Facultative Apomixis in Garcinia atroviridis (Clusiaceae) and Effects of Different Pollination Regimes on Reproductive Success

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1 Tropicl Life Sciences Reserch, 20(2), , 2009 Fculttive Apomixis in Grcini troviridis (Clusicee) nd Effects of Different Pollintion Regimes on Reproductive Success 1 Ssithorn PngsunF* F, 2 Noprt Bmroongrugs, 2 Kmnoon Knchnpoom nd 3 Chrssri Nulsri 1 Deprtment of Science, Fculty of Science Technology nd Agriculture, Yl Rjht University, Thilnd 2 Deprtment of Biology, Fculty of Science, Prince of Songkhl University, Thilnd 3 Deprtment of Plnt Science, Fculty of Nturl Resources, Prince of Songkl University, Thilnd Astrk: Pelgi spek kejyn dlm pemikn Grcini troviridis Griff. telh dikji. Eksperimen pendeungn terkwl telh dijlnkn di dlm seuh keun di wilyh Songkhl, seltn Thilnd, dri Feruri hingg Juli Kelnjutn usi gi ung, reseptiviti stigm dn keoleh hidupn deung telh diperiks seelum eksperimen dijlnkn. Tid perezn yng signifikn dlm set uh wl ntr tig jenis rwtn pendeungn, iitu pendeungn teruk, pendeungn dieg dn mnul, dn dieg tnp pendeungn (pogmi). Wlu gimnpun, pendeungn dieg dn mnul telh menghsilkn kdr jtuh uh yng juh leih signifikn dripd pogmi dn pendeungn semuljdi, yng dikesn stu minggu seleps ung-ung terseut telh dideungkn. Rwtn pogmi mempunyi kdr jtuh uh yng leih esr ernding pendeungn semuljdi dn mnul seelum uh msk. Dlm pd itu, keolehn memuh sert siz uh didpti kurng dlm ung-ung yng dieg tnp pendeungn, ernding dengn ung-ung pendeungn semuljdi dn pendeungn silng. Wlupun uh dripd ung-ung yng tidk dideungkn oleh menjlni pementukn enih secr seksul, uh-uh ini menghsilkn nomor purt enih yng kurng, dn kuliti enih yng teruk dn kurng memuskn (didefinsikn segi ert ersih purt dn kdr percmhn) ernding dengn rwtn-rwtn yng lin. Pemikn seksul dn seksul jug dikji menggunkn nlisis Rndom Amplifiction of Polymorphic DNA (RAPD) dn mellui pemezn corkcork jlur yng dihsilkn dripd DNA yng diekstrk dripd k uh yng melkukn pendeungn silng secr semul jdi. Secr purt, 58% dripd k terseut mempunyi konstitusi genetik yng sm dengn induk (dri 36% 87%), yng menunjukkn d di ntr k dihsilkn tnp penyuurn. Penemun ini mencdngkn hw pomisis fkulttif telh erlku dlm populsi yng dikji. Wlugimnpun, sutu residul seksul dlh penting untuk penghsiln uh, siz uh, set iji enih norml dn kuliti enih. Kt kunci: Apomisis Fkulttif, Kejyn dlm Pemikn, RAPD 0BAstrct: Vrious spects of the reproductive success of Grcini troviridis Griff. were studied. Controlled pollintion experiments were crried out in n orchrd locted in Songkhl province, southern Thilnd, from Ferury to July Florl longevity, stigm receptivity, nd pollen viility were exmined efore crrying out the experiments. Three pollintion tretments were compred: open pollintion, mnul pollintion with gs, nd gs without pollintion (pogmy). Although there ws no significnt difference in the initil fruit set, gged nd mnul pollintion produced significntly greter fruit drop rte thn pogmy or nturl pollintion t one week fter the flowers hd een pollinted. On the other hnd, the pogmy tretment hd greter fruit drop rte thn nturl nd * Corresponding uthor: Hstmppriy@yhoo.com 89

