The Role of Nectar in the Germination of Pollen in ...

GAZ.150(3):266-270. 1989. (C)1989 by The Universityof Chicago. All rightsreserved. 0006-8071/89/5003-0008$02.00 BOT.

THE ROLEOF NECTARIN THE GERMINATIONOF POLLENIN ASCLEPIASSYRIACAL. P. G. KEVAN,* D. EISIKOWITCH,AND B. RATHWELL* *Departmentof EnvironmentalBiology, Universityof Guelph,Guelph,Ontario,CanadaN1G 2W1 and tDepartmentof Botany, Tel-Aviv University,Israel The nectaryof Asclepias syriaca is in the stigmaticchamber.Nectar flows from there by a capillary system to the nectarreservoirs(cuculli). The fluid connectionfrom the stigmaticchamberto the cuculli allows quick mixing of liquidsin the system. Nectarin the stigmaticchamberis the germinationmedium for the pollen. Polliniado not germinatein drystigmaticchambers.The nectarmustcontain5%-30%sugars for pollen germination.The nectarin the cuculli is the rewardtakenby insect pollinatorsand may become alteredby physical(e.g., evaporationor dilution)or biotic (e.g., microbes,enzymes)factors.

Material and methods Rhizomes of differentclones of Asclepias syrPlants of the family Asclepiadaceae are highly iaca were collected in the field near Guelph, Onspecialized for pollination. Nevertheless, pollinatario, in the springof 1987. They were plantedin tion in some species is effected by a wide variety 1887; MACIOR1965; woodencontainersand grew outsidethroughoutthe of anthophiles (ROBERTSON summerof 1987. To maximizethe growthof the WILLSONand BERTIN 1979; KEPHART1983; plants, all flowers were removed as the infloresSOUTHWICK 1983; MORSE1982, 1985). The pollen cences developed. The plantsdied back in the auin Asclepias is produced in paired packages, pollinia, which are removed from the flowers by intumn.The boxes were then packedwith strawand left outside over winter. In 1988, healthy plants sects whose limbs enter the flowers so that the corwere available for experimentation.When they pusculum (a sticky clasp) of the pollinial pair bloomed, flowers were removed from the plants attaches thereto (WYATT1978). On subsequent visand taken to the laboratory.The pedunclesof the its to another flower, the insects insert the polliflowers were insertedthroughholes puncturedin nium into the stigmatic chamber where it remains the lids of sterile, plastic petridishes, which were to affect pollination. Despite nearly 200 years of 1793; DELPINO1865; BROWN filled with water so that the peduncles were imresearch (SPRENGEL 1883; CORRY1883) the mechanism of pollination was protected mersedin the water.The preparation and fertilization has not been fully understood. The by anotherinvertedbottomof a petridish. During experimentspolliniawere removedfrom the flownectaries are the walls of the stigmatic chamber ers andused for experimentson pollengermination. (STADLER 1886) (but not also or just the cuculli as 1793, BROWN1883; To confirmthat the capillarysystem that links previously thought [SPRENGEL and the stigmaticchamberwith the cuculli (GALIL 1886]). The CORRY1883] or conjectured [STADLER ZERONI1965) operatesin A. syriaca, we placed nectar flows from there by an elaborate capillary wicks of small slips of filter paper into the stigsystem (GALILand ZERONI1965) to the cuculli, matic chamberand added 3 >1 of neutralred in which act as nectar reservoirs and serve as part of 70% ethanolto each of the five cuculli (fig. 1). the visual attraction for flower visiting insects. It has escaped suspicion, until recently, that the necTo examinepollen andpollinialgermination,we transferredpollinia directly to drops of 5 >1 sutar, which is a solution of almost pure sucrose et al. 1981), may be the natural gercrose solutionson the inside of lids of clean petri (SOUTHWICK dishes (3 cm diameter).The bottom of the petri mination medium for the pollen inserted into the stigmatic chamber during pollination (EEIKOWITCH dish containeda reservoirof sugarsolutionto buffer and WYATT(1986) state that the 1986). SHANNON the effects of atmospherichumidityandto keep the sugarconcentrationin the dropsmore or less conpollen can germinate in nectar, but we know of no stantduringa given experiment.The methodis a deliberate attempts to prove experimentally that 1900) and variationof the hangingdrop(JORGENSEN nectar is the natural germination medium. The aim accommodatesup to 25 dropsperlid. Pollinialgerof our paper is to elucidate experimentally the role minationwas scored as successful if, on microof nectar in the natural germination of pollen in A. scopic examination,pollentubeswere seen to have syriaca. grownfromthe pollinium.We also examinedpollinia within stigmaticchambers,and, 6 h afterinManuscript received September 1988; revised manuscript resertion, they were scored in the same manneras ceived March 1989. above afterthe pollinia had been withdrawnfrom Addressfor correspondenceand reprints:Dr. P. G. KEVAN, the stigmaticchambersandtransferredto a droplet Departmentof EnvironmentalBiology, Universityof Guelph, of 10%sucrosein water. Guelph,OntarioN 1G 2W1, Canada. Introduction

