to retention time in GC

Constituents Pop 1 Pop 2 Pop 3

a-pinene t t 05sabinene t t ndashb-pinene t t ndashmyrcene t t ndashb-phellandrene t 86 17camphene ndash ndash 06(Z)- b-ocimene ndash 6 ndash(E)- b-ocimene t 75 ndashlinalool t t ndashterpinen-4-ol t t ndasha-terpineol t t ndasha-cubebene t t 05a-copaene t t ndashb-bourbonene t t 04b-elemene t t 11(E)-caryophyllene 134 19 102b-cubebene ndash ndash 86a-elemene ndash ndash 11a-humulene 72 102 48a-muurolene ndash ndash 10germacrene D 48 246 ndashbicyclogermacrene 53 167 ndashnaphthalene 1 2 3 4 4a 5 6 8a-octahydroxyl ndash ndash 40g-cadinene t t 13d-cadinene 60 t 37naphthalene 1 2 3 4 4a 7-hexahydroxyl ndash ndash 13spathulenol 35 72 65caryophyllene oxide t t 29g-gurjunene ndash ndash 09a-cadinol 104 t 37eudesma-4(15)7-dien-1-beta-ol 60 t ndashcaryophyllenol-11 ndash ndash 06aromadendrenepoxide ndash ndash 21octacosane ndash ndash 05nonahexacontanoic acid ndash ndash 05cyclopentane ndash ndash 45eicosane ndash ndash 852-pentadecanone 6 10 14-trimethyl ndash ndash 2512-benzenedicarboxylic acid ndash ndash 25

Total of identified compounds 998 998 754

Botanica Helvetica 116 2006 165

Discussion

Non-glandular and glandular trichomes of S verticillata leaves were similar to thetrichome types described previously for Lamiaceae (Tab 3) The number of head cellsin peltate trichomes varied from four to 16 in different Salvia species (Tab 3) Accor-ding to our findings peltate trichomes of S verticillata leaves had a four-celled secretoryhead Although the structure of trichomes should be of taxonomic value and constantfor the species (Uphof and Hummel 1962) this disagreed with the findings of Remenyi(1997a) who reported 6- 8- and 12-celled heads of sessile glandular trichomes Four-celled head had also been recorded for S blepharophylla and S glutinosa (Tab 3)Capitate glandular trichomes of three typeswere recorded for S verticillataTypes I andII with a unicellular stalk and a bi- and unicellular secretory head respectively cor-responded to trichomes described for S blepharophylla S tomentosa and S officinalis(Tab 3)Glandular trichomes of type III of S verticillatawere similar to those describedby Corsi and Bottega (1999) for S officinalis type III and IV Despite the overallsimilarity of trichome structures the combination of a four-celled head on peltatetrichomes together with the occurrence of types I II and III for capitate trichomes hasnot yet been described for another Salvia species suggesting that it might be a criterionfor the identification of S verticillata However information about trichomes is stillmissing for the majority of the wild-growing Serbian Salvia species so that we cannotexclude that other species have the same trichome characteristics as S verticillataAnother taxonomically important finding is that trichome morphology did not differbetween leaves and calyces

Capitate glandular trichomes were more numerous than peltate trichomes Com-pared with S glutinosa S tomentosa and S officinalis (Tab 3) S verticillata seems tohave a lower total number of glandular trichomes on the abaxial epidermis and a highernumber on the adaxial epidermis of leaves However the number of glandular tri-chomes is generally a very variable character which is strongly influenced by externalfactors such as temperature and light intensity (Uphof and Hummel 1962) so thatinterspecific comparisons based on a small number of populations must be consideredwith caution

