floral nectar secretion and ploidy in brassica rapa and b. napus

Annals of Botany 77 : 223–234, 1996

Floral Nectar Secretion and Ploidy in Brassica rapa and B. napus (Brassicaceae).

II. Quantified Variability of Nectary Structure and Function in Rapid-cycling

Lines

A. R. DAVIS*‡, L. C. FOWKE*, V. K. SAWHNEY* and N. H. LOW†

*Department of Biology, and †Department of Applied Microbiology and Food Science,

Uni�ersity of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5E2

Received: 3 August 1995 Accepted: 2 October 1995

Haploid, diploid and tetraploid lines of Brassica rapa L. (syn. campestris), and allotetraploid B. napus L., wereexamined to determine the influence of ploidy on floral features, particularly nectary morphology and anatomy, andto relate nectary structure to nectar production capacity. Except for haploids, all lines were rapid-cycling. Averageflower dry weight, and petal length and width, were in the descending order B. napus"B. rapa (4n)" 2n" n. Pollengrains of 4n plants were larger than those of 2n plants ; haploids lacked pollen.

All lines developed nectaries. Typically, each flower produced two pairs of nectaries, of different types and nectarproduction capacity. Normally, each lateral gland was located above the base of a short stamen, and together thispair yielded most of a flower’s nectar carbohydrate. Each median nectary arose at the outer junction of the bases oftwo adjacent long stamens. All lateral nectaries received a vascular supply of phloem alone, but median glandsreceived reduced amounts of phloem, or lacked vasculature altogether. Most nectaries were solitary, but 14% of allflowers, and especially those of 2n B. rapa, had at least one median and lateral gland connected.

Obvious variation existed in nectary morphology between ploidy levels, between flowers of the same plant, and evenwithin flowers. Ten forms of each nectary type were recognized. Plants producing the most nectar carbohydrate hadhigh frequencies of lateral nectaries which were symmetrical, unfurrowed swellings. Tetraploids of B. rapa had boththe highest frequencies of furrowed lateral glands, and of isolated segments of nectarial tissue at that position. Eventhese separated nectarial outgrowths received phloem and produced a nectar droplet. At the median location,nectaries were commonly of two forms: peg- or fan-shaped. Lobes on median nectaries, up to four per nectary, weredetected in almost half of glands of 4n flowers examined; lobes were absent in haploids.

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Key words : Brassica rapa, Brassica napus, flower size, nectar production, nectary variability, petal size, phloem,ploidy, pollen, rapeseed.

INTRODUCTION

Floral nectar often attracts flower visitors. In theBrassicaceae, floral nectaries—the glands which secretenectar—are situated at the base of the androecium (Bayer,1905; Schweidler, 1911; von Hayek, 1911; Schulz, 1936;Norris, 1941; Clemente Munoz and Hernandez Bermejo,1978; Davis, Peterson and Shuel, 1986). As a result, insectswithdrawing nectar often inadvertently pick up, anddisperse, pollen. The attraction of floral nectar to potentialpollinators of Brassica, a genus of many economically-important species, is well known (see Davis et al., 1994).Little is known, however, about the constancy of nectarymorphology, and the relationship between nectary form andnectar-carbohydrate production, within a single species ofBrassica.

Earlier this century, Bayer (1905), Villani (1905),Schweidler (1911) and von Hayek (1911) emphasized thevalue of differences in nectary morphology and location ascharacters to distinguish taxa within the Brassicaceae. Davisand Heywood (1963), however, questioned the taxonomic

‡ For correspondence.

value of nectaries because of the lack of information on themorphological variability of nectarial tissue within in-dividual species. Subsequently, by the presentation of limitednumbers of line drawings, Dvora! k (1965, 1967, 1968,1970), Dvora! k and Uhlı!rova! (1967), and especiallyAvetisyan (1979), demonstrated variability in morphologyof the nectarial tissue within species of Arabis, Deilosma,Hesperis, Lunaria, Malcolmia, Matthiola and Turritis. How-ever, the frequency of different forms was not quantified,nor were they correlated with nectar production.