2 Ssithorn Pngsun et l. mnul pollintion tretments efore fruit mturtion. In ddition, unpollinted gged flowers ore fewer nd smller fruit thn nturlly nd mnully cross-pollinted flowers. Although the fruits from unpollinted flowers were cple of sexul seed formtion, they produced fewer seeds nd hd poorer seed qulity (defined s verge fresh weight nd germintion rte) thn those from the other tretments. The occurrence of sexul nd sexul reproduction ws lso studied using Rndom Amplifiction of Polymorphic DNA (RAPD) nlysis nd y compring the ptterns of nds produced from DNA extrcted from the offspring of the nturlly cross-pollinted fruits. On verge, 58% of the offspring hd genetic constitution identicl to tht of the mternl prent (rnging from 36% to 87%), indicting tht some offspring were produced without prior fertilistion. However, the reminder showed polymorphism, demonstrting the occurrence of sexul reproduction. These findings indicte tht fculttive pomixis occurred in the study popultion. However, residul sexulity ws importnt for fruit production, fruit size, norml seed set nd seed qulity. Keywords: Fculttive Apomixis, Reproductive Success, RAPD 2BINTRODUCTION The Clusicee (Guttifere) fmily of trees is nturlly distriuted in tropicl zones etween the ltitudes of 10 north nd 10 south (Cox 1976). Grcini is the iggest genus in this fmily, with over 400 species. G. troviridis Griff. ex T. Anders is one of the est known fruit trees of this genus. This is n endemic species in peninsulr Mlysi, nd preprtions from it hve een widely used y ethnootnists nd ethnophrmcists s preservtive, for sesoning nd for medicinl purposes. Its dried fruit hs een used to improve lood circultion, s n expectornt, for tretment of coughs, nd s lxtive (Ypwttnphun et l. 2002). In ddition, phytochemicl studies of the fruit s rind hve estlished the presence of commercil sustnce ((-)-hydroxycitric cid (HCA)) tht inhiits ATP-dependent citrte lyse, key enzyme tht diverts crohydrte metolites to ftty cid nd cholesterol synthesis (Lewis & Neelkntn 1965). HCA is used in mny populr over-the-counter weight loss formultions, nd the demnd for the fruit is thus incresing. However its horticulturl potentil hs only een poorly developed (Suhdrndhu 2001). In prticulr, its reproductive iology is still not well understood. A deeper knowledge of this could revel rriers to seed nd fruit set, increse fruit production, ccelerte reeding progrms, nd promote its conservtion. G. troviridis is gynodioecious (Pngsun et l. 2007) nd is commonly propgted y seeds. The trees egin to flower 5 6 yers fter plnting, nd mximum fruit yield is usully ttined fter 6 7 yers. The femle trees produce fruits, while the hermphrodite trees sometimes er only few fruits or hve no fruit-set (Pngsun et l. 2007). Hence, the growers prefer to plnt femle trees rther thn hermphrodite trees to optimise fruit production. However, the gender selection of the trees poses serious prolem to the growers s the gender cnnot e determined until the trees flower (Suhdrndhu 2001). The growers usully resort to grfting or they inrch ud wood of femle trees onto seedlings or existing hermphrodite trees to ensure fruit production. By this pproch, hermphrodite trees, nd hence pternity, tend to decline nd e limited in 90

3 Fculttive Apomixis in Grcini troviridis numers in the popultion. Thus, knowledge of G. troviridis reproductive ehviour is urgently required ecuse it is importnt for growers to design suitle reeding strtegies. Theoreticlly, the mjority of dioecious tropicl trees should exhiit mleised secondry sex rtios, given the reduced lloction of resources required for successful reproduction y mles (Thoms 1996; Opler & Bw 1978). However, mny Grcini species hve femle-ised sex rtios, especilly in southest Asin forests (Thoms 1997). One explntion for the existence of the femle-ised sex rtio is their reproductive ehviour, which hs een proposed to e y gmospermy (pomixis y seed) (Thoms 1997; Richrds 1997). More thn 300 species (from more thn 35 fmilies) of ngiosperms hve een descried s pomictic (Koltunow et l. 1995). Mny species of Grcini re in this ctegory, including G. livingstonii (Puri 1939), G. mngostn (Kur et l. 1978), G. prviflor (H et l. 1988), G. scortechinii (Thoms 1997), G. homronin (Richrds 1990), nd G. gummi-gtt (Ri 2003). However, G. cntleyn is not n pomict. Hence, not ll species of Grcini re pomictic (Richrds 1997). Likewise, Richrds (1990) hs suggested tht G. troviridis might e fculttive pomict on the sis of its sex rtio. However, his suggestion hs not een confirmed. Here we present the results of the first comprehensive investigtion of the reproductive ehviour of G. troviridis. The first issue investigted ws whether this species, which is known to reproduce y pomixis, could set unfertilised seed in unpollinted flowers under controlled pollintion conditions. In order to scertin the occurrence of sexul versus sexul reproduction, we lso tested whether offspring would express n identicl DNA fingerprint to the mternl prent in RAPD mrker survey. Any non-mternl RAPD fingerprint in the offspring would indicte tht zygotic offspring were formed vi fertilistion. These investigtions lso hd the potentil to revel influences of reproductive ehviour on other importnt mesures of reproductive success, such s fruit setting, fruit growth, seediness nd seed qulity. MATERIALS AND METHODS Study Site nd Plnt Mterils The study site for this work ws n orchrd locted in Songkhl province, Tmon Pien in the south of Thilnd (N7.13 E100.53) t n ltitude of pproximtely 200 m. Annul rinfll in this region rnged from 29.2 mm in Jnury to mm in Novemer. The verge temperture vried from 22.5 C to 24.6 C, nd reltive humidity vried from 50.3% to 97.3% (Pttni Meteorologicl Sttion 2003). The trees er fruit nnully, with flowering nd fruiting occurring once yer in the summer (Ferury to June). The conspicuous flower hs long pedicel with four wrm yellow sepls. Its petls re lrge, right crimson, oriculr nd/or oovte. The stigms seem to e convex. There is no cler seprtion etween stigm nd style. The femle flowers re usully solitry, ut the hermphrodite flowers re present in compound cymes (Whitmore 1973). Most femle flowers hve ovules per flower, wheres the hermphrodite flowers hve 9 12 ovules per 91