The yield of the essential oil (040ndash042) permits the assignment of this species tooil-poor representatives of Lamiaceae as the widely accepted borderline between oil-poor and oil-rich representatives of Salvia is 1 (Mathe et al 1992 Senatore and deFusco 1997 Malencic et al 2004) Sesquiterpenes were present in all populationsconsistent with previous reports that oil-poor species of Lamiaceae possess oil rich insesquiterpene compounds (Malencic et al 2004) There were striking differences in theessential oil composition of the three populations Monoterpenes were well reACHTUNGTRENNUNGpresent-ACHTUNGTRENNUNGed in populations 2 and 3 but only present in traces in population 1Hydrocarbonsweremissing in populations 1 and 2 from the North of Serbia while population 3 fromsouthwest mountainous regions was rich in them The number of compounds found inindividual populations (23ndash27) was similar to the number of compounds found in awild-growing population in Iran (Sefidkon and Khajavi 1999) Two dominant compo-nents in Iran (E)-caryophyllene (247) and a-humulene (78) were also dominantcomponents in all three Serbian populations suggesting that these are generally presentin S verticillata Of the two other components that were present in significant amountsin the Iranian population a-muurolene (228) and limonene (89) only a-muu-rolene was recorded in Serbia and only in population 3

166 Lana Krstic Djordje Malencic and Goran Anackov

Both environmental factors (Mathe et al 1992) and genetic differences (Werker etal 1985) have been found to cause variation in oil composition in other species ofLamiaceae The number of populations investigated in this study does not yet allowconclusions about factors determining the essential oil composition of S verticillataHowever our results suggest that low oil content and high intraspecific variability in oilcomposition may restrict the commercial use of wild-growing S verticillata plants

This work was financially supported by theMinistry of Science andEnvironmental Protection of Serbia andMontenegro Grant No 143037 We would like to thank Mr Milos Bokorov University Center for ElectronMicroscopy Novi Sad for his technical assistance and SEM micrographs

References

Bisio A Corallo A Gastaldo P Romussi G Ciarallo G Fontana N de Tommasi N andProfumoP 1999Glandular hairs and secretedmaterial in Salvia blepharophyllaBrandegeeex Epling grown in Italy Ann Bot 83 441 ndash 452

Bosabalidis AM and Kokkini S 1997 Infraspecific variation of leaf anatomy in Origanumvulgare grown wild in Greece Bot J Linn Soc 123 353 ndash 362

Bosabalidis AM 1990 Glandular trichomes in Satureja thymbra leaves Ann Bot 65 71 ndash 78

Tab 3 Non-glandular and glandular trichome characteristics of various Salvia species (adeadaxial epidermis abe abaxial epidermis ndash not determined)

Species Non-glandulartrichomesnr of cells

Peltatetrichomesnr of headcells

Capitatetrichome type

Meannumber ofglandulartrichomesmm2

References

ade abe

S verticillata 3ndash6 4 I II and III 57 53 This studyS verticillata ndash 6 8 and 12 ndash ndash ndash Remenyi (1997a)S aethiops 3 or more 16 ndash ndash ndash Remenyi (1997b)S aurea ndash 6ndash8 I ndash ndash Serrato-Valenti et

al (1997)S blepharo-

phylla1 4ndash5 or more 4 I and II ndash ndash Bisio et al (1999)

S glutinosa 2ndash7 4 I 80 245 Krstic et al (2001)S officinalis 3ndash4 12 I and II 30 63 Venkatachalam et

al (1984)Corsi and Bottega(1999)

2ndash4 III and IV Chakalova et al(1993)

S officinalis faurea

2ndash4 12 48 87 Chakalova et al(1993)

S tomentosa 2ndash4 12 I and II 066 106 Chakalova et al(1993)

plus trichomes with multicellular stalk and unicellular head

Botanica Helvetica 116 2006 167

Chakalova ES Genova EM and Thuy NT 1993 Comparative anatomical investigations onleaves of Salvia officinalis L and S tomentosa Mill Fitologija 46 3 ndash 11

Corsi G and Bottega S 1999 Glandular hairs of Salvia officinalis new data on morphologylocalization and histochemistry in relation to function Ann Bot 84 657 ndash 664

Demissev S and Harley MM 1992 Trichome seed surface and pollen characters in Stachys(Lamioidae Labiatae) in tropical Africa In Harley R M and Reynolds T (eds) Ad-vances in Labiate Science Royal Botanic Gardens Kew 149 ndash 166

Dickison WC 2000 Integrative Plant Anatomy Academic Press San Diego 297 ndash 307Diklic N 1974 Rod Salvia L In Josifovic M (ed) Flora SR Srbije VI SANU Beograd 432 ndash

453Duke SO 1994 Glandular trichomes ndash focal point of chemical and structural interactions Int