In his classification system, Norris (1941) distinguishedthree categories of nectaries in mature Brassicacean flowers,according to the location and degree of isolation of nectarialtissue: (a) the annular type, a continuous zone of nectarialtissue around the receptacle ; (b) the four-nectary type; and(c) the two-nectary type. Brassica campestris, a speciesexamined in the present study, was listed by Norris asbelonging to the second group. More recently, ClementeMunoz and Hernandez Bermejo (1978) and Davis et al.(1986, 1994) have upheld this designation by referring totwo pairs of nectaries in flowers of Brassica spp., the lateral(inner) and median (outer) pairs (Figs 1 and 2).

Generally, in studies of floral development and mor-

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224 Da�is et al.—Influence of Ploidy on Nectary Structure in Brassica

F. 1. Stylized diagram of the flowers of B. rapa and B. napus, in telescoping view, showing location of the lateral (LN) and median (MN)nectaries. The dashed black lines connecting adjacent nectaries denote the occasional occurrence of nectarial confluence (see text).

F. 2. SEM micrograph of flower base of B. napus, showing lateral nectary (LN) surrounded by insertion points of long stamens (LS), petals(P), and a short stamen (SS). Two elongated median nectaries (MN) are evident at opposite sides of the flower. Gynoecium (G) with valve (V)

of silique; lateral sepal (Sp) ; pedicel (Pd). Bar¯ 0±5 mm.

F. 3. Newly-flowering racemes of n, 2n and 4n plants of B. rapa, and of B. napus. Bar¯ 2 cm.

phology, it is common for nectaries to be treated superficiallyor neglected entirely. In fact, no comprehensive studydocumenting themorphological variability of floral nectarialtissue, within an indi�idual species, has been published forany angiosperm. Association of nectar carbohydrate pro-duction with nectary size and morphology, within a species,is also rare (Beutler, 1953; Davis and Gunning, 1991).Furthermore, the influence of ploidy on nectary morphologyhas not been investigated in any taxon. Accordingly, theaims of this study were to examine haploid, diploid andtetraploid plants of B. rapa L. (syn. campestris), through theuse of light and scanning-electron microscopy (SEM), (a) tocategorize, both qualitatively and quantitatively, variabilityin the structure of both lateral and median pairs of the floralnectaries of rapid-cycling lines, and (b) to relate thisvariability to nectar production capacity. Allotetraploidplants of B. napus L. were included in the study for tworeasons: this species is derived from B. rapa (and B. oleraceaL.), and its ploidy almost matches that of tetraploid B. rapa(see below).

MATERIALS AND METHODS

Plant material

Haploid plants of Brassica rapa L. (n¯ 10) were derivedfrom microspore culture (Baillie et al., 1992) and were a giftfrom Dr A. M. R. Ferrie (Kristjansson Biotechnology Com-plex, Saskatoon, Saskatchewan, Canada). The four plantsreceived were already flowering and were grown in pots ina greenhouse under controlled conditions: 16 h light(20 °C)}8 h dark (15 °C), minimum light 200 µE m−#.

Rapid-cycling lines (Williams and Hill, 1986) of diploid(2n¯ 20) (Serial g1-1) and tetraploid (4n¯ 40) (g1-32) B.rapa, and allotetraploid Brassica napus L. (4n¯ 38; 2n¯ 20from B. rapa and 2n¯ 18 from B. oleracea) (g5-1), wereobtained from Dr P. H. Williams (Crucifer Genetics Co-operative, Madison, Wisconsin, USA). Plants were grownfrom seed in pots containing Sunshine Mix (FisonsHorticulture Inc., Vancouver, B.C., Canada) in a growthchamber at 16 h light}8 h dark, and 22 °C (constant).Lighting was provided by fluorescent tubes at a fluence rateof 200 µE m−#.

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Da�is et al.—Influence of Ploidy on Nectary Structure in Brassica 225

T 1. Influence of ploidy on �arious floral characteristics, including size of the lateral nectaries, in plants of Brassica rapaand B. napus

Flower dryPetal

Pollen grain Lateral nectary

SpeciesPloidylevel

weight (mg)xa ³s.e. (no.)

Length (mm)xa ³s.e. (no.)

Width (mm)xa ³s.e. (no.)

length (µm)xa ³s.e. (no.)

volume (¬10−$ mm$)xa ³s.e. (no.)