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FIG. 1.- Flowersof Asclepiassyriaca. Fig. 1A, normal,untreatedflower. Fig. 1B, flower with wick in stigmaticchamberto demonstratefluidconnectionbetweennectaryandcuculli. Fig. 1C, flowerwith wick in cucullusto demonstrateeffect of removing nectarfrom the stigmaticchamber.

To demonstratethatsugar(sucrose)alone in solutionwas enoughto cause germinationof the polliniaandto determinewhichconcentrations of sugar supportedmaximumgermination,we prepareda series of six solutions (0%-40% w/w) and preparedhangingdropsof one solutionper petridish. We transferredpolliniadirectlyfromthe anthersof one clone to the drops and examined and scored them 4 h later. We scored the numberof pollinia with germinatedpollen as follows: 0, no pollen tubes;1, 1-5 tubes;2, 6-10 tubes;3, 11-15 tubes; 4, 16-20 tubes, and 5, 21 tubes (EISIKOWITCH et al. 1987) andthe vigor of germinationas: 1, pollen tubes < half widthof polliniumin length;2, pollen tubesbetweenhalf and full widthof the pollinium; 3, pollen tubes > widthof polliniumin length. We took the productof those scores, and any value of 10, 12, or 15 was deemedexcellent, and values of 6, 8, 9 were deemedgood. The excellentandgood values were combinedto give a categoryof successful germination.Values less than 6 were consideredto be mediocreat best. Three techniqueswere used to test the role of nectarin pollen germinationin naturalconditions: 1. We insertedthe pollinia in reverse into the stigmaticchambersand comparedtheir germination with those insertedin theircorrectorientation (fig. 2). The germinationof the pollen is restricted to the convex side of the pollinium(BROWN1883; GALILand ZERONI1969). Cross-pollinationswere made becauseA. syriaca is mostly self-incompatible (SPARROW and PEARSON1948). 2. We insertedslips of filterpapersto act as nectar wicks in the cuculli, therebywithdrawingnectar as it was secretedby the stigmaticcavity. The wicks were replaced every 20 min. We inserted

pollinia, in their correct orientation, into the stigmatic chambers of flowers with and without wicks and compared the success of pollinial germination. Cross-pollinations were made. 3. We allowed nectar to build up in flowers so that it flooded into the pollinial cavities and soaked

FIG. 2. Crosssectionof the areaof two stigmaticchambers of a flowerof Asclepiassyriaca. CP = polliniuminsertedcorrectly, pollen tubes growing into stigmaticsurface;E = entranceto stigmaticchamber;N = nectarwithin the stigmatic chamber;P = polliniumwithin the anthersac; PT = pollen tube;RP = polliniuminsertedin reversedposition;SC = walls of stigmaticchamber.


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bag with holes and (b) the cover to the preparation not sealed down but supportedajar.