J Plant Sci 155 617 ndash 620Gersbach PV Wyllie SG and Sarafis V 2001 A new histochemical method for localization of

the site of monoterpene phenol accumulation in plant secretory structures Ann Bot 88521 ndash 525

Hedge IC 1972 Genus Salvia L In Tutin et al (eds) Flora Europaea Vol 3 CambridgeUniversity Press London p 192

Krstic L Malencic Dj Merkulov Lj Popovic M Adamovic D and Boza P 2001 A con-tribution to themorpho-anatomical study ofwild-growing and cultivated Salvia glutinosaLMed Plant Rep 8 (8) 38 ndash 47 (in Serbian English summary)

Liu J Zapp J and Becker H 1995 Comparative phytochemical investigation of Salvia mil-tiorrhiza and Salvia triloba Planta Med 61 453 ndash 455

Malencic Dj 2001 Biochemical investigations of selected wild-growing Salvia species fromVojvodina province PhD Thesis University of Novi Sad Faculty of Science (In SerbianEnglish summary)

Malencic Dj Couladis M Mimica-Dukic N Popovic M and Boza P 2004 Essential oils ofthree Salvia species from the Pannonian part of Serbia Flavor Fragr J 19 225 ndash 228

Marin P 1996 Orasice i trihome u familiji Lamiaceae Bioloski fakultet BelgradeMathe JrI Olah L Mathe A Miklossy V Bernath J Blunden G Patel A and Mathe I

1992 Changes in the essential oil production of Salvia officinalis under climatic conditionsof the temperate belt Planta Med 58 680 ndash 686

Metcalfe CR and Chalk L 1950 Anatomy of the dicotyledons Vol II Clarendon PressOxford

PharmacopM EuropMenne vol 1 1983 Maisonneuve SA Sainte-RuffineRemMnyi ML 1997a Research on epidermal appendices of Salvia aethiops L Magyar Nccedil-

vMnyanatOmiai SzimpOzium Abstracts Szeged 89RemMnyi ML 1997b Variations in tissue structures on the epidermis of Salvia verticillata L

Magyar NccedilvMnyanatOmiai SzimpOzium Abstracts Szeged 91Sefidkon F and Khajavi MS 1999 Chemical composition of the essential oils of two Salvia

species from Iran Salvia verticillata L and Salvia santolinifolia Boiss Flavor Fragr J 1477 ndash 78

Senatore F and de Fusco R 1997 Essential oils from Salvia spp (Lamiaceae) I Chemicalcomposition of the essential oils from Salvia glutinosa L growing wild in Southern Italy JEssent Oil Res 9 151 ndash 157

Serrato-Valenti G Bisio A Cornara L and Ciarallo G 1997 Structural and histochemicalinvestigation of the glandular trichomes of Salvia aurea L leaves and chemical analysis ofthe essential oil Ann Bot 79 329 ndash 336

Uphof JCT and Hummel K 1962 Plant Hairs Borntraeger BerlinValkama E Salminen J-P Koricheva J and Pihlaja K 2004 Changes in leaf trichomes and

epicuticular flavonoids during leaf development in three birch taxaAnnBot 94 233 ndash 242Venkatachalam KV Kjonaas R and Crotean R 1984 Development and essential oil content

of secretory glands of sage (Salvia officinalis) Plant Physiol 76 148 ndash 150Werker E Putievsky E and Rivid V 1985 The essential oils and glandular hairs in different