4 Ssithorn Pngsun et l. flower (Pngsun et l. 2007). The trees used for determining flowering performnce in this study were rndomly selected ccording to ccessiility. Florl Longevity The femle trees flower synchronously in Ferury. Florl longevity ws estimted on three selected femle trees. In order to ctegorise the developmentl stges of the flowers, 10 florl uds per tree were rndomly chosen nd individully tgged. The uds re conceled t the ses of the uppermost lef stlks. No externl fetures distinguish the florl uds from the vegettive uds until the two sutending rcts spred into horizontl position due to the incresing size of the immture florl ud hed. Developmentl stges of the flowers were monitored dily from the initition of the visile florl ud until the formtion of fruit. Morphologicl chnges nd the time intervls etween chnges were recorded. Stigm Receptivity Stigm receptivity ws ssessed y two pproches; the ppernce of exudte nd detection of peroxidse ctivity on the stigms under nturl conditions (Kerns & Inouye 1993; Klisz et l. 1999). Oservtions were mde on the three selected femle trees. Ten flowers t ech different stge were tested for peroxidse ctivity y plcing them into 3% solution of hydrogen peroxide (H 2 O 2 ) nd wtching for oxygen uling ctivity. Stigms were scored s positive for peroxidse ctivity only if vigorous uling ws oserved cross the entire surfce of the stigm within one minute. The stigms showing wek uling fter the ppliction of the H 2 O 2 were presumed to e flse positives nd were scored s unreceptive (modified from Klisz et l. 1999). The stigmtic exudte ws lso evluted for its viscosity nd glistening ppernce. Pollen Germintion Pollen viility ws determined y n in vitro germintion test using germintion medium consisting of 10% sucrose + Brewker nd Kwck s Medium (Shivnn & Rngswmy 1992). Ten hermphrodite flower uds were collected prior to flower opening. Before plcing these selected flower uds in Petri dishes, their sepls nd petls were removed. In this wy, their nthers could dehisce during the next dy without disturnce of their stigmtic exudte. Eventully, their pollen grins were pooled nd mintined under room temperture to evlute their in vitro germintion dily. A suitle numer of pollen grins were dispersed in 200 μl of culture medium in Eppendorf tues nd mde into homogeneous suspension with needle. The cultures were incuted under mient conditions (~25%) for 48 h. This suspension ws then drwn into Psteur pipette nd trnsferred onto clen microslide. A drop of 2% cetocrmine ws dded to terminte pollen development. The germintion test ws performed with three replictes. Three slides of ech replicte were prepred for monitoring. A pollen grin ws considered to hve germinted when the length of its tue ws greter thn the grin s dimeter (Shivnn & Rngswmy 1992). For ech slide, grins from 9 rndomly selected fields were counted t 100X mgnifiction using light microscope. 92

5 Fculttive Apomixis in Grcini troviridis Controlled Pollintion Four femle trees were selected (with dimeters t rest height of 9.64, 7.96, 8.28 nd 8.76 cm). The experiment ws crried out under Completely Rndomised Design model. Controlled pollintion ws conducted during the flowering seson (Ferury 2003). For the purposes of the study, it ws ssumed tht the position of the flower in the tree nd the prthenocrpic ility of the fruit did not significntly influence the result. Femle flowers selected for the gging tretments were locted on lower rnches tht could e reched from the ground. In preliminry study, some flowers were gged with cellophne gs to ensure tht this process did not hrm the flowers or lter fruit development. The wether ws dry during the dy of the experimentl pollintion. The femle flowers of G. troviridis did not require emscultion (Pngsun et l. 2007). Ten helthy florl uds per tretment were lelled. Three types of tretment were pplied to ech selected tree, sed on Dfni (1992). For nturl pollintion, the florl uds were left nturlly exposed. For mnul pollintion, ech ud ws protected from nturl pollintion y cellophne g. When the uds reched suitle stge, the gs were crefully opened to llow mnul pollintion with the picked hermphrodite flowers, fter which the pollinted flowers were regged. For pogmy, the florl uds were gged without pollintion. To minimise the effects of pollen source, the pollen used for mnul pollintion ws collected from the hermphrodite plnt used in the ove study. All gs for tretments (2) nd (3) were retined until the flowers senesced or the initil fruit set ws oserved (i.e., when n ovry recommenced growth, c mm). Finlly, developing fruits from tretments (2) nd (3) were relesed from their gs to chieve similr conditions to those from tretment (1). Four replictes were used for ech tretment. Fruit setting ws recorded. Developing fruits were counted dily throughout the period of heviest fruit loss (the first seven dys fter pollintion), nd then t two-week intervls until the fruits were fully mture. The fruit drop rte (the numer of fruits tht scised during tht week) ws clculted (Truemn & Wllce 1999). The dimeters of the fruits retined were mesured with vernier clipper. Ripe fruits were recognised y the chnge in colour of their rind from green to ornge. After hrvest, the ripe fruits were immeditely tken to the lortory to determine their dimeter nd weight. The numer of seeds per ripe fruit ws counted, nd the men fresh weight of the seeds ws lso determined efore they were plced in the greenhouse for germintion (locted t the Fculty of Nturl Resources, PSU, Songkhl, Thilnd). All fresh seeds were immeditely plnted in pots filled with snd mixture nd then plced in the shde. Seed germintion rte (%) ws clculted fter one, two nd three months. 3BDNA Extrction nd Moleculr Anlysis Mture seeds from nturlly cross-pollinted fruits of three femle trees were used in this nlysis. The fruits were rndomly hrvested. Seeds from ech ripe fruit were sown singly in the greenhouse (Fculty of Nturl Resources, PSU, Songkhl, Thilnd). The genetic nlyses were crried out on 25 intct seedlings from ech femle tree. Genomic DNA ws isolted from young fresh leves 93