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FIG.3. Ratesof pollen germinationin solutionsof various concentrationsof sucrose. For explanationof excellent and successful, see text.

the pollinia in situ. To create that situationtwo methodswere used: (a) newly opened flowers on inflorescenceson plantsin the field were placedin polyethylenebags for 48 h and (b) cut flowerswere used and the cover for the preparationwas sealed with vaselineandleft for 48 h. The resultsof those experimentswere comparedwith their respective controls,which allowedfor the nectarto evaporate and so not flood the pollinia: (a) a polyethylene

Results We confirmedthatthereis an effective capillary connectionfrom the stigmaticchamber(nectary) and the cuculli. Within5 sec of addingthe neutral red to the cuculli, the wicks in the stigmaticchambers (no. = 15 flowers) becamestainedpink (fig. 1B), showing a direct liquid connectionbetween the stigmaticchamberandcuculli. Rapiddiffusion takes place in the fluid. The sucrose concentrationsupportingmaximal pollinialgerminationin 4 h was between 11%and 15%(fig. 3). Sugarconcentrationsbelow 5% did not supportgermination,and above 30%, germinationwas slow. Polliniainsertedin the correctand reversedpositions in the stigmaticchambersshowed no differences in pollen germination(table 1, fig. 2, X2 = .22; P = .64. Those pollinia that failed to producegerminatedpollen, no matterwhat their position,were in stigmaticchambersthatremained dry duringthe experiment. The effect of the wicks in removingnectar(fig. 1C)was marked(table 1). About50%of the wicks in the cuculli removed nectar from the stigmatic chambers(X2= 7.33; p = .007; Ho wicks have no

iBLE 1 THE GERMINATION OFPOLLEN OF ASCLEPIASSYRIACAUNDER VARIOUS EXPERIMENTAL CONDITIONS

No. pollinia germinated

A. Pollinialorientation: Correct ......................... Reversed........................ B. Nectarwithdrawal: No wick and nectar ............... Wick and nectar.................. No wick and no nectar............. Wick and no nectar ............... C. Nectarflooding: Bags: Nonperforated *. PerforatedT.................... Petridish: Sealed ........................ Not sealed..................... Sealed and flooded.............. Sealed and not flooded .......... Not sealed and flooded .......... Not sealed and not flooded .......

18

No. pollinia not germinated 10

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NoTE.-A.-pollinia insertedinto stigmaticchambersin correctandreversed orientation(fig. 2). B.-pollinia insertedinto stigmaticchambersaffectedby wicks (in the cuculli) (fig. 1) intendedto remove nectar. C.-pollinia left in situ in antherchambersin flowers,baggedor in petridishes, for effects of nectar flooding. * No. = 26 flowers. t No. = 20 flowers.


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effect on presenceof nectarin the stigmaticchamber) and, in all of these, the polliniafailed to germinate,as they did in the threestigmaticchambers withoutwicks and withoutnectar. The wicks had a signiElcant effecton pollengermination (X2= 9.94; P = .002) andthe overallx2for germinationversus nongermination for the four treatments(wicks and nectarto no wicks and no nectar)was 57.6; P < lo-8.

In the experimentsin which we allowed flowers containedin petridishes to fill with nectarto soak the polliniain the antherchamber,only the flooded flowers supportedpollen germination(51 pollinia in 17 flowers), whereasall 129 pollinia in flowers thatdid not flood failed to germinate,regardlessof whetheror not the petri dish was sealed (table 1) (x2=

