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Seed Science Reseárch (1995) 5, 161-170 161 )

Ecophysiology ÏÉ germination ßnthe aromatic plants thyme, savory and oregano (Labiatae)

Costas Á. Thanos*, Costas c. Kadis and Flora Skarou

Departmentof Botany,University of Athens,Athens 15784,Greece

,.

, 'É Abstract estabIishment are prevented during the early phase ~ .. . . of the rainy period, which is usuaIIy interrupted by '. EcophysloIogIcaI aspects of seed germInatlon were drought speIIs ßçthe Mediterranean cIimate. investigated ßç the wideIy distributed Med- iterranean-endemic, aromatic plants thyme Keywords: Coridothymus capitatus, Satureja (Coridothymus capitatus), savory (Satureja thymbra, Origanum vu/gare, seed germination, thymbra) and oregano (Origanum vu/gare subsp. essentiaI oiIs, persistent calyx, phytochrome hirtum). Thyme seed germination is Iight indifferent, whereas oregano seeds have an absoIute Iight requirement; their germination can be promoted even by green safeIight ÏÃ far-red Iight. lç savory, a lntroduction portion of the seeds germinates ßç the dark and germination can be either increased ÏÃ decreased Aromatic and medicinal plants constitute a con- by the appropriate iIIumination. ÁÉÉthree species siderableportion óßthe flora and the vegetation óßthe show more germination at a relatively low tem- Mediteuanean Rim. Éç the so-called cradle óß civ- perature range, a Mediterranean characteristic, ilization óßthe Eastem Mediterranean region, in par- with an optimum around 15-20°C. The rate of ger- ticular, aromatic and medicinal plants have been mination is considerabIy higher ßç the tiny seeds collectedfrom the wild and also cultivated for several of thyme and oregano than ßçthe larger seeds of millennia. Theophrastus (4th century B.C.) in his savory. lç the Iatter species, germination is also Enquiry into Plants (HistoriáPlántárum, ÇÑ) compiled dependent õñïç the age of the seeds; oId seeds the accumulated botanical knowledge during Greek germinate to a higher percentage than fresh ones, ClassicalAntiquity; seedgeunination holds an eminent - as aIready observed by Theophrastus, possibIy as position among the vaÞóus aspectsóß cultivation ïß , a resuIt of the voIatiIization of the essentiaI oils pot-herbs.Á considerablenumber óß aromatic plants é É. present ïç the nutlet coat. Seeds of thyme, savory are described (e.g. ÇÑ 6.2), including the three taxa ;! and oregano are dispersed within the persistent investigated in the present study. Á whole book (ÇÑ - fruiting calyces and the seeds eventuaIIy germinate 7) is devoted to pot-herbs and similar wild-growing within it. Essential oiIs ßçthe calyx strongly inhibit plants. germination of the encIosed seeds; germination is Thyme (Coridothymuscápitátus) is a dwarf calcicole much suppressed ßçthyme and to a lesser extent shrub and an important element óß the xeromorphic ßç savory and oregano. This diaspore dormancy 'phryganic vegetation. It is endemic ßç the Med- caused by essential oiIs apparently is overcome iteuanean Rim (including Portugal). It grows near the under natural conditions by leaching of the inhibi- sea (0-700 m altitude) and, as indicated by Theo- tors with rainwater. It is suggested that this dor- phrastus (ÇÑ 6.2.4),'the ÑecuéiaÞtyabout thyme is that mancy operates as an adaptation strategy that it cannotgrow ßça region without a seabreeze'. Savory delays germination by acting as a rain gauge. lç this (Sáturejáthymbrá) is a dwarf phryganic shrub endemic way, seed germination and subsequent seedling to the eastem Mediteuanean (Sardinia to Israel). Oregano(Origánum vulgáre subsp. hirtum, formerly Ï. herácleoticum)is a woody rhizomatousperennial. It also "CïðeSÑïndence is a Mediteuanean endemic, but with a Balkan dis- 162 C.Á. Thanosetá/.