6 Ssithorn Pngsun et l. ccording to Doyle nd Doyle (1987). Lef smples (pproximtely mg) were ground into powder with liquid nitrogen in mortr. The mteril ws thoroughly mixed with 2.5 ml of pre-wrmed (60 C) extrction uffer (20 mm N 2 EDTA ph 8.0, 100 mm Tris-HCl (ph 8.0), 1.4 M NCl,1% PVP-40, 2% CTAB, 2% β-mercptoethnol v/v) nd ground further, then trnsferred to 5 ml centrifuge tue nd incuted for 5 h t 60 C. One volume of phenol: chloroform: isomyl lcohol (25: 24: 1 v/v) ws dded to the tues nd mixed thoroughly. The mixture ws centrifuged for 15 min t 4000 rpm. The top lyer ws trnsferred to fresh tue nd one volume of chloroform: isomyl lcohol (24: 1 v/v) ws dded nd mixed gin. The mixture ws gin centrifuged for 15 min t 4000 rpm. The top lyer ws trnsferred to 1.5 ml Eppendorf tue. One volume of ice-cold isopropnol ws dded to precipitte the DNA. This ws pelleted y centrifugtion for 10 min t 10,000 rpm, nd the superntnt ws crefully poured off. The DNA pellet ws wshed twice with 800 μl of 70% ethnol nd centrifuged for 3 min t 10,000 rpm. The superntnt ws discrded nd the pellet ir-dried. The DNA ws then resuspended in 30 μl of utoclved doule-distilled wter (DDW). The DNA concentrtion ws checked, nd the DNA ws then seprted y electrophoresis on n 0.7% (w/v) grose gel in 1X Tris-cetic cid-edta (TAE) nd stined in 1.5 μl/ml ethidium romide solution efore compring the nds to rnge of concentrtions of lmd high-moleculr-weight DNA (Sigm, USA). In previous study (Nkkuntod 1998), 12 primers from Operon Technologies Inc. (Almed, Cliforni) were successfully used in RAPD nlysis for txonomic study of the genus Grcini. Therefore, ll these primers were tested in the present study. However, two of these primers filed to give reproducile nds. Thus, only the following 10 primers (OPAA17: 5 - GAGCCCGACT-3, OPAD04: 5 -GTAGGCCTCA-3, OPAD05: 5 -ACCGCATGGG- 3, OPAD06: 5 -AAGTGCACGG-3, OPAD08: 5 -GGCAGGCAAG-3, OPAD09: 5 - TCGCTTCTCC-3, OPAD10: 5 -AAGAGGCCAG-3, OPAD11: 5 -CAATCGGGTC- 3, OPAD12: 5 -AAGAGGGCGT-3, OPAD15: 5 -TTTGCCCCGT-3 ) were used in this RAPD nlysis. The RAPD rections were crried out with 15 μl of Tq polymerse (0.5 U/μl); Tq uffer X1 (20 mm Tris-HCl, 50 mm KCl, ph 8.4); dntps (2 mm ech); MgCl 2 (2.5 mm); primer (5 pmole/μl) nd 50 ng of templte DNA. Polymerse Chin Rection (PCR) conditions were s follows: 3 min t 94 C; 94 cycles of : 1 min t 94 C, 1 min t 35 C, 2 min t 72 C; nd s lst step 5 min t 72 C. Amplifictions were crried out using PCR Sprint (Hyid, USA). The mplifiction products were resolved on 1.5% (w/v) grose gels in 0.5X Tris-oric cid-edta (TBE) nd stined in 1.5 μl/ml ethidium romide solution. After ethidium romide stining, the nding ptterns were recorded y photogrphy (Gel-documenttion) nd nlysed lter. The reproduciility of the RAPD methodology used in this study ws checked very crefully. Differences in RAPD fingerprints were only ccepted if they could e confirmed in two replictes. 1BDt Anlysis The mens nd stndrd errors were clculted for ll mesurements. Pollen viility ws clculted y dividing the numer of germinted pollen grins y the totl numer of pollen grins in the field of view nd expressed s percentge. Dt from the controlled pollintion experiment were nlysed using the non- 94