B. rapa n 1±65a³0±11 (60) 4±79a³0±17 (20) 2±50a³0±09 (20) — 6±44a³0±53 (60)2n 3±23b³0±10 (60) 6±98b³0±16 (20) 3±31b³0±13 (20) 25±90a³0±03 (80) 16±84b³1±05 (80)4n 4±63c³0±08 (60) 7±59c³0±15 (20) 4±35c³0±12 (20) 32±26c³0±18 (80) 25±81c³1±82 (80)

B. napus 4n 6±87d³0±11 (60) 10±26d³0±15 (20) 4±67d³0±04 (20) 29±41b³0±11 (80) 17±88b³0±72 (80)

In columns, means having the same superscript letter are not significantly different (Two-tailed t tests, α¯ 0±05).

Flower characteristics

For the purpose of comparing overall flower size withfloral-nectary size, flower dry weights and other measure-ments were taken. Fresh, fully-open flowers (includingpedicels) bearing nectar were removed from rapid-cyclingplants for dry-weight determination. These flowers wereamong the first ten produced on the primary inflorescences.Haploid flowers were collected similarly, but from manyracemes. Without nectar removal, flowers were placed inPetri dishes and transferred to a 60 °C oven for 24 h; flowerdry weight remained unchanged beyond 24 h. Sixty flowerswere weighed from four to eleven plants representingeach ploidy level, and means compared by two-tailed t tests(P¯ 0±05).

Other floral parameters, such as sizes of pollen grains andpetals, have served as diagnostic features of ploidy in theBrassicaceae (Renard and Dosba, 1980; Altmann et al.,1994). From the anther of one long (tetradynamous) stamenof each of four plants per ploidy level in B. rapa, 20 pollengrains were photographed by SEM (see below) at 2100¬,and grain length measured from the micrographs. For onepetal from each of 20 flowers total taken from four to sixplants per ploidy level, maximum length and width (at thebroadest part) were determined after taping petals to glassmicroscope slides, and examining with a dissecting micro-scope (15¬). Means were calculated for these morphologicalcharacteristics, and compared statistically, as above.

Scanning electron microscopy (SEM ) of floral nectaries

Four plants (numbered 1–4) of each rapid-cycling line,and three plants (5–49, 5–63, 5–93) of the B. rapa haploids,were used for SEM studies. Ten open, nectar-bearingflowers from each plant were chosen randomly and fixed,dehydrated and critical-point dried as described in Davis etal. (1994). Both pairs of lateral and median nectaries (Daviset al., 1986, 1994) were examined using a Philips SEM 505at 30 kV, and observations recorded either on high densityprinting paper (UPP-110HD Type II) using a Sony UP-860Video Graphic Printer, or on Polaroid 665 positive}negativefilm.

SEM micrographs (145¬) of lateral nectaries photo-graphed in polar view (Fig. 4), and then perpendicular (Fig.

21, right) to the adaxial, secretory surfaces, were used tocompute nectary volumes (length¬width¬height). Themedian glands, which are much poorer nectar yielders, werephotographed only in their longitudinal plane, along theadaxial surface, for comparisons of their morphology.

Light microscopy of floral nectaries

Flowers from which nectar had been collected were thenfixed, dehydrated, embedded in LR White plastic resin,sectioned (2±0 µm) on a Reichert Ultramicrotome OmU3,stained with toluidine blue O, and examined (Davis et al.,1994). Photographs were taken with a Zeiss AxioplanUniversal light microscope, using Tech Pan film.

Determination of nectar carbohydrate production

Nectar samples collected using filter-paper wicks wereeluted in known volumes (0±5–1±0 ml) of distilled water,before analysis using a Waters 625 metal-free gradient high-performance liquid chromatograph (see Davis et al., 1994).In cases where individual droplets of nectar were analysedseparately, the total nectar carbohydrate per flower wascalculated later by summation of those samples.

RESULTS

Sizes of flowers, petals and pollen grains

Flowers of 4n plants of B. rapa were visibly larger thanthose of 2n, and especially n, plants (Fig. 3). The average dryweight of 4n flowers of B. rapa exceeded the n and 2n flowersby 2±8 and 1±4 times, respectively (Table 1). However,overall, flowers of B. napus were largest (Fig. 3), andoutweighed the n, 2n and 4n flowers of B. rapa by 4±2, 2±1and 1±5 times, respectively (Table 1). The size of floral partscontributed most to this weight disparity. Petal dimensions,both length and width, were significantly larger as ploidy inB. rapa increased; petals of B. napus were largest (Table 1).Quantities of nectar sugar affected flower dry weight only ina minor way. In B. napus and 2n B. rapa, slightly over 5%of the flower dry weight was attributable to nectar, whereasthe exudate accounted for 3±3 and 4±2% of the dry weight offlowers of n and 4n B. rapa, respectively (Davis et al., 1995).