180;P 40%) (PUNCHIHEWA

1984), which would inhibit pollen germination.Only when the sugar concentration in the nectar falls below about 30% (i.e., in the evening until the next morning) can pollen in pollinia inserted into the stigmatic chambers actually germinate. SOUTH WICK(1983) has shown that the highest rate of nectar secretion occurs between 2000 and 0800 at the time when evaporation would be minimal. That observation may explain the findings of PUNCHIHEWA(1984) and WILLSON and BERTIN(1979) that nocturnal pollination is just as effective as diurnal pollination despite the relatively short duration of activity of nocturnal pollinators and their relatively low numbers vis-a-vis diurnal pollination and pollinators. Other environmental conditions that affect nectar are chemical and microbial contamination. For example, FRITZand MORSE(1981) and PUNCHIHEWA(1984) noted that the flowers of A. syriaca are attended by ants, which may, by their antibiotic properties, inhibit germination of pollen grains (BEATrIEet al. 1985). Some pollen, which may contaminatenectar, has allelopathic effects on other pollen (THOMSON et al. 1981). Further, invertase may be added to nectar (sucrose solution in A. syriaca) by the feeding of insects, which would result in the nectarbecoming richerin hexoses. The change in sugar ratio could affect the pollinator guild, favoring short-tonguedinsects (see BAKERand BAKER 1983). Microbial contamination of nectar can take place readily from pollinators and from the atmosphere and occurs in A. syriaca (EISIKOWITCH et al., in press), but some nectars appear to have antimicrobial properties (GILLIAM et al. 1983). We have shown that the nectar of A. syriaca is a dynamic and complex system. Pollinators alter the amount of nectar present in flowers and contaminate it with enzymes and microbes. Environmental conditions affect the sugar concentration in nectar. Thus, pollinators and environmental conditions have both positive and negative influences on the whole process of sexual reproduction in A. syriaca.

Acknowledgments This research was funded by Natural Sciences and Engineering Research Council of Canada (grant A8098) and the Ontario Pesticides Advisory Committee of the Ontario Ministry of the Environment. We gratefully acknowledge the technical assistance of S. WILLIS.

LITERATURE CITED BAKER,H. G., and I. BAKER.1983. A brief historicalreview of the chemistryof floralnectar.Pages 126-152 in B. BENTLY and T. ELIAS,eds. The biology of nectaries.ColumbiaUni-

versityPress, New York. BEATrIE,A. J., C. TURNBULL, T. HOUGH,S. JOBSON,and R. B. KNOX. 1985. The vulnerabilityof pollen and fungal

sporesto ant secretions:evidenceandsome evolutionaryimplications.Am. J. Bot. 72:606-614. BROWN, R. 1883. On the organsand mode of fecundationin Orchideaeand Asclepiadeae. Trans. Linn. Soc. London 16:685-745. CORRY, T. H. 1883. On the structureand developmentof the


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bumblebees and implications for outcrossing. Oecologia 53: 187-196. MORSE,D. H. 1985. Milkweeds and their visitors, Sci. Am. 253(7): 112-119. PUNCHIHEWA, R. W. K. 1984. Anthophilous insects and the pollination ecology of Asclepias syriaca. L. and Asclepias incarnata L. in southern Ontano. M.Sc. thesis. University of Guelph, Ontario, Canada. ROBERTSON, C. 1887. Insect relations of certain Asclepiads. I. and II. BOT. GAZ. 12:207-216, 244-250. SHANNON , T . R ., and R . W . WYATT . 1986. Pollen germinability of Asclepias exaltata: effects of flower age, drying time, and pollen source. Syst. Bot. 11:322-325. SOUTHWICK, E. E. 1983. Nectar biology and nectar feeders of common milkweed, Asclepias syriaca L. Bull. Torrey Bot. Club 110:324-334. SOUTHWICK, E. E., G. M. LOPER,and S. SADWICK. 1981. Nectar production, composition, energetics and pollinator attractiveness in spring flowers of western New York. Am. J. Bot. 68:994-1002. SPARROW , F . K ., and N . L . PEARSON . 1948. Pollen compatibility in Asclepias syriaca. J. Agric. Res. 77:187-199. SPRENGEL, C. K. 1793. Das entdeckte Geheimniss der Natur in Bau und in der Befruchtung der Blumen. Vieweg dem Aeltern, Berlin. STADLER, S. 1886. Beitrage zur Kenntnis der Nektarien und Biologie der Bluten. Freidlander, Berlin. THOMSON, J. D., B. J. ANDREWS, and R. C. PLOWRIGHT. 1981. The effect of the foreign pollen on ovule development in Diervilla lonicera (Caprifoliaceae). New Phytol. 90:777-783. WILLSON, M. F., and R. I. BERTIN.1979. Flower visitors, nectar production and inflorescence size of Asclepias syriaca L. Can. J. Bot. S7:1380-1388. WYATT,R. W. 1978. Experimental evidence concerning the role of the corpusculum in Asclepias pollination. Syst. Bot. 3:313-321.