tribution, inc1udingAnatolia a,ndCyprus. Thesethree (2nd 10t,mean weight of 20 nut1ets:6.67:t 0.09mg, n = p1antsare mainly used as oreganospice and as a high- 51) and 0.41 mg (3rd 10t; mean weight of 20 nut1ets: content source of essentia1oi1s (Philianos et aÉ.,1982; 8.21:t 0.06 mg, n = 50); and ï. vulgare subsp. hirtum, Kokkini and Vokou, 1989;Bosabalidis, 1990; Kanias and 61.4 ìg (mean weight of 20 nut1ets:1.23:t 0.02mg, n = Loukis, 1992;Kokkini et al., 1991). 50). The percentage of unsound seeds (revea1edby Severa1specific accounts of Theophrastusconcern dissectionunder a stereomicroscope)in S. thymbrawas seedgermination of aromaticp1ants. For examp1e,there 62,26 and ca.10% in eachof the three 10ts,respective1y, is a commentïç the 10wgermination rate ('savory and but only about 10%in C. capitatusand ï. vulgaresubsp. marjoram take more than thirty days to germinate' ÇÑ hirtum. The number of nut1ets per ca1yxwas deter- 7.1.3)as well as its enhancementin the older seedsof mined under a stereomicroscope.The thymo1 and savory ('some p1antsgerminate quicker from old seed, carvacro1chemotypes of thyme showed a remarkab1e as . . . savory, marjoram . . ., unless the fresh seeds have similaÞty (means of 1.8:t 0.2 and 1.6:t 0.2 seeds per been previous1y soaked in water or milk' ÇÑ 7.1.6). ca1yx,n=50, respective1y);mean numbers for savory Furthermore, in contrast to savory, which produces (3rd 10t) and oregano were 2.4:t 0.1 (ç = 290) and sizab1e,germinab1e seed (ÇÑ 6.2.3), thyme is pro- 2.1:t 0.1 (ç = 100),respective1y. claimed to generate inconspicuous seeds (which 'cannot be discerned visually' ÇÑ 1.5.3), but are Germination conditions . 'somehow mixed up with the flower for when the flower is sown p1antscome up from it' (ÇÑ 6.2.3). Seed germination was carÞed out with nut1ets Apart from certain generalru1es for 1aboratoryger- (hereafter called seeds) and persistent ca1yces ,,- mination (e.g.AOSA, 1981;E1lis et al., 1985;ISTA, 1993), (containing up to 4 nut1etseach). Seeds were extracted information is scarce concerning aromatic p1ants in from their ca1ycesmanually by carefu1rubbing. Five general and the three taxa mentioned above in par- rep1icatesof 20 or 25 seedsor ca1yceswere sown ïç ticular. Éç the context of the potentia1use of aromatic moistened fi1ter paper discs in Petri dishes. The cÞ- p1antsas a1ternativecrops in margina11ands,the eco- teÞïn of germination was radic1eprotrusion, and after physio1ogy of seed germination was investigated in eachcount germinated seedswere discarded.The tests thyme, savory and oregano. were terminated when çï additiona1seeds germinated. Á ca1yxwas scored 'germinated' (and consequent1y discarded) when one or more of the seedsin it had Materials and methods germinated. Tsois the time neededfor 50% of the fina1 germination va1ue,and is ca1cu1atedby 1inearinterp- P/ant materia/ olation from the two germination va1uesc1osest to median germination. At the end of each e×ÑeÞment Seedswere collectedfrom the wild, most1yin Crete. ungerminated seedswere dissectedand inspectedfor Coridothymuscapitatus (é.) Reichenb. fi1. (thyme); a soundness under a stereomicroscope.Consequent1y, carvacrï1-Þchchemotype collected at ÔheÞsï Gorge, the data have beencouected for sound seeds.In exper- Chania in August 1993was used in most experiments iments ïç the ro1eof the ca1yx,a number of ca1yces while a second10t, from a thymï1-Þch popu1ation,was were p1acedin the centre of the dish while the seeds collected at Agia ÉÞçß,Chania, in September1993 and were sown a1onga circ1e,so that the distance of each was used in the e×ÑeÞmentsreported in Figure 8. Sat- seedfrom the centra1source of essentia1oils was about . urejathymbra É. (savory);three 10tscollected at Masom, 2 cm. Simu1ationof rain-mediated 1eachingof essentia1 Ka1ymnosIs1and in Ju1y1988, at Mournies, Chania in oils was cauied out with 100ca1yces immersed in dis- September1991 and at Mouzouras, Chania in August til1ed water in a 0.5-1beaker. The water was slow1y 1993,respective1y. Origanum vulgareÉ. subsp.hirtum stiued by a magneticstiuer, at 15°Cand under con- , (Link) Ietswaart (oregano)was collected at Mournies, tinuous white 1ight. Chania in October1992. Nomenc1ature follows Greuter For germination e×ÑeÞmentsin darkness,seeds or et al. (1986)and dßstÞbutiïn data are based ïç both ca1yceswere incubated within 1ight-proof,meta1 con- Flora Europaea (Tutin et al., 1972)and Med-Checklist tainers in cabinets(Mode1 ÂÊ 5060ÅÉ, W.C. Heraeus (Greuter et aÉ.,1986). The two thyme chemotypeshave GmbH, Germany) maintained at constant tem- been identified by colleagues at the Mediteuanean peratures. Seed germination experiments under Agronomic Institute of Chania, Crete (Ç. Kargio1aki vaÞïus light regimeswere performed in temperature- and Ì. Skou1a). and light-programmab1egrowth benches(mode1 GB48, Seedweight was as follows: C. capitatus,0.20 mg Conviron, Canada)equipped with a 1ampcanopy of 48 (first 10t;mean weight of 20 nut1ets:3.94:t 0.03mg, n = incandescentbu1bs (Sy1vania 50Á19, 50 W, 227 V) and 50)and 0.23mg (second10t; mean weight of 20 nut1ets: 10 fluorescent tubes (Sy1vania Coo1 White 4.68:t 0.10mg, n = 20);S. thymbra, 0.61 mg (1st10t, mean FR96TI2/CW /VHO-235/1). weight of 25 nut1ets:15.28:t 0.20 mg, n = 25), 0.33 mg White 1ight(W) had a valueof æ(660/730 nm photon Ecophysiologyof germinationin aromaticplants 163