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T 2. Influence of ploidy on the proportion of Brassica flowers possessing a narrow strand of glandular tissue that mergesa lateral nectary with an adjacent median nectary, and on the proportion of flowers bearing two small nectar droplets at one

or both lateral sides, as opposed to one large droplet

Percentage of flowersshowing nectary

confluence at

Percentage ofpotential

junction zones

Percentage of total flowers havingtwo small nectar droplets

SpeciesPloidylevel

at least onepotential

junction zone

showing confluencebetween lateral andmedian nectaries

At at least onelateral side

of the flower

At bothlateral sidesof the flower

B. rapa n 6±7a (30) 1±7a (120) 0a (53) 0a (53)2n 30±0b (40) 15±0b (160) 2±4a (82) 1±2a (82)4n 2±5a (40) 1±9a (160) 47±4c (78) 21±8b (78)

B. napus 4n 15±0ab (40) 5±6a (160) 16±9b (83) 6±0a (83)

Data in brackets denote numbers of flowers or junctions examined; each flower has a possibility of four such zones of junction. A total of fourplants was examined for each ploidy level, except for the haploids, three.

In columns, means with the same letter as superscript are not significantly different (Central Limit Theorem, two-tailed Z test, α¯ 0±05).

The length of pollen grains of 4n plants of B. rapaexceeded those of 2n plants and those of B. napus (Table 1).Haploid plants produced no pollen.

Nectary morphology and anatomy

Regardless of ploidy level, all plants bore floral nectarieswhich were generally in the normal positions for the genus(Norris, 1941; Clemente Munoz and Hernandez Bermejo,1978; Davis et al., 1986). In B. napus and B. rapa, typicallyeach flower bears two pairs of receptacular nectaries at thebase of its six stamens. Each lateral (inner) gland is locatedabove the base of a short stamen, whereas each median(outer) nectary is found external to the bases of theadjacent, long stamens (Figs 2 and 21).

Until now, each Brassica flower has been considered topossess four solitary glands. Microscopic examination of thezones where the filaments of long stamens meet the claws ofpetals revealed that, in a minority of cases (Table 2), lateralglands actually were connected, by narrow strands ofnectarial tissue, with adjacent median glands (Figs 7, 8, 27,37). Overall, 14% of these 150 flowers exhibited at least onesuch confluence, but less than 7% of all lateral glands wereconnected to a median gland (Table 2). However, thesenectarial connections did not occur randomly across allploidy lines ; 30% of the 2n flowers, but less than 3% of 4nflowers, bore such a connection (Table 2).

Lateral nectaries

Two lateral nectaries were present in all of the 150 flowersexamined; however, their morphology in both species ofBrassica was highly variable. Moreover, within a ploidylevel, nectary morphology differed between flowers ofdifferent plants, between flowers of the same plant, andsometimes even at opposite sides of the same flower (Figs 7and 8). Ten forms were recognized.

On the whole, the most common form of lateral nectary

was a symmetrical, multicellular, uniform swelling oroutgrowth situated above the insertion point of the shortstamen (Figs 2 and 4). These glands were supplied richlywith a direct vasculature consisting of phloem alone (Fig.16). On average, this form accounted for 86±7% (n), 88±8%(2n) and 50±0% (4n) of lateral nectaries in B. rapa, and65±0% in B. napus (Fig. 20). Less often, the outgrowth wasasymmetrical (Figs 5, 17 and 20). In other flowers, especiallyin B. napus (Fig. 20), the lateral gland exhibited a centralfurrow, whereas in haploids, any furrow was always shallow(Fig. 6). Furrows were not central in other glands (Figs 8and 9), particularly those in tetraploids (Fig. 20). A largephloem supply was detected in this morph, as well (Fig. 18).Only in 2n and 4n plants of B. rapa were glands above theshort stamen detected that had two furrows (Figs 7 and 20) ;none had more than two.

In very rare instances, limited to 4n plants of B. rapa andB. napus, the glandular tissue partially encircled the inner(Figs 12 and 20) or the outer (Figs 13 and 20) base of theshort stamen. Multiple furrows were observed in thesenectaries, which consisted of uninterrupted glandular tissue.