7 Fculttive Apomixis in Grcini troviridis prmetric Kruskl-Wllis Test, since the dt tht ws not distriuted normlly even fter log or rcsine squre-root trnsformtions nd the vrince differed significntly. Lter, the post-hoc Duncn s New Multiple Rnge Test ws used to ssess comprisons mong tretments (Smuels & Witmer 2003). Moleculr dt nlysis for identifying the pomictic seedlings ws performed y compring the nding ptterns etween ech mternl prent nd their seedlings. The RAPD fingerprints were considered polymorphic if they showed either fewer or more nds thn their mternl prent nd monomorphic if the nds were identicl. The results were expressed s the percentge of genotypiclly mternl seedlings. All sttisticl nlyses were performed with SPSS v. 10. RESULTS AND DISCUSSION Florl Longevity The inception of the flower primordi occurred t the end of Jnury. Blooming commenced during lte Ferury, progressed through Mrch nd terminted in April, nd occurred synchronously mong individul trees. The flower uds opened t night, hd ttrctive colours (pink to red), were odourless, nd secreted smll mount of stigmtic exudte, like other Grcini species. The period etween the commencement of florl uds nd fruit setting verged out 22 dys, wheres the florl longevity (defined s the time from opening to shrivelling of the petls) ws out 3 dys. The chnges in florl colour nd form re descried in detil in Tle 1. The development of the flowers ws determined on the sis of their morphology nd exudte, nd ws divided into five distinct stges (S 0 S 4 ): (1) closed uds (S 0 ) = uds tht re still enclosed y the outer sepls nd without ny exudte; (2) open uds (S 1 ) = expnded sepls nd flower petls (defined s petls open to n ngle of less thn 45 with the pedicel) with smll mount of exudte ppering over the stigms; (3) mture flowers (S 2 ) = hlfopened flowers (defined s petls open to n ngle of more thn 45 nd less thn 90 with the pedicel) with stigmtic exudte covering the entire stigmtic surfce; (4) post-nthesis (S 3 ) = fully opened flowers (defined s petls open to n ngle of 90 or more with the pedicel) with dried exudte; nd (5) fruit setting (S 4 ) = the ovry hs recommenced growth, fruit development hs egun, petls re shrivelled nd the exudte hs disppered. 95

8 Ssithorn Pngsun et l. Tle 1: Developmentl stges of the femle flower of G. troviridis. Stge* Dys Description Externl fetures S 0 1 pink stigm, exudte sent, swollen flower ud S 1 0 pink to red stigm, little exudte present, sepls nd petls strt opening S 2 ** 1 reddish stigm, exudte covers entire stigm surfce, sepls nd petls hlf open nd extremely expnded S 3 2 crimson stigm, exudte egins to dry, flower fully expnded, ovry swollen S 4 3 lck-red stigm, exudte sent, sepls nd petls strt shrivelling nd colour hs fded, fruit setting Notes: * See detils in text. ** Florl stge selected for controlled pollintion. Scle r: 0.5 cm Stigm Receptivity On the sis of the peroxidse test, stigms of the flower uds t S 0 were nonreceptive, ecuse vigorous uling ws not oserved. However, vigorous uling ws oserved to occur on the stigms s flowers developed further (S 1 S 4 ). Interestingly ll stigms in the hlf-opened flowers (S 2 ) were recognised s receptive (Fig. 1). Similrly, stigmtic exudte ws not oserved in the S 0 stge (Tle 1), while smll mount of exudte ws first oserved on the centrl region of the stigms in the S 1 stge. Next, the viscous exudte grdully incresed on the stigmtic surfces (nd strted to glisten) until the entire stigmtic surfce ws covered in the hlf-opened flowers (S 2 ). Finlly, the 96

9 Fculttive Apomixis in Grcini troviridis stigmtic exudte egn to dry in the fully opened flower (S 3 ) stge nd towrd the eginning of the initil fruit set stge (S 4 ). These results indicte tht the receptivity of the stigms (on the sis of the peroxidse test) ws concomitnt with the secretion of the exudte. The mximum mount of exudte in the S 2 stge coincides with the competence to support pollen hydrtion nd germintion during the pollintion process. Thus, flowers in the S 2 stge were selected for the controlled pollintion experiment. 100 Stigm receptivity (%) S1 S2 S3 S4 Florl developmentl stges Figure 1: Frequencies of receptive stigms of G. troviridis femle flowers (n = 30 flowers per stge). Ech stge is descried in the text. The verticl single lines represent the stndrd error (SE). Pollen Germintion The hermphrodite flowers were orne in compound cymes, in which terminl centrl flower ws first formed nd lterl flowers developed sequentilly. This sequentil flowering in the hermphrodite flowers ment tht ech flower cluster could present pollen over severl dys. The developmentl fetures of the individul flowers were the sme s in the femle flower. However, ech flower contined mny nthers tht rndomly dehisced s soon s the florl ud roke. On the sis of the in vitro germintion ssy, pollen viility ws high (79.5%) on the first dy fter nther dehiscence nd hd hlf-life (50.0% loss of pollen viility) of 17 dys (Fig. 2). By 25 dys fter nther dehiscence, none of the pollen grins could germinte. Therefore, to ensure efficient cross-pollintion, pollen grins to e used s donors in the controlled pollintion experiment were hrvested on the first dy fter nther dehiscence. 97