ratio) equal to 1.119,establishing a calculated phy- 100 't'!c;IX tochrome photostationary state ratio (ö= [Pfr]/[Ptot]);! ~ ïß 0.641. The total photon flux densif); in the visible range (400-800 nm) was 103.0 ìmïl m - s -É.U sing blue 80 and red plexiglas filters, four types ïß light regimes ~ were established: red (R), blue/far-red (Â) and two c- types ïß 'canopy simulating' far-red Þch light (FRÉ and ~ 60 FR 11).For each ïß these ßïáé light sources,the values .S ïß æ, Ö and total flux density (ìmïl m-2 S-I), respect- ~ ively,were the fo11owing;R: 1.081,0.593 and 45.2;Â: ~ 40 0.001,0.095and 20.9;FR É:0.001, 0.062 and 13.6;FR 11: ~ 0.229,0.320and 20.7.The spectral dßstÞbutiïç curves for the above light sources are presented elsewhere 20 (Thanos and Doussi, 1995). Apart from a few cases where complete darkness is explicitly stated, most manipulations ïß imbibed seeds and calyces were 00 5 10 15 20 35 . camed out under a dim, green safelight (ù and total Temperature,C flux density were 0.322 and 0.8 ìmïl m-2 S-I, respectively). This dim gre~n light ~as obtained with Figure 1. Germination ïß Coridothymuscápitátus seeds in the . one green fluorescent tube filtered Wlth layers ïß green dark as a function ïß temperature.Circled numbers are Ô50in and orange plexiglas. Nearly 39% ïß the total amount days. Final germination values are shown by solid squares ïß photons were emitted in the 'green' band 500-600 connectedwith a straight line. apen squaresrepresent sub- nm and 54% ßç the 'far-red' 700-800 nm, and ïçlÕ 6% sequent germination for the ungerminated seed samples in the 'red' 600-700 nm with less than 1% in the 'blue' (samesamples shown ßçsolid squares)after eventualtransfer 400-500 nm. Photon flux densities were measured with to 15°C,under continuouswhite light. Vertical bars represent a spectroradiometer (ISCO SR, USA); calculations ïß % SE (standarderror ïß the mean)values. flux densitiesand valuesïß æand Ö weremade with a previously described software application (Thanos et light at 15°C. Germination was completed within 20 áÉ.,1994). days, in a11four cases,with Tsovalues between 8 and 11days. The minute seedsïß oregano did not germinate ßç Results darkness at any temperature but did so after illumi- .. nation with a green safelight when effects ïß tem- The effect ïß temperature ïç final, dark gernunation perature were revealed (Table 1). percentageïß thyme seeds is. illustrated in !igure .1. When thyme, savory and oregano seeds were Alm~st a.11ïß the seeds germmated a~ 5-20 C, w~e imbibed under various continuous light regimes (Fig. gernun~t1onwas suI;1pre.ssedabove 20 C. O~ the bas1S 3), different responseswere obtained. Éç thyme, ger- ïß the time to genmnation (Tsovalues), optimal tem- mination was particularly fast in a11cases and apart perature was 15.oCwhere. germination was completed from a slight deviation in the time to germinati~n (Tso . ßçone week while at 5°C rt took more than a month for the seeds to germinate. Seeds imbibing for a relatively long ÑeÞïd at an inhibitory temperature could fu11y Table 1. Interactlonbetween temperature and greensafelight germinate upon subsequent transfer to the optimal ïç germination ïß Origánum vu/gáre subsp. hirtum seeds. . temperature; nevertheless, seeds previously at 25°C Seedswere imbibed in darknessand were checkedfor ger- completed their germination within 7 days while those mination every other day under a dim, green safelight (Ô50 pretreated at 30°C required more than 2 weeks. values correspondto the time required for half ïß final ger- Dark germination as a function ïß temperature ßç mination; SE:standard eðïr ïß the mean)

Ô savory( seeds. (Fig. 2) was related to their age.The oldest (OC) F. 1G . ti Ô seeds Flg. 2Á) genmnat. ed optima. 11 Õ at 5-25°C, w hile .emperature. ma (ï/ßermma SE) ïç (d 50) those ïß intermediate age (Fig. 2Â) showed a con- 0% ays siderable decreaseat both ends ïß the temperature 5 30.0% 5.0 35 range,most markedly at 25°C.Freshly harve~tedseeds 10 39.0% 11.1 17 (Fig. 2C) were considerably ~lower to gernun~te than 15 72.0% 5.6 10 the other two at 5-10°C, while they were partlcularly 20 72.0%5.2 8 dormant at higher te~peratu~s.. ~evertheless, seeds 25 33.1% 5.1 15 imbibing for long penods at inhlbltory temperatu~s 30 0- could sti1lbe induced to germinateby continuouswhite 1 . ;",~.."

164 C. Á. Thanoset á/.

100 100 Á Á

80 80

~ C ~ 260 c'60 éó ï .-c +Ïéó Å c ~ ¸ DI 40 óé~ 40 -éó (!J c ii:

20 20

Ï Ï Ï 5 10 15 20 25 30 Ï 5 10 15 20 Temperature, C Tlme, d

100 100 ~ Â Â

80 80 ... ~ - ,ï C ï' .2 60 C 60 10 ï .-C +Ïéó Å c ~ ¸ DI 40 ~ 40 óé -éó (!J c ii: @

20 20

Ï 5 10 15 20 25 30 Ï 10 20 30 Temperature, C Tlme, d

100 100 C C ~ ~ 80 80 ~ É ~ É "c ~ 9. 60 ïC 60 -éó .- .-c -éó . Å ..5 ~ Å DI 40 ~ 40 m (!J c ii: . 20 20