Two completely separated nectarial lobes were found toflank the base of the short stamen in plants of B. napus, butmore commonly in 4n plants of B. rapa (Fig. 20). Theselobes at the lateral position could be dissimilar (Fig. 10) orsimilar (Figs 11 and 19) in size. Each separated lobe had itsown traces of phloem (Fig. 19).

Overall, the proportion of lateral glands that had two ormore lobes, whether separated or not, was highest for 4nplants of B. rapa (42%), followed by B. napus (24%), 2nplants of B. rapa (10%), and n plants of B. rapa (5%) (seeFig. 20).

In extreme cases, where a reduction in normal numbers ofstamens or petals occurred (only B. napus, in this study),nectarial tissue was even more aberrant. In a flower withonly five stamens, the gland was skewed and below theabnormally-large stamen at the lateral position (Fig. 14). Ina flower with only three petals and five stamens, anasymmetrically-aligned mound of glandular tissue occurredon one lateral side of that flower (Fig. 15).

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F 4–15. SEM of lateral nectaries, normal to their secretory surfaces, in n, 2n and 4n lines of B. rapa, and in B. napus. Abbreviations : G,gynoecium; LS, long stamen; MN, median nectary; P, petal ; S, stamen; SS, short stamen; Sp, lateral sepal. Bars¯ 100 µm for Figs 4–13, and

0±5 mm for Figs 14 and 15.

F. 4. Gland symmetrical (B. rapa, 2n, plant 2).

F. 5. Gland asymmetrical (B. rapa, n, plant 5-63).

F. 6. Gland with central furrow (B. rapa, n, plant 5-93).

F. 7, 8. Opposite sides of same flower, showing one (Fig. 8) or two (Fig. 7) non-central furrows. Note the narrow strands of nectarial tissue(arrows) that connect with the median nectaries (B. rapa, 2n, plant 1).

F. 9. Gland constriction between the short and a long stamen (B. napus, plant 2).

F. 10. Separated nectarial tissue of unequal size (B. rapa, 4n, plant 2).

F. 11. Equal-sized lobes of separated glandular tissue (B. rapa, 4n, plant 1).

F. 12. Insertion point of short stamen surrounded, from above, by united nectarial tissue (B. rapa, 4n, plant 3).

F. 13. United, lobed glandular tissue enclosing short stamen from below (B. napus, plant 1).

F. 14. Formation of only five stamens has led to skewed nectarial tissue (arrows). Large stamen (S) near lateral position (B. napus, plant 1).

F. 15. Isolated, displaced mound of nectarial tissue (arrow) in flower lacking a petal and a stamen (B. napus, plant 2).

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F. 16–19. Light microscopy of sectioned lateral nectaries in B. rapa.Abbreviations : G, gynoecium; LS, long stamen; P, petal ; SS, shortstamen; Sp, lateral sepal. Bars¯ 100 µm for Figs 16–18, and 250 µm

for Fig. 19.

F. 16. Near-transverse section through flower showing symmetricallateral nectary and its supply of phloem (arrows). (n, plant 5-49).

F. 17. Asymmetrical lateral gland in flower sectioned transversely.Nectary stomata sectioned obliquely (arrowheads). (4n, plant 3).

F. 18. Longitudinally-sectioned flower bearing a lateral nectary withnon-central furrow. Note nectary phloem (arrows). (4n, plant 1).

F. 19. Flower sectioned in longitudinal plane to reveal two isolatedoutgrowths of nectarial tissue flanking filament of short stamen (SS).

Phloem in nectary interior (arrows). (4n, plant 1).

4nB. rapa

B. napus

nB. rapa

2nB. rapa

F. 20. Frequencies of different forms of lateral nectaries in B. rapa (n,2n, 4n) and B. napus. For haploids, three plants were studied; for otherlines, four plants each. The data represent the summation of 10 flowers(20 glands) for each plant. *, Symmetrical outgrowth above shortstamen (Figs 2, 4, 16, 21 and 33) ; 7, asymmetrical outgrowth aboveshort stamen (Figs 5 and 17) ; 7, lobed, with one central furrow inoutgrowth above short stamen (Fig. 6) ; 8, lobed, with one non-centralfurrow in gland above short stamen (Figs 8, 9 and 18) ; 4, lobed withtwo furrows in outgrowth above short stamen (Fig. 7) ; +, unitedglandular tissue encircling short stamen from above (Fig. 12) ; 7,united glandular tissue encircling short stamen from below (Figs 13 and14) ; 9, two separated lobes of glandular tissue, of different size (Fig.10) ; 8, two separated lobes of glandular tissue, of similar size (Figs 11and 19) ; 5, one asymmetrically-aligned lobe of glandular tissue

(Fig. 15).