chemotypes of Origanum vulgare L Ann Bot 55 793 ndash 801

168 Lana Krstic Djordje Malencic and Goran Anackov

Discussion

Non-glandular and glandular trichomes of S verticillata leaves were similar to thetrichome types described previously for Lamiaceae (Tab 3) The number of head cellsin peltate trichomes varied from four to 16 in different Salvia species (Tab 3) Accor-ding to our findings peltate trichomes of S verticillata leaves had a four-celled secretoryhead Although the structure of trichomes should be of taxonomic value and constantfor the species (Uphof and Hummel 1962) this disagreed with the findings of Remenyi(1997a) who reported 6- 8- and 12-celled heads of sessile glandular trichomes Four-celled head had also been recorded for S blepharophylla and S glutinosa (Tab 3)Capitate glandular trichomes of three typeswere recorded for S verticillataTypes I andII with a unicellular stalk and a bi- and unicellular secretory head respectively cor-responded to trichomes described for S blepharophylla S tomentosa and S officinalis(Tab 3)Glandular trichomes of type III of S verticillatawere similar to those describedby Corsi and Bottega (1999) for S officinalis type III and IV Despite the overallsimilarity of trichome structures the combination of a four-celled head on peltatetrichomes together with the occurrence of types I II and III for capitate trichomes hasnot yet been described for another Salvia species suggesting that it might be a criterionfor the identification of S verticillata However information about trichomes is stillmissing for the majority of the wild-growing Serbian Salvia species so that we cannotexclude that other species have the same trichome characteristics as S verticillataAnother taxonomically important finding is that trichome morphology did not differbetween leaves and calyces

Capitate glandular trichomes were more numerous than peltate trichomes Com-pared with S glutinosa S tomentosa and S officinalis (Tab 3) S verticillata seems tohave a lower total number of glandular trichomes on the abaxial epidermis and a highernumber on the adaxial epidermis of leaves However the number of glandular tri-chomes is generally a very variable character which is strongly influenced by externalfactors such as temperature and light intensity (Uphof and Hummel 1962) so thatinterspecific comparisons based on a small number of populations must be consideredwith caution

The yield of the essential oil (040ndash042) permits the assignment of this species tooil-poor representatives of Lamiaceae as the widely accepted borderline between oil-poor and oil-rich representatives of Salvia is 1 (Mathe et al 1992 Senatore and deFusco 1997 Malencic et al 2004) Sesquiterpenes were present in all populationsconsistent with previous reports that oil-poor species of Lamiaceae possess oil rich insesquiterpene compounds (Malencic et al 2004) There were striking differences in theessential oil composition of the three populations Monoterpenes were well reACHTUNGTRENNUNGpresent-ACHTUNGTRENNUNGed in populations 2 and 3 but only present in traces in population 1Hydrocarbonsweremissing in populations 1 and 2 from the North of Serbia while population 3 fromsouthwest mountainous regions was rich in them The number of compounds found inindividual populations (23ndash27) was similar to the number of compounds found in awild-growing population in Iran (Sefidkon and Khajavi 1999) Two dominant compo-nents in Iran (E)-caryophyllene (247) and a-humulene (78) were also dominantcomponents in all three Serbian populations suggesting that these are generally presentin S verticillata Of the two other components that were present in significant amountsin the Iranian population a-muurolene (228) and limonene (89) only a-muu-rolene was recorded in Serbia and only in population 3

166 Lana Krstic Djordje Malencic and Goran Anackov

Both environmental factors (Mathe et al 1992) and genetic differences (Werker etal 1985) have been found to cause variation in oil composition in other species ofLamiaceae The number of populations investigated in this study does not yet allowconclusions about factors determining the essential oil composition of S verticillataHowever our results suggest that low oil content and high intraspecific variability in oilcomposition may restrict the commercial use of wild-growing S verticillata plants

This work was financially supported by theMinistry of Science andEnvironmental Protection of Serbia andMontenegro Grant No 143037 We would like to thank Mr Milos Bokorov University Center for ElectronMicroscopy Novi Sad for his technical assistance and SEM micrographs

References

Bisio A Corallo A Gastaldo P Romussi G Ciarallo G Fontana N de Tommasi N andProfumoP 1999Glandular hairs and secretedmaterial in Salvia blepharophyllaBrandegeeex Epling grown in Italy Ann Bot 83 441 ndash 452

Bosabalidis AM and Kokkini S 1997 Infraspecific variation of leaf anatomy in Origanumvulgare grown wild in Greece Bot J Linn Soc 123 353 ndash 362

Bosabalidis AM 1990 Glandular trichomes in Satureja thymbra leaves Ann Bot 65 71 ndash 78

Tab 3 Non-glandular and glandular trichome characteristics of various Salvia species (adeadaxial epidermis abe abaxial epidermis ndash not determined)