Ï Ï 5 10 15 20 25 30 35 Ï 2 4 6 8 10 Temperature, C Tlme, d Figure 2. Final germination percentages ïß Satureja thymbra Figure 3. Time course ïß seed germination at 15°C and under seeds ßç the darkas a function ïß temperature. Graphs Á, Â, vmous continuous light regimes (White light, ï; Red: 181; and C refer to seedlots collectedßç 1988,1991 and 1993, Blue/far-red:..; Far-RedÉ: ÷; Far-Red11: ×; Darkness:.). Á: respectively,tested for germination 4 years, 1 year and 3 Coridothymuscapitatus; Â: Saturejathymbra; and C: Origanum months after harvest,respectively. Circled numbers are Tsoßç vulgaresubsp. hirtum. days. apen squares(ßç C) represent final germination per- centagesfor the ungerminated seed samples(same samples shown ßç solid squares)after their transfer to 15°C, under continuouswhite light. Verticalbars represent:É: SE (standard error ïß the mean)values. Ecophysiologyïß gerrnination in aromatic plants 165

 Á

C .. 1mm Ï " ; :'::

Figure The seedand the dispersal unit (dry fruiting calyx) ïß Coridothymuscápitátus (Á), Sáturejáthymbrá (Â) and Origánum -é vu/gáre4.subsp. hirtum (C). The fruiting calyx ïß thyme (lateral view) is shown closed(upper) and opened (lower). Little 'bubbles' ïç the calyx surfaces(as well as the tiny ones at the ÑïsteÞïr end ïß savory seed)portray the essentialoil-containing heads ïß the secretoryglands.

values ranged between 4 and 6 days), it was completed slightly increased germination obtained under white within 10 days. Nevertheless, çó statistically significant illumination over complete darkness might be attÞ- differences were observed in the final germination buted to a partial removal óß essential oi1s from the values among the six light treatments (Fig. 3Á). lç exposed dishes (while those in darkness were main- savory (Fig. 3Â), lightwas particularly importantto ger- tained within tightly closed metalcontainers). Note also mination:whiteandredlightpromoted,whileblue/far- that the percentages óß 'germinating' calyces were red and both far-red regimes inhibited germination. clearly overestimated ßçcomparison with seedsbecause Both blue/ far-red and far-red É(intense far-red) supp- each calyx was considered as one although it contained ressed germination entirely; however when these seeds nearly two seeds óç average. This was equally true ßç and those imbibing ßç darkness were subsequently the following cases for savory and oregano. Á notable traçsfeðed to continuous white light (data not presen- de1ayóß germination, as well as a considerable decrease ted), germination reached about 80% in 14 days. lç ore- óß final germination was al~D obtained in savory (Fig. gano seeds (Fig. 3C), darkness was entirely inhibitory, 5Â) and oregano (Fig. 5C). Furthermore, a particularly while fu1l induction óßgermination was obtained by red strong induction óß calyx 'germination' by white light and white light. Á gradually increasing amount óßger- was observed ßç both species, the germination in dark . mination (ßç positive relationship to the Ö values óßthe contro1s being virtually fully inhibited. This differential light regimes) resulted under far-red É,blue/ far-red and response was similar to the differences obtained in iso- far-red 11.Rate óß oregano seed germination was high, lated seeds óßthese species. witha Tsoaround5days,irrespectiveoflightconditions. When thyme seeds were imbibed ßç the presence óß When ungerminated seeds were eventually traçsfeðed a gradually increasing number óßthyme calyces within to continuous white light (data not presented), full pro- the saôçe dish, a statistically significant delay óß seed motion óßgermination óccuðed within 15 days. germination took place (Fig. 6). Although çó inhibition Dispersal óß seeds ßç thyme, savory and oregano óß final germination amount was observed with õñ to takes place within the persistent fruiting calyces, the 20 calyces, the higher the number óßcalyces present the opening óß which is brought about by an increase in slower the germination rate óßnearby seeds; 20 calyces moisture (hygrochasy). Nevertheless, the seeds remain caused a delay óß almost 5 days, and even one calyx within the calyx where they eventually germinate (Fig. per dish produced a certain effect. 4). The young roots and stems óß the seedlings may Ôó simulate the leaching action óß rain, calyces óß either emerge through the 'collar' óß the calyx, or ßç thyme, savory and oregano were continuously sooed most casespierce it at vaÞóus points. Éç sharp contrast ßç distilled water at 15°C under continuous white light. to isolated thyme seeds, germination within the calyx At the end óß the treatment, the calyces were placed in was sporadically spread over a very long ÑeÞód and it dishes and left to germinate under the same conditions reached óçÉÕ a very low final amount (Fig. 5Á); the óß temperature and light. Even 1 day óß washing can 166 C. Á. Thanoset á/.

100 100 Á

80 80

~ c' 60 ~ g C 60 céó .-ï ¸ Ö óé~ 40 .-c CI i 40 " 20 20

Ï 10 20 30 40 50 60 Tlme,d Ï Ï 5 10 15 20 (

Tlme,d 80 Â Figure6. TlInecourse ïß germinationfor Coridothymuscap- itatus seedsat 15°Cunder continuouswhite light, in the pres- , ence ïß 1 (0),5 (JEI),10 (÷) and 20 (Á) thyme calyces per Petri 60 dish. Controls (çï calyces within the dishes): ..

~ c ï ~ 40 increasedramatically both the rate and the final per- ¸ centageïß germination in thyme and savory (Figs 7Á ~ and 7Â, respectively).' Éç oregano, germination enhancementwas much less;washing the calycesfor 3 20 days decreasedtime to germination only slightly (Fig. 7C). When seedsïß the two carvacrol- and thymol-rich chemotypesïß thyme were reciprocally incubated in Ï 10 20 ÔÉ30 d 40 50 60 the presenceïß calycesïß either ïß the two chemotypes, me, fuIl germination was attained ßçaIl four combinations ïß seedsand calyces(Fig. 8). Moreover, for eachone ïß 100 the two seed lots used, a set ïß two, almost identical, curves was obtained (Fig. 8).