10 Ssithorn Pngsun et l. 90 Pollen viility (%) y = x x R 2 = Time fter nthers dehiscence (Dys) Figure 2: Viility of mture pollen grins from G. troviridis hermphrodite plnts s ssessed y in vitro germintion test t vrious times fter nther dehiscence (n = 10). Controlled Pollintion The numer of fruits otined from the vrious controlled pollintion tretments did not differ significntly (Kruskl Wllis, H = 1.494, d.f. = 2, P-vlue = 0.342) (Fig. 3). This result indicted tht the initil fruit set ws not dependent on pollintion. Thus, it might e ssumed tht unpollinted flowers cn continue into fruit set vi sexul reproduction (pomixis). However, further moleculr nlysis ws needed to test this ssumption. One week fter the flowers hd een sujected to the different tretments, significnt differences were oserved in fruit drop (H = 6.870, d.f. = 2, P-vlue < 0.05). The heviest scissions occurred fter mnul pollintion, while the nturlly pollinted flowers did not shed fruits (Fig. 4). Possily, the initil high fruit drop rte in the mnully pollinted flowers reltive to the other tretments ws due to dmge during mnul pollintion. Dt on the fruit drop rte collected t the third nd fifth weeks fter fruit setting showed no significnt differences (H = 4.094, d.f. = 2, P-vlue = nd H = 5.301, d.f. = 2, P- vlue = 0.071, respectively). Interestingly, the records tken 7 to 11 weeks efore the fruit were ripe showed tht gret numer of unpollinted fruits hd fllen from their trees (H = 6.488, d.f. = 2, P-vlue < 0.05). These results indicte tht the nturl thinning of premture fruits seems to selectively remove unpollinted fruits. This might e due to internl competition mong fruits within the trees. As result of this process, the verge dimeter of the fruits from unpollinted flowers ws significntly lower thn for the other tretments (Fig. 5). The retention of fruits growing from nturlly nd mnully pollinted flowers ws not significntly different (Tle 2). 98

11 Fculttive Apomixis in Grcini troviridis 100 Initil fruit set (%) ns ns ns 20 0 Nturl pollintion Mnul pollintion Apogmy Tretments Figure 3: Men (± SE) initil fruit set following three controlled pollintion tretments in G. troviridis (n = 40 treted flowers). Significnt differences were ssessed with the Kruskl-Wllis test. ns indictes no significnt difference Fruit drop rte Initil fruit set Nturl pollintion ns ns ns ns ns ns w1 w3 w5 w7 w9 w11 Week(s) Nturl pollintion Mnul pollintion Apogmy Figure 4: Men (± SE) rtes of fruit drop (numer of fruits tht scised per week) in G. troviridis t vrious stges of development. Significnt differences were ssessed with the Kruskl-Wllis test. Points with the different letters show significnt differences t the 95% level. ns indictes no significnt difference. 99

12 Ssithorn Pngsun et l. 60 Nturl pollintion Mnul pollintion Fruit size (mm) Apogmy w1 w3 w5 w7 w9 w11 Week(s) fter pollintion Figure 5: Men (± SE) fruit enlrgement in G. troviridis t vrious stges of development. Significnt differences were ssessed with the Kruskl-Wllis test. Points with different letter show significnt differences t the 95% level. Tle 2: Kruskl-Wllis proility levels of differences in fruit dimeters mesured during development (from Fig. 5). Fruit development (weeks) H d.f P-vlue <0.010 <0.010 <0.010 <0.010 <0.010 <0.010 The fruits ecme ripe weeks fter fruit setting. Hrvesting ws done when their rinds showed full ornge colour. It is importnt to highlight the fct tht unpollinted flowers were le to er fruit (Tle 3), nd these fruits were le to set seeds. These results support the previous ssumption tht pomixis occurs in these flowers. Also, the seeds from the different pollintion regimes were morphologiclly similr. Thus, it ws not possile to visully distinguish whether they were produced sexully or vi pomixis. The verge numer of seeds in ripe fruits following mnul pollintion ws greter thn the numer produced y pogmic or nturl pollintion (H = 15.50, d.f. = 2, P-vlue < 0.05). This indictes tht the seediness my hve een regulted y the mount of pollintion. Interestingly, the lower seed set in the nturlly crosspollinted fruits indictes tht there either ws pollen limittion or lck of 100