Species Non-glandulartrichomesnr of cells

Peltatetrichomesnr of headcells

Capitatetrichome type

Meannumber ofglandulartrichomesmm2

References

ade abe

S verticillata 3ndash6 4 I II and III 57 53 This studyS verticillata ndash 6 8 and 12 ndash ndash ndash Remenyi (1997a)S aethiops 3 or more 16 ndash ndash ndash Remenyi (1997b)S aurea ndash 6ndash8 I ndash ndash Serrato-Valenti et

al (1997)S blepharo-

phylla1 4ndash5 or more 4 I and II ndash ndash Bisio et al (1999)

S glutinosa 2ndash7 4 I 80 245 Krstic et al (2001)S officinalis 3ndash4 12 I and II 30 63 Venkatachalam et

al (1984)Corsi and Bottega(1999)

2ndash4 III and IV Chakalova et al(1993)

S officinalis faurea

2ndash4 12 48 87 Chakalova et al(1993)

S tomentosa 2ndash4 12 I and II 066 106 Chakalova et al(1993)

plus trichomes with multicellular stalk and unicellular head

Botanica Helvetica 116 2006 167

Chakalova ES Genova EM and Thuy NT 1993 Comparative anatomical investigations onleaves of Salvia officinalis L and S tomentosa Mill Fitologija 46 3 ndash 11

Corsi G and Bottega S 1999 Glandular hairs of Salvia officinalis new data on morphologylocalization and histochemistry in relation to function Ann Bot 84 657 ndash 664

Demissev S and Harley MM 1992 Trichome seed surface and pollen characters in Stachys(Lamioidae Labiatae) in tropical Africa In Harley R M and Reynolds T (eds) Ad-vances in Labiate Science Royal Botanic Gardens Kew 149 ndash 166

Dickison WC 2000 Integrative Plant Anatomy Academic Press San Diego 297 ndash 307Diklic N 1974 Rod Salvia L In Josifovic M (ed) Flora SR Srbije VI SANU Beograd 432 ndash

453Duke SO 1994 Glandular trichomes ndash focal point of chemical and structural interactions Int

J Plant Sci 155 617 ndash 620Gersbach PV Wyllie SG and Sarafis V 2001 A new histochemical method for localization of

the site of monoterpene phenol accumulation in plant secretory structures Ann Bot 88521 ndash 525

Hedge IC 1972 Genus Salvia L In Tutin et al (eds) Flora Europaea Vol 3 CambridgeUniversity Press London p 192

Krstic L Malencic Dj Merkulov Lj Popovic M Adamovic D and Boza P 2001 A con-tribution to themorpho-anatomical study ofwild-growing and cultivated Salvia glutinosaLMed Plant Rep 8 (8) 38 ndash 47 (in Serbian English summary)

Liu J Zapp J and Becker H 1995 Comparative phytochemical investigation of Salvia mil-tiorrhiza and Salvia triloba Planta Med 61 453 ndash 455

Malencic Dj 2001 Biochemical investigations of selected wild-growing Salvia species fromVojvodina province PhD Thesis University of Novi Sad Faculty of Science (In SerbianEnglish summary)

Malencic Dj Couladis M Mimica-Dukic N Popovic M and Boza P 2004 Essential oils ofthree Salvia species from the Pannonian part of Serbia Flavor Fragr J 19 225 ndash 228

Marin P 1996 Orasice i trihome u familiji Lamiaceae Bioloski fakultet BelgradeMathe JrI Olah L Mathe A Miklossy V Bernath J Blunden G Patel A and Mathe I

1992 Changes in the essential oil production of Salvia officinalis under climatic conditionsof the temperate belt Planta Med 58 680 ndash 686

Metcalfe CR and Chalk L 1950 Anatomy of the dicotyledons Vol II Clarendon PressOxford

PharmacopM EuropMenne vol 1 1983 Maisonneuve SA Sainte-RuffineRemMnyi ML 1997a Research on epidermal appendices of Salvia aethiops L Magyar Nccedil-

vMnyanatOmiai SzimpOzium Abstracts Szeged 89RemMnyi ML 1997b Variations in tissue structures on the epidermis of Salvia verticillata L

Magyar NccedilvMnyanatOmiai SzimpOzium Abstracts Szeged 91Sefidkon F and Khajavi MS 1999 Chemical composition of the essential oils of two Salvia

species from Iran Salvia verticillata L and Salvia santolinifolia Boiss Flavor Fragr J 1477 ndash 78