80

.. ~ 60 Discussion ï ~ Dark germination as a function ïß temperature in Å 40 isolated thyme seeds seems to be typicaIly Med- é";' itenanean (Thompson, 1970;Thanos, 1993).VirtuaIly germination ïß aIl seedstook place at relatively low 20 temperatures (5-20°C), with an optimum at 15°C. Although the rate ïß germination was somewhat slower than those previously reported (Argyris, 1977; 20 30 Vokou, 1983), it was still relatively rapid for Med- Tlme,d 40 itenanean plants (Thanos, unpublished). Continuous Figure5. TlInecourse ïß germinationfor isolatedseeds (., Ï) ~umination. under vari°:Us~ght regim.esrevealed çï and seedswithin calyces(Á, ÷) at 15°Cand under continuous dxfferences ~ the germmation behaVlour ïß thyme white light (ï, ÷) or darkness(., Á). Á: Coridothymuscap- seeds;thus, rt may be concluded that thyme seedsare itatus;Â: Saturejathymbra; and C: Origanumvulgare subsp. totaIly indifferent to light, unexpectedßç such a tiny- hirtum. Seedsand calycesirnbibing in darknesswere seededspecies. inspectedunder dirn safelight. Savory seed germinability as a function ïß tem- Ecophysiologyof germination in aromatic plants 167

100 100 Á

80 80

~ ïc- 60 ï~ ~ C 60 c ï ¸ ;

40 ca ~ óé C (:J ¸ ~ 40 " 20

20 Ï Ï 10 20 30 40 50 .. Tlme,d Ï Ï 10 15 80 Tlme, d  Figure 8. Time course ïß seed gerrnination in two chemotypes ïß Coridothymus capitátus (thymol-type: Á, ×; carvacrol-type: " ï) at 15°C, under continuous white light in the presence 60 ïß 10 calyces per dish ïß each chemotype (thymïl-Þch calyces: Á, ï; carvacrïl-Þch calyces: ×, .). ~ r:: .2 ~ 4 perature seems to depend upon the age ïß the seed, as ~ Theophrastus had already noticed two millennia ago. ~ Éç particu1ar, freshly harvested seeds were deeply 20 dormant at higher temperatures, but they cïþd be induced to germinate by continuous white light. More- over, the relatively slow germination' rates also depended upon the age ïß the seeds. Light was import- 00 10 20 30 40 50 ant for savory seed germination. Under white or red