13 Fculttive Apomixis in Grcini troviridis pollintors t the study site. However, the seeds from the unfertilised fruits hd lower verge fresh weight thn those from the other tretments (H = 8.733, d.f. = 2, P-vlue < 0.05). Possily, the genetic qulity of these unfertilised seeds ws compromised. Consequently, they hd slower germintion rte thn the fertilised seeds from the nturlly nd mnully cross-pollinted fruits (H = 6.338, d.f. = 2, P-vlue < 0.05) (Fig. 6). Thus, we speculte tht fertilistion improved the genetic mke-up of the seeds, giving them more vigour nd fitness. Finlly, seeds from ll the three tretments hd successfully germinted y three months. They followed the grcini-type of germintion, in which the primry root nd shoot emerged from opposite ends of the seed. This clerly indictes tht pomixis occurred, since fertile seeds were produced without pollintion or fertilistion. Tle 3: Fruit set nd seed set in G. troviridis fter three controlled pollintion tretments. Numers in prentheses indicte the numer of smples. Significnt differences were ssessed with the Kruskl-Wllis test. Numers with different letters indicte significnt differences t the 95% level. Tretment No. of treted flowers Fruit set (%) Averge numer of seeds per ripe fruit Averge seed fresh weight (mg) Nturl pollintion ± 0.71(21) ± 2.06(71) Mnul pollintion ± 0.83(24) ± 2.42(81) Apogmy ± 0.99(7) ± 4.48(23) 100 Seed germintion (%) Nturl pollintion 20 Mnul pollintion Apogmy Month(s) Figure 6: Seed germintion (men ± SE) following three controlled pollintion tretments in G. troviridis. Significnt differences were ssessed with the Kruskl-Wllis test. Points with different letters indicte significnt differences t the 95% level. 101

14 Ssithorn Pngsun et l. Moleculr Mrkers Using RAPD Anlysis A totl of ten 10 nt ritrry primers were used to screen the 3 femle trees, s well s set of 25 offspring from ech. From the entire set of primers, four (OPAD 04, OPAD 09, OPAD 10, nd OPAD 12) filed to give polymorphic nds. Thus, these were omitted ecuse the nding pttern did not help to discriminte etween pomictic nd zygotic seedlings. However, the other 6 primers did produce totl of 319 nds. The size of the polymorphic nds vried from 200 to 1000 p, s shown in Figure 7. These nds were useful for screening pomictic nd zygotic seedlings, ecuse distinguishing these seedlings ws mde difficult y their very similr outwrd fetures. This use of RAPD fingerprints clerly demonstrtes their utility for discriminting etween pomictic nd zygotic seedlings. For the entire set of primers, the mjority of the RAPD mrkers showed monomorphism (87.5%), wheres the reminder of the nds clerly showed polymorphisms (12.5%). The results indicted tht 58% of the offspring were sexully derived, with nd pttern identicl to tht of the mternl prent. However, some seedlings cme from mixed prentge, s their RAPD ptterns were distinct from their mternl plnts. These results indicte tht under nturl conditions, seedlings were produced oth y pomixis nd from zygotes fter fertilistion. As result, the nturl reproductive ehviour rnged from 36% pomixis ( high mount of sexul reproduction) to 87% pomixis ( high mount of sexul reproduction) within the smpled plnts. These results verified the occurrence of fculttive pomixis in the study popultion. Offspring Figure 7: RAPD profiles of n individul mternl plnt nd its offspring otined y mplifiction of DNA smples with the primer OPAD 08. From left to right, the first lne is 100 p ldder (Operon, USA) frgment size mrker (M), followed y the mternl prent (P) nd 25 offspring. Numers correspond to the code numers of the offspring. The sterisks ( ) indicte dignostic RAPD mrkers used for the discrimintion of zygotic seedlings (Nos. 1, 2, 11 13, 16 18, 20, 22, 24, 31, 33) nd pomictic seedlings (Nos. 3 8, 10, 14, 21, 25, 30, 32). 102