Senatore F and de Fusco R 1997 Essential oils from Salvia spp (Lamiaceae) I Chemicalcomposition of the essential oils from Salvia glutinosa L growing wild in Southern Italy JEssent Oil Res 9 151 ndash 157

Serrato-Valenti G Bisio A Cornara L and Ciarallo G 1997 Structural and histochemicalinvestigation of the glandular trichomes of Salvia aurea L leaves and chemical analysis ofthe essential oil Ann Bot 79 329 ndash 336

Uphof JCT and Hummel K 1962 Plant Hairs Borntraeger BerlinValkama E Salminen J-P Koricheva J and Pihlaja K 2004 Changes in leaf trichomes and

epicuticular flavonoids during leaf development in three birch taxaAnnBot 94 233 ndash 242Venkatachalam KV Kjonaas R and Crotean R 1984 Development and essential oil content

of secretory glands of sage (Salvia officinalis) Plant Physiol 76 148 ndash 150Werker E Putievsky E and Rivid V 1985 The essential oils and glandular hairs in different

chemotypes of Origanum vulgare L Ann Bot 55 793 ndash 801

168 Lana Krstic Djordje Malencic and Goran Anackov

Both environmental factors (Mathe et al 1992) and genetic differences (Werker etal 1985) have been found to cause variation in oil composition in other species ofLamiaceae The number of populations investigated in this study does not yet allowconclusions about factors determining the essential oil composition of S verticillataHowever our results suggest that low oil content and high intraspecific variability in oilcomposition may restrict the commercial use of wild-growing S verticillata plants

This work was financially supported by theMinistry of Science andEnvironmental Protection of Serbia andMontenegro Grant No 143037 We would like to thank Mr Milos Bokorov University Center for ElectronMicroscopy Novi Sad for his technical assistance and SEM micrographs

References

Bisio A Corallo A Gastaldo P Romussi G Ciarallo G Fontana N de Tommasi N andProfumoP 1999Glandular hairs and secretedmaterial in Salvia blepharophyllaBrandegeeex Epling grown in Italy Ann Bot 83 441 ndash 452

Bosabalidis AM and Kokkini S 1997 Infraspecific variation of leaf anatomy in Origanumvulgare grown wild in Greece Bot J Linn Soc 123 353 ndash 362

Bosabalidis AM 1990 Glandular trichomes in Satureja thymbra leaves Ann Bot 65 71 ndash 78

Tab 3 Non-glandular and glandular trichome characteristics of various Salvia species (adeadaxial epidermis abe abaxial epidermis ndash not determined)

Species Non-glandulartrichomesnr of cells

Peltatetrichomesnr of headcells

Capitatetrichome type

Meannumber ofglandulartrichomesmm2

References

ade abe

S verticillata 3ndash6 4 I II and III 57 53 This studyS verticillata ndash 6 8 and 12 ndash ndash ndash Remenyi (1997a)S aethiops 3 or more 16 ndash ndash ndash Remenyi (1997b)S aurea ndash 6ndash8 I ndash ndash Serrato-Valenti et

al (1997)S blepharo-

phylla1 4ndash5 or more 4 I and II ndash ndash Bisio et al (1999)

S glutinosa 2ndash7 4 I 80 245 Krstic et al (2001)S officinalis 3ndash4 12 I and II 30 63 Venkatachalam et

al (1984)Corsi and Bottega(1999)

2ndash4 III and IV Chakalova et al(1993)

S officinalis faurea

2ndash4 12 48 87 Chakalova et al(1993)

S tomentosa 2ndash4 12 I and II 066 106 Chakalova et al(1993)

plus trichomes with multicellular stalk and unicellular head

Botanica Helvetica 116 2006 167

Chakalova ES Genova EM and Thuy NT 1993 Comparative anatomical investigations onleaves of Salvia officinalis L and S tomentosa Mill Fitologija 46 3 ndash 11

Corsi G and Bottega S 1999 Glandular hairs of Salvia officinalis new data on morphologylocalization and histochemistry in relation to function Ann Bot 84 657 ndash 664

Demissev S and Harley MM 1992 Trichome seed surface and pollen characters in Stachys(Lamioidae Labiatae) in tropical Africa In Harley R M and Reynolds T (eds) Ad-vances in Labiate Science Royal Botanic Gardens Kew 149 ndash 166