TIme, d light, which establish calculated Ö values ïß 0.6, approximately 70% germination occuued, whereas there was only 20% germination in seeds treated with 80 C FRII (Ö= 0.32). Since germination ßç darkness was about 40% Ö in 'dark' seeds must be between 0.32 and 0.6. Light which establishes Ö below 0.01 (similar to that . 60 under dense leaf canopies) ßä understandably inhibi- tory ïß germination ( and FRI ßç Fig. 3Â). The partial ~ dormancy in fresh savory seeds may be caused by .g essential oils, which are either present ïç the nutlet .s 40 coats or are simply diffused within the seed from the ~ suuounding calyx tissues. Éç a micromorphological (:J study ïß certain representatives ïß the tribe Saturejeae, 20 Husain et áÉ (1990) found both trichomes and sessile ïßé glands at the posterior end ïß the nutlets ïß Satureja horvatii and S. pillosa (but not in S. montana, S. subspicata Ï and S. cuneifolia). Á similar, preliminary observation Ï 5 10 15 20 25 30 has been made ïç S. thymbra under a stereomicroscope TIme, d (cf. Fig. 4Â) and this may explain the statement by Figure 7. Time course ïß gerrnination ïß Coridothymus cáp- Theop~ras~ that older seeds germinate better: the itátus (Á) Sáturejá thymbrá (Â) and Origánum vulgáre subsp. es~eçé.ßaÉ 011s .present ïç the. ~utlet coat suppress ger- hirtum (C) seeds within calyces at 15°C under continuous Inlnation until they are volatilized. , white light. Calyces previously soaked in distilled water (for Germination ïß the minute seeds ïß oregano was 1, 1 and 3 days, respectively): Á; controls: .. clearly light-requiring. Even a few short exposures to a 168 C. Á. Thanoset á/. dim, green safelight strongly promoted germination, enhancementïß germination by a rain-simulating treat- suggesting a very low threshold level ïß photo- ment ïß calyces.Nevertheless, actual field observation sensitivity. Á similar response has previously been and documentationneed to be performed beforereach- reported ßç several cases,e.g. in Stellariamedia seeds ing a final conclusion.The conceptïß a rain gaugewas (Baskin and Baskin, 1979).Full induction ïß oregano initially postu1atedby Went (1955)for seedsïß desert seedgermination was obtained by red and white light, annuals,which germinatewhen eno~ghrain hasfallen and even the far-red light partially promoted ger- to leach out the inhibitors contained in the seedcover- mination. Thus, in oreganoseeds imbibing under com- ings. However, little evidencefor breaking ïß seeddor- pletedarkness the establishedÖ is presumablybelow mancy by leachingïß chemicalinhibitors has been 0.06 (the level established by far-red É). The rate ïß produced ßçthe meantime,and thisinteresting hypoth- germination ïß illuminated oreganoseeds was remark- esishas not been proved under field conditions. ably high, similar to that ïß thyme seeds. Seedlingsïß thyme are minute and very difficult Origanum vulgare is a taxonomically difficult and to count under natural conditions. However, Ar~ variable specieswith severalsubspecies (Kokkini et al., (1977)found that emergencetakes place a1mostexclus- 1991).Previous experiments ïç seed germination ïß ively during November and December,with a certain oregano subspecies(unspecified but assumed to be delay ßç respectto the onset ïß the rainy season.The different to the one used in the present study) have number ïß seedlings recorded was much lower than reached contradictory conclusions. Thus, oregano the estimatedseed density. It still hasnot beenresolved seedswere believed to germinate equally well ßçdark- as to whether limited seedling emergenceshould be ness or under white light (Macchia et al., 1983;Grime attributed to high mortality ïß seedlings or to an . et aÉ.,1988). Éç addition, only a partial suppressionïß enforced dormancy. Simi1arly to its eastem Med- germination was obtained when seedsimbibed under iterraneancounterpart, Coridothymuscapitatus, postfire a layer ïß leaves(Silvertown, 1980)or bryophytes (van recolonization by Thymusvulgaris takes place through Tooren,1990). However, ßçagreement with the results both resprouting and reseeding(Belhassen et al., 1987). ïß the present study, when experiments were per- Furthermore, seedsïß Ô. vulgaris can withstand tem- formed ßçcomplete darkness, germination was almost peratures up to 100°C, although they are not hard- nil (Pons, 1991a).Á red light pulse induced full ger- seeded. Therefore, it might be. worth testing the mination, and even a far-red one promoted ger- possibility that thyme seedsare able to form either a mination to around 40%. Upon successive transient or a short-lived persistentsoil seedbank, as a illuminations, the final irradiation determinedthe level result ïß their tiny size and presenceïß essentialoils. ïß germination, a proof for phytochrome mediation One noteworthy characteristic ïß certain types ïß (Pons,1991a,b). Mediterranean vegetation (chaparral and coastalsage Éç neither ïß the three speciesused in the present in Califomia, phrygana ßçEast Mediterranean) is either study, was any secondary dormancy found to be the depauperate herbaceous vegetation under the imposed by light or high temperatures. Absence ïß chaparral canopy (McPhersonand Muller, 1969)or the secondarydormancy (photodormancy) induction was bare zonesaround the dominant, aromatic shrubs,e.g. noted ßçsavory and oreganoseeds, which germinated Salvia leucophylla,Artemisia californica(Muller et al., promptly under favourable light conditions and irres- 1964)and Coridothymuscapitatus (Vokou, 1983;Katz et pective ïß a previous adverse light pretreatment; ßç al., 1987),respectively. This scarcity ïß vegetation has thyme seedsçï thermodormancywas induced. been attributed, inter alia, to toxins produced by the Seeddispersal in thyme, savory and oreganotakes leaves ïß the establishedmature plants, which in this place within the persistent fruiting calyces (Stopp, way prevent new germination and growth. Allelopathy 1952), the opening ïß which is brought about by was also suggested ßç the case ïß the essential oils increasedmoisture. Nevertheless,despite opening ïß produced by aromatict phryganic shrubs (e.g. Vokou, .. the calyx the seedsremain inside and eventually ger- 1983;Katz et aÉ.,1987; Friedman, 1995).óç the other minate within it (Thanos, 1993).óç the other hand, hand, allelopathy, or chemical inhibition by the shrub the presenceïß the essential oils ßç the calyx tissues canopy,was consideredan unlikely explanation ïß veg- strongly inhibits germination ïß the enclosedseeds, as etation suppression(Keeley and Keeley,1989). Despite shown in the present work and by deductions from the voluminous laboratory evidence,the ecologicalsig- numerous previous reports (e.g.Vokou and Margaris, nificance ïß allelochemicals is still strongly ques- 1986). Seed germination within the calyx was dra- tionable (e.g.Friedman, 1995). matically suppressedßç thyme and to a lesserextent in The major constituent ïß the essentialïil in all three savory and oregano.It was, moreover, spread over a plants studied ßçthe presentwork is carvacrol(Kokkini very long period. Therefore, this diasporedormancy and Vokou, 1989), although several cases ïß popu- causedby the essentialïil is suggestedto be relieved lations (or chemotypes)rich ßç thymol (the isomer ïß under natural conditions by leaching with rainwater. carvacrol) have been reported. The effects ïß the indi- This hypothesis is supported by the spectacular vidual constituentsïß thyme essentialïßl (thymol was Ecophysiologyof germination ßçaromatic plants 169

not inc1uded)have been previous1ytested and carva- References cro1was found among the most inhibitory substances (Vokou,1983), The experimenta1protoco1 ïß the present ÁrgyÞs,J.P, (1977) Se~d ecol?gy ï! somephryganic species (ßç study was adopted ßç order to de1ay (instead ïß inhibit) Greek), Ph,D, Thesls, t!mverslty ïß Athens, Greece, germination and ßç this way to revea1 any differences ÁÏ5Á (1981)Ru1es for testing seeds,Journal ï[ SeedTechnology