15 Fculttive Apomixis in Grcini troviridis DISCUSSION We conducted the first comprehensive study of the reproductive iology of G. troviridis. The results provide evidentil support for the suggestion of Richrds (1990, 1997) tht this plnt might e fculttive pomict. Although identifying n effective pollintion period ws not n ojective of this study, successful fertilistion in sexul reproduction depends on three fctors: stigm receptivity, pollen germintion nd pollen tue kinetics, nd ovule longevity (Snzol & Herrero 2001). Therefore, identifying the optimum conditions for stigm receptivity nd pollen viility re importnt spects of ny evlution of reproductive ehviour (Dfni 1992; Espinoz et l. 2002). This work estlishes tht there is correltion etween stigm receptivity s mesured y the peroxidse test nd the timing of secretion of the stigm exudte. This provides optimum conditions llowing nturl pollintion to occur over the whole stigm. The stigmtic exudte hs three importnt functions: the ttchment of pollen t the stigmtic surfce, the rehydrtion of pollen for germintion nd pollen tue entry into the style, nd s nectr rewrd to ny visiting pollintor (Richrds 1997). Little is known out pollen viility in G. troviridis. According to the literture, hrvested pollen grins of mny Grcini species hve high viility s ssessed y their stinility; e.g., 99.4% for G. corymos, 92.5% for G. foresii, nd 85% for G. cf. foresii (H et l. 1988). Bsed on our in vitro germintion test, we report similr result for G. troviridis. This chrcteristic might e conserved property of this genus. The results of the controlled pollintion nd RAPD nlysis in this study indicte tht fculttive pomixis occurs in G. troviridis. This reproductive ehviour is dvntgeous for the femle trees, s it ensures reproduction nd llows G. troviridis to escpe extinction even in the sence of hermphrodite trees. However, Richrds (2003) lso stted tht sexul seed formtion provides opportunities for the ccumultion of recessive mutnts. Our findings lend support to this rgument. In this study, hyridistion improved fruit production, fruit size, seediness nd seed qulity. It is of interest tht mnully pollinted flowers potentilly produced more highly fertilised ovules thn did unpollinted or openpollinted flowers. Frequently, seeds in developing fruits produce hormones necessry for fruit development (Sedgley & Griffin 1989; Goldwin 1992). For instnce, in Aridopsis, ovries without seeds exhiited norml fruit development, ut the ovries were smller thn those with seeds (Cox & Swin 2006). Fertilised Aridopsis fruits with lrger numer of seeds were igger in size thn those with smller numer of seeds. In our cse, the fruits from unpollinted flowers were smller in size nd hd lower verge numer of seeds thn fruits from pollinted flowers. A similr result ws oserved in Ruus rmenicus. Fruits from unpollinted flowers were smller nd the numer of seeds ws significntly lower reltive to fruits from open-pollinted flowers (Kollmnn et l. 2000). There re two possile explntions for this. First, the fruit size might e regulted y endogenous fctors cting on unfertilised ovules. This ide is supported y severl oservtions. For instnce, less GA 1 ws synthesised y unpollinted thn y pollinted pericrps nd/or ovules of Pisum stivum nd Juglns regi (Grcí-Mrtínez et l. 1991; 103

16 Ssithorn Pngsun et l. Tdeo et l. 1994). Secondly, there were fewer unfertilised ovules in the multiovulte flowers tht developed into mture seeds. This indictes tht perhps some growth-regulting hormones produced y the seeds were insufficient to induce fruit development. As result of this process, the fruits with fewer developing seeds were likely to e smller in size nd dropped efore reching mturity (Ho 1992; Kollmnn et l. 2000). It seems resonle to ssume tht the pternl genomes from pollen grins improved seed qulity in this crop ecuse the gmospermous seeds hd lighter verge fresh weights nd required longer incution time for germintion thn did the fertilised seeds. These oservtions re similr to those reported in G. mngostn, in which the germintion percentge ws directly relted to the weight of the gmospermous seeds (Morton 1987). The complex genetics of seeds should e tken into ccount when determining wht it is tht regultes seed weight. Vritions in the rtio of mternl to pternl genomes could produce spectrum of phenotypes in the emryo nd endosperm, nd could ffect chrcters such s seed viility, seed weight, rte of mitosis in the endosperm, nd timing of endosperm cellulristion (Scott et l. 1998). For exmple, pollen (s the pternl genome) in R. rmenicus cused positive heterosis during endosperm formtion in cross-pollinted seeds, cusing fertilised seeds to e hevier thn unfertilised ones (Kollmnn et l. 2000). By contrst, oligte pomixis gives rise to fertile seeds with emryos derived only from the mternl genome. However, fculttive pomicts produce two types of fertile seeds: (1) pomictic emryos geneticlly identicl to the mternl genotype, nd (2) zygotic emryos geneticlly different from the mternl genotype. Consequently, their different genetic mke-up my ffect other physiologicl properties of these seedlings, s oserved with Opunti spp Cctcee, in which zygotic emryos emerge erlier thn pomictic seedlings (Jcoo 2001). There re two viewpoints to consider. First, there my e competition etween the hyrid genomes in the developing emryos, resulting in more vigour nd greter competition thn occurs mong the pomictic emryos (Koltunow et l. 2002). On the other hnd, the pomictic progeny my compenste for their lower genetic vrition y dopting slower germintion progrm tht will ensure optiml growth. However, in order to confirm this suggestion, the ploidy levels of the progeny should first e determined (Jcoo 2001). Also, comprison of their RAPD mrkers will provide evidence of mternl inheritnce even when the pollen source is unknown. CONCLUSION Femle G. troviridis trees do not normlly outreed ecuse their flowers cn produce unfertilised seeds without pollintion, nd oth fertilised nd unfertilised seeds cn germinte into seedlings. Therefore, their reproductive ehviour is clssified s fculttive pomixis. This type of reproduction hs the dvntge tht femle plnts successfully produce progeny even when they exist in mlefree popultion. However, fertilistion with pternl genome does enhnce fruit yield. Fruits produced y different pollintion regimes showed considerle 104

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