Dickison WC 2000 Integrative Plant Anatomy Academic Press San Diego 297 ndash 307Diklic N 1974 Rod Salvia L In Josifovic M (ed) Flora SR Srbije VI SANU Beograd 432 ndash

453Duke SO 1994 Glandular trichomes ndash focal point of chemical and structural interactions Int

J Plant Sci 155 617 ndash 620Gersbach PV Wyllie SG and Sarafis V 2001 A new histochemical method for localization of

the site of monoterpene phenol accumulation in plant secretory structures Ann Bot 88521 ndash 525

Hedge IC 1972 Genus Salvia L In Tutin et al (eds) Flora Europaea Vol 3 CambridgeUniversity Press London p 192

Krstic L Malencic Dj Merkulov Lj Popovic M Adamovic D and Boza P 2001 A con-tribution to themorpho-anatomical study ofwild-growing and cultivated Salvia glutinosaLMed Plant Rep 8 (8) 38 ndash 47 (in Serbian English summary)

Liu J Zapp J and Becker H 1995 Comparative phytochemical investigation of Salvia mil-tiorrhiza and Salvia triloba Planta Med 61 453 ndash 455

Malencic Dj 2001 Biochemical investigations of selected wild-growing Salvia species fromVojvodina province PhD Thesis University of Novi Sad Faculty of Science (In SerbianEnglish summary)

Malencic Dj Couladis M Mimica-Dukic N Popovic M and Boza P 2004 Essential oils ofthree Salvia species from the Pannonian part of Serbia Flavor Fragr J 19 225 ndash 228

Marin P 1996 Orasice i trihome u familiji Lamiaceae Bioloski fakultet BelgradeMathe JrI Olah L Mathe A Miklossy V Bernath J Blunden G Patel A and Mathe I

1992 Changes in the essential oil production of Salvia officinalis under climatic conditionsof the temperate belt Planta Med 58 680 ndash 686

Metcalfe CR and Chalk L 1950 Anatomy of the dicotyledons Vol II Clarendon PressOxford

PharmacopM EuropMenne vol 1 1983 Maisonneuve SA Sainte-RuffineRemMnyi ML 1997a Research on epidermal appendices of Salvia aethiops L Magyar Nccedil-

vMnyanatOmiai SzimpOzium Abstracts Szeged 89RemMnyi ML 1997b Variations in tissue structures on the epidermis of Salvia verticillata L

Magyar NccedilvMnyanatOmiai SzimpOzium Abstracts Szeged 91Sefidkon F and Khajavi MS 1999 Chemical composition of the essential oils of two Salvia

species from Iran Salvia verticillata L and Salvia santolinifolia Boiss Flavor Fragr J 1477 ndash 78

Senatore F and de Fusco R 1997 Essential oils from Salvia spp (Lamiaceae) I Chemicalcomposition of the essential oils from Salvia glutinosa L growing wild in Southern Italy JEssent Oil Res 9 151 ndash 157

Serrato-Valenti G Bisio A Cornara L and Ciarallo G 1997 Structural and histochemicalinvestigation of the glandular trichomes of Salvia aurea L leaves and chemical analysis ofthe essential oil Ann Bot 79 329 ndash 336

Uphof JCT and Hummel K 1962 Plant Hairs Borntraeger BerlinValkama E Salminen J-P Koricheva J and Pihlaja K 2004 Changes in leaf trichomes and

epicuticular flavonoids during leaf development in three birch taxaAnnBot 94 233 ndash 242Venkatachalam KV Kjonaas R and Crotean R 1984 Development and essential oil content

of secretory glands of sage (Salvia officinalis) Plant Physiol 76 148 ndash 150Werker E Putievsky E and Rivid V 1985 The essential oils and glandular hairs in different

chemotypes of Origanum vulgare L Ann Bot 55 793 ndash 801

168 Lana Krstic Djordje Malencic and Goran Anackov

Chakalova ES Genova EM and Thuy NT 1993 Comparative anatomical investigations onleaves of Salvia officinalis L and S tomentosa Mill Fitologija 46 3 ndash 11

Corsi G and Bottega S 1999 Glandular hairs of Salvia officinalis new data on morphologylocalization and histochemistry in relation to function Ann Bot 84 657 ndash 664