that lnlg' ht eX1S' t m. seed ger1nlna' ti' ïç beh aVlour. and/ or 6,1-125, ' nh' b' ßß f h ' 1 il b h Âaski ç, J.Ì . and Âaskin , C..C (1979)Ñromoti ïç ï f ger- 1 1 ltory e ect ï t e essentia ï s, etween t e carva- mination ïß Ste//ariamedia seeds by light from a greensafe cro1 and thymo1 chemotypes, The results obtained lamp, New Phytologist82, 381-383, revea~edé:é~ inter~ch~~otype differences in either seed Belhassen, Å., Pomente, D., Trabaud, L. and Goyon, Ñ.Ç. germmability or mhibltory effect ïß the ca1yces,Thus, (1987)Recolonisation apres incendie chez Thymus vulgaris it may be conc1uded that thymo1 and carvacro1 isomers (L,): resistancedes graines aux temperatureselevees, Acta are equa11yeffective ßç thyme seed autopathy, Á simi1ar Oecologica,Oecologia Plantarum 8, 135-141, conc1usionwas recent1yreached for Thymusvulgáris; Bosabalidis,Á.Ì. (1990)Glandu1ar trichomes ßç Satureja thymo1and carvacro1(either in pure form or in crushed .thymbra leaves, Annals ï[ Botany65, 71-78, 1eavesïß the cïðeSÑïçdßçg chemotypes)were equally EIhs,R.H., Hong, T.D. and Roberts,Å.Ç. (1985) Handbook ï[ ' ' seed technology pendium O 1' nh 1.b 1.t ory ïç seed germma ti.ïç ï f bï th Ârách ÕÑÏd lum " [or'" genebanks,Volume 11, Com . f É , ,. specific germmatlon m[ormatlon and test recommendatlons. phoenlcoldesand thyme ltse1f (Tarayre et áÉ" 1995). Rome International Board f É hib . , t G eti R f d " b . , l ' or Plaç en c esources, ç éÉÉïç ï see germmation Õ lts own essentia FÞedman, J. (1995) AlIelopath}\ autotoxicit}\ and ger- ïßéin Coridothymuscápitátus is an examp1eïß autopathy mination,ññ 629-644ßç Kigel, 1, and Galili, G, (Eds)Seed (Vokou, 1983)and not autotoxicity (as refeðÏÏ to by developmentand germination, New York, Marcel Dekker Friedman, 1995), since çï toxic effect remains after Inc, remova1 ïß the essentia1oils. This autopathic effect has Greuter, W., Burdet, Ç.Ì. and Long, G. (Eds) (1986)Med- been suggested to be a mechanism ïß se1f- regu1ating Chec,klist,Vo~. 3. Genev~,Editions des Conservatoire et the density ïß thyme p1ants in the fie1d (Vokou, 1992). . Jardm Botamquede la ville de Geneve, , Since the ca1ycesare quite rich in essentia1 oils known Gnme, J.P.,Hodgson, ~.G. and Hunt, R. (1988)~~mparat~ve to l' nhib ' t both eï.; ti' ç d d. 1 th th plant ecology:á functlonal approachto commonBntlsh specles, 1 g ...una Ï an ra lC e grow, e L d U ' d ' hi h ' 1 ( f ïç ïç, nwm Çyman, 1 f d ' í Ispersa ï see s wlt ç t elr ca yces a act not Hus~;n 5 Æ Mann. D D 5é,lé.' C Q . Ì d Ñ t ., " ~, . ., ,C.. c,., alser, . an e coVlC appr~clat,edby ~rgyrlS, 19~ and Vokou, 1~83)?e1ays Â. (1990)Á micromorphologicalstudy ïß some rep- ger1nlnationunti1 the 1eachingïß the essentia1011s. We resentativegenera ßç the tribe Saturejeae(Lamiaceae) , suggest that such a device is an eco10gica1adaptation BotanicalJournal ï[ theLinnean Society 103, 59-80, to the unpredictab1e rainfall pattern during the ear1y 15ÔÁ (1993),International ru1es for seed testing, Rules & part ïß the rainy period in the Ìedßteðaçeaç c1imate. Annexes1993. Seed Science and Technology,Supp/ement 21, It ensuresthe de1ayïß germination until well within 1-288, the wet season.Such a de1ayis a common trait among Kanias,~.D. ,andLoukis, Á. (19~2~Statistic~ analysis ïß Ìedßteðaçeaç p1antsand is illustrated by 10w ger- essentialïél percentagecomposltion ïß Cortdothymuscap-

' Essential ' t ' t hi h b tt b t d t th itatus Reichb, f. and Satureja thymbra L. Journal ï[ lnlna 10çra es,w c may e a n u e ï e proper- 0 ' , f 'th h d h b Th f ' é1 Researc,h 4 577- 584' ti~s ï el er t e see . co~t or t e em ryo, ere ore~m Katz, D.A., 5neh, Â. and FÞedmaç,J. (1987)The allelopathic this ~ay seed germmation an? subsequent seed1mg potential ïß Coridothymuscapitatus L, (Labiatae), Pre- estab11Shmentare secured agamst the dry spe11sfre- Iiminary studiesïç the roles ïß the shrub ßçthe inhibition . quent1yencountered during the ear1yphase ïß the rainy ïß annuals germination and/ or to promote alle- period in the Ìedßteðaçeaç climate, Recent results ïç lopathically active actinomycetes, Plant and Soil 98, Thymus vulgáris support this postulation: the observed 53-66, waning ïß the inhibitory effect ïß essentia1 oi1s over Kee~ey,J.E. and Keeley, 5.C. (1989).ÁÉÉeÉÏÑathÕ and the fire- time would imp1y that if such an effect really occurs in md~c~ herb cycle, ññ 6?-72 m Kee,ley,S,C, (Ed,) The the fie1dit wou1d only be ïß a temporary characterand Californta chaparral, Paradtgms reexammed, Los Angeles, thus represent a 'temporary inhibition' adaptation to Nat;ural History Museum ïß Los Angeles COUllty, Science