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F 21–33. For legend see p. 230.

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Median nectaries

In all 150 flowers examined, a nectarial outgrowth wasfound at both median positions. Ten forms were designated,of which two predominated. Together, these two mostcommon forms composed 88±3% (n), 62±5% (2n) and 52±5%(4n) of median glands in B. rapa, and 60±0% in B. napus. Apeg-shaped structure (Figs 25 and 33, right) was the mostcommon in B. napus (Fig. 38), whereas a fan-shaped morph(Figs 21 bottom, and 26) predominated in B. rapa,particularly in haploids (Fig. 38). In many glands, a weaksupply of phloem was identified at the base (Fig. 34). Theonly other forms detected in haploids, that were notabundant throughout this survey (Fig. 38), were oblong(Fig. 22) and crescent-shaped (Fig. 24).

Median nectaries whose length exceeded their width tolesser (Fig. 2) or greater (Fig. 33, left) extents were uniqueto B. napus (Fig. 38). Figure 33 illustrates the great disparityin nectary size that can occur at the median positions withinan individual flower.

All but the haploid plants possessed median nectaries thatwere sometimes lobed. The degree of lobing ranged fromslight (Figs 15, left ; 27, 28 and 35) to moderate (Figs 23, 29and 36) in B. napus and in 2n and 4n B. rapa. Sometimes anobvious trace of phloem penetrated the nectary interior(Fig. 36). In 4n B. rapa, two distinct but united lobes couldbe found where the furrow extended to the nectary base(Fig. 30). All but one 4n plant exhibited multiple lobes (Figs31, 32 and 37) on their median nectaries ; within tetraploids,this averaged almost 10% of all median glands (Fig. 38).Overall, the proportion of median nectaries having at leasttwo lobes was highest for plants of B. rapa (4n, 45%; 2n,28%), followed by B. napus (25%). Unlike the case forlateral locations, separated lobes of nectarial tissue at themedian locations were never observed.

Variability in size of lateral nectaries

Within B. rapa, average size of the lateral nectariesincreased with ploidy (Table 1). However, mean volume of

F 21–33. SEM of median nectaries (and lateral nectaries ; Figs 21 and 33) in mature flowers of B. rapa and B. napus, viewed from above alongtheir adaxial surfaces. Abbreviations : G, gynoecium; LN, lateral nectary, LS, long stamen; MN, median nectary; P, petal. Bars¯ 100 µm for

Figs 21–32, and 1 mm for Fig. 33.

F. 21. Telescoping view of median and lateral nectaries adjacent to bases of long stamens, petals and gynoecium (B. rapa, n, plant 5-49).

F. 22. Oblong nectary (B. rapa, 2n, plant 4).

F. 23. Bilobed gland (B. rapa, 4n, plant 2).

F. 24. Crescent-shaped gland (B. napus, plant 4).

F. 25. Peg-shaped nectary (B. rapa, 2n, plant 1).

F. 26. Fan-shaped nectary (B. rapa, 4n, plant 1).

F. 27. Slight lobing of nectary (*). The arrow denotes glandular tissue connecting the median and lateral nectaries (B. rapa, 2n, plant 1).

F. 28. Nectary with obvious lobe (B. napus, plant 1).

F. 29. Accentuated lobing of nectary. This is the left-most gland of Fig. 14 (B. napus, plant 1).

F. 30. Gland with different sized lobes (B. rapa, 4n, plant 3).

F. 31. Gland with three lobes (B. rapa, 4n, plant 3).

F. 32. Gland with four lobes (B. rapa, 4n, plant 3).

F. 33. At left, the median nectary is elongate, but peg-shaped at right (B. napus, plant 2).

the lateral nectaries in B. napus was similar to 2n B. rapa,but significantly less than 4n B. rapa. Within the latter,interplant differences in lateral nectary volume were par-ticularly large; plant 4 averaged 48±4³7±7 (s.e.) (¬10−$ mm$,n¯ 20 glands), whereas together, plants 1–3 averaged18±3³1±0 (¬10−$ mm$ ; n¯ 60; P! 0±01), very similar tothe mean sizes of lateral nectaries of 2n B. rapa and B. napus(Table 1).