Demissev S and Harley MM 1992 Trichome seed surface and pollen characters in Stachys(Lamioidae Labiatae) in tropical Africa In Harley R M and Reynolds T (eds) Ad-vances in Labiate Science Royal Botanic Gardens Kew 149 ndash 166

Dickison WC 2000 Integrative Plant Anatomy Academic Press San Diego 297 ndash 307Diklic N 1974 Rod Salvia L In Josifovic M (ed) Flora SR Srbije VI SANU Beograd 432 ndash

453Duke SO 1994 Glandular trichomes ndash focal point of chemical and structural interactions Int

J Plant Sci 155 617 ndash 620Gersbach PV Wyllie SG and Sarafis V 2001 A new histochemical method for localization of

the site of monoterpene phenol accumulation in plant secretory structures Ann Bot 88521 ndash 525

Hedge IC 1972 Genus Salvia L In Tutin et al (eds) Flora Europaea Vol 3 CambridgeUniversity Press London p 192

Krstic L Malencic Dj Merkulov Lj Popovic M Adamovic D and Boza P 2001 A con-tribution to themorpho-anatomical study ofwild-growing and cultivated Salvia glutinosaLMed Plant Rep 8 (8) 38 ndash 47 (in Serbian English summary)

Liu J Zapp J and Becker H 1995 Comparative phytochemical investigation of Salvia mil-tiorrhiza and Salvia triloba Planta Med 61 453 ndash 455

Malencic Dj 2001 Biochemical investigations of selected wild-growing Salvia species fromVojvodina province PhD Thesis University of Novi Sad Faculty of Science (In SerbianEnglish summary)

Malencic Dj Couladis M Mimica-Dukic N Popovic M and Boza P 2004 Essential oils ofthree Salvia species from the Pannonian part of Serbia Flavor Fragr J 19 225 ndash 228

Marin P 1996 Orasice i trihome u familiji Lamiaceae Bioloski fakultet BelgradeMathe JrI Olah L Mathe A Miklossy V Bernath J Blunden G Patel A and Mathe I

1992 Changes in the essential oil production of Salvia officinalis under climatic conditionsof the temperate belt Planta Med 58 680 ndash 686

Metcalfe CR and Chalk L 1950 Anatomy of the dicotyledons Vol II Clarendon PressOxford

PharmacopM EuropMenne vol 1 1983 Maisonneuve SA Sainte-RuffineRemMnyi ML 1997a Research on epidermal appendices of Salvia aethiops L Magyar Nccedil-

vMnyanatOmiai SzimpOzium Abstracts Szeged 89RemMnyi ML 1997b Variations in tissue structures on the epidermis of Salvia verticillata L

Magyar NccedilvMnyanatOmiai SzimpOzium Abstracts Szeged 91Sefidkon F and Khajavi MS 1999 Chemical composition of the essential oils of two Salvia

species from Iran Salvia verticillata L and Salvia santolinifolia Boiss Flavor Fragr J 1477 ndash 78

Senatore F and de Fusco R 1997 Essential oils from Salvia spp (Lamiaceae) I Chemicalcomposition of the essential oils from Salvia glutinosa L growing wild in Southern Italy JEssent Oil Res 9 151 ndash 157

Serrato-Valenti G Bisio A Cornara L and Ciarallo G 1997 Structural and histochemicalinvestigation of the glandular trichomes of Salvia aurea L leaves and chemical analysis ofthe essential oil Ann Bot 79 329 ndash 336

Uphof JCT and Hummel K 1962 Plant Hairs Borntraeger BerlinValkama E Salminen J-P Koricheva J and Pihlaja K 2004 Changes in leaf trichomes and

epicuticular flavonoids during leaf development in three birch taxaAnnBot 94 233 ndash 242Venkatachalam KV Kjonaas R and Crotean R 1984 Development and essential oil content

of secretory glands of sage (Salvia officinalis) Plant Physiol 76 148 ndash 150Werker E Putievsky E and Rivid V 1985 The essential oils and glandular hairs in different

chemotypes of Origanum vulgare L Ann Bot 55 793 ndash 801

168 Lana Krstic Djordje Malencic and Goran Anackov