h Ì d ' 1. (11 É ) Senes Íï, 34, t e e lterraneanc lInate arayreet á º 1995, Kokkini, 5. and Vokou,D. (1989)CarvacroÉ-Þch plants ßç Greece,Flavour and Fragrance Journal4, 1-7, Kokkini, 5., Vokou, D. and Karousou, R. (1991) Mor- phological and chemicalvaÞatiïç ïß Origanumvulgare L, Acknowledgements ßçGreece, Botanica Chronica 10,337-346, McPherson,J.K. and Muller, C.H. (1969)AlIelopathic effects The present work was ful1y supported through a ïß Adenostomafa~cicu/atum, 'chamise', ßç the California European õçßïç funded researchproject (Directorate chaÑaðal,Ecologtcal Monographs 39,177-198, General VÉ CAMAR-PL900422 Íï 8001-CT91-O104 Macchia, Ì., Benvenuti, Á. and Angelini, L. (1983) Car- Wi h nkJ '  ¿çß ful '. d ' atteÞstiche germinative di alcune sementi di specie et a erry as oruse commentsan sugges- ffi ' É, R ' ' t ,+é fr tt ' It It É, tions, ïç an earher'.' verSl0nïß the manuscnpt. ï190. cma É, lVlS á dellaOrt °J,oro u lco ura á éáïï67' 165- 170 C.A.ThanosetáÉ.

Muller, C.H., Muller, W.H. and Haines, Â.É. (1964)Volati1e Thanos, C.A., Georghiou, Ê. and Delipetrou, Ñ.(1994) Pho- growth inhibitors produced by aromatic shrubs. Science toinhibition ïß seed germination ßç the maÞtime plant 143,471-473. Ìátthiolá tricuspidátá.Annáls ÏÉ Botány 73, 639-644. Philianos, 5.Ì., AndÞÏÑïulïu-Áthanassïula, Ô. and Thompson, Ñ.Á. (1970)Characterization ïß the germination Loukis, Á. (1982)Sur les constituants de l'essencedu responseto temperatureïß speciesand ecotypes.Náture thym capite (Thymus cápitátus Hoffm. et Link, Cori- 225,827-831. dothymuscápitátus Reichb. f.) de diverses regions de la Theophrastus. Enquiry into plánts, íïé. Ð. Hort, A.F. Grece.Biologia Gállo-Hellenicá 9, 285-289. (translator) (1926).CambÞdge, Mass., London, Harvard Pons, Ô.É.(1991a) Induction ïß dark dormancy ßç seeds:its University Pressand William Heinemann Ltd. importancefor the seedbank ßçthe soil.Functional Ecology Tutin, T.G., Heywood, V.H., Burges Í .Á., Moore, D.M., Val- 5,669-675. entine D.H., Walters 5.Ì. and Webb, D.A. (Eds) (1972) Pons, Ô.É.(1991b) Dormancy, germination and morta1ity ïß Florá Europáeá,Volume 3. CambÞdge, CambÞdge Uni- seedsßç a chalk-grasslandflora. JournalÏÉ Ecology 79, 765- versity Press. 780. van Tooren, B.F. (1990)Effects ïß a bryophyte layer ïç the 5ß1vertown,J.W. (1980) Leaf-canopy-induced seed dormancy emergenceïß seedlingsïß chalk grassland SÑeÜes.Actá ßça grasslandflora. New Phytologist85,109-118. Oecologicá11, 155-163. 5topp, Ê. (1952) Morphologische und verbreit- Vokou, D. (1983)Volátile oils ánd their role in phrygániceco- ungsbiologische Untersuchungen uber persistierende systems(ßç Greek). Ph.D. Thesis, University ïß Thes- :1t

Bltitenkelche. Abhandlungen der Ìáthematisch-Nát- saloniki, Greece. É urwissenschaftlichenKlásse, Akademie der Wissenschaften und Vokou, D. (1992)The allelopathicpotential ïß aromaticshrubs . der Literátur in Máinz 12,903-971. ßç phryganic (East Ìedßteðaçeaç) ecosystems.ññ 303-II,f Tarayre, Ì., Thompson, J.D., Escarre,J. and Linhart, ã.Â. 320 ßç Rizvi, S.J.H.and Rizvi, V. (Eds) Allelopáthy:básic ~ (1995)Intra-specific vaÞatiïn ßç the inhibitory effectsïß ándápplied áspects. London, Chapman & Hall. Thymusvulgáris (Labiatae) monoterpenesïç seed ger- Vokou, D. and Margms, Í.5. (1986) Autoallelopathy ïß mination. Oecologia101, 110-118. Thymuscapitátus. Actá Oecologicá,Oecologia Plántárum 7, Thanos, C.A. (1993) Germination ecophysiology ïß Med- 157-163. ßteðaneanaromatic plants. ññ 281-287in Cäme, D. and Went, F.W. (1955)The ecologyïß desertplants. ScientificAmer- Corbineau, F. (Eds) ProceedingsÏÉ theFourth International icán192, 68-75. Workshopon Seeds.Básic ánd Applied Aspects ÏÉ Seed Biology. Angers,Fránce 20-24 July, 1992.íïé. 1. ÑaÞs,ASFlS. Received13 March 1995,accepted after revision 24 May 1995 Thanos,germination C.A. and ßç Doussi,aromatic Ì.Á. labiates (1995)ïß Ecophysiology Crete.Isráel Journal ïß seedÏÉ @CAB INTERNATIONAL' 1995 Plánt Sciences(ßç press.)

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