Similarly, the greatest range in morphology of lateralnectaries existed in flowers of 4n B. rapa (Fig. 20). In plant1, an extreme range in lateral nectary form was foundamongst and within flowers, and so this plant will bepresented in detail. Of its ten flowers examined by SEM,three groupings can be made. Only four flowers (Group 1)did not have isolated nectarial lobes at a lateral position.Instead, flowers of Group 1 had symmetrical outgrowths(Figs 2 and 4) that averaged 22±22³1±65 (s.e.)¬10−$ mm$,n¯ 8 lateral sides) and were of similar size within a flower(mean constancy 1±08). In three other flowers (Group 2) thathad isolated nectarial segments at only one lateral side, thetwo separated lobes were of disparate size [7±93 �s. 4±28;10±66 �s. 2±16; 7±16 �s. 3±47 (¬10−$ mm$)] and, whencombined, averaged much less [11±89³0±66 (¬10−$ mm$ ;n¯ 3)] than the size of the symmetrical gland on the oppositeside of the flower [17±48³2±52 (¬10−$ mm$ ; n¯ 3) ;P! 0±05]. Finally, three flowers (Group 3) bore isolatedlobes of nectarial tissue at both lateral sides, and thevolumes of these lobes were unequal : 3±82, 0±67 �s. 2±21, 1±26(¬10−$ mm$) ; 3±40, 2±15 �s. 2±72, 0±13 (¬10−$ mm$) ; and5±15, 2±89 �s. 6±15, 4±05 (¬10−$ mm$). In Group 3, combinednectarial tissue per lateral position averaged only 5±77³1±16(¬10−$ mm$, n¯ 6; P! 0±01), a four-fold reduction fromthe symmetrical outgrowths of Group 1.

Relationship between lateral nectary morphology and size,and nectar-carbohydrate production

Previously it was discovered that Brassica plants whichproduced the greatest quantities of nectar carbohydratedisplayed high constancy in lateral nectary size within each

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F 34–37. Light micrographs of median nectaries in mature flowerssectioned transversely. Abbreviations : LN, lateral nectary; LS, longstamen; MN, median nectary; P, petal ; SS, short stamen. Bars¯

100 µm for Figs 34–36, and 250 µm for Fig. 37.

F. 34. Fan-shaped gland (B. rapa, n, plant 5-49).

F. 35. Bilobed nectary. Stoma sectioned obliquely (arrowhead)(B. rapa, 4n, plant 3).

F. 36. Bilobed nectary. Note the vascular trace of phloem alone,penetrating the gland interior (arrows) (B. rapa, 4n, plant 3).

F. 37. Multi-lobed gland, at base showing confluence with lateralnectary (arrows) (B. napus, plant 3).

4nB. rapa

B. napus

nB. rapa

2nB. rapa

F. 38. Frequencies of different forms of median nectaries in B. rapa(n, 2n, 4n) and B. napus. For haploids, three plants were studied; forother lines, four plants each. The data represent the summation of 10flowers (20 glands) for each plant. 7, Gland peg-shaped (Figs 25 and33, right) ; *, gland fan-shaped (Figs 21 and 26) ; 7, gland crescent-shaped (Figs 24 and 34) ; +, gland oblong (Fig. 22) ; 8, gland elongate(Fig. 2) ; 7, gland filamentous (Fig. 33 left) ; 4, gland slightly lobed(Figs 15 left, 27, 28 and 35) ; 9, gland with accentuated lobing (Figs 23,29 and 36) ; 8, gland with two distinct lobes, furrow reaching nectarybase (Fig. 30) ; 5, gland with three or more lobes (Figs 31, 32 and 37).

flower (Davis et al., 1994). In these flowers, it was usual foreach lateral nectary to bear, on its adaxial surface, a single,relatively large nectar droplet. Each lateral location inhaploid flowers of B. rapa, which always bore unseparatednectarial tissue (Fig. 20), never bore two droplets of exudate.Flowers of 4n B. rapa, however, possessed the highestpercentage of isolated lobes of nectarial tissue at the lateralposition (Fig. 20), and this accounts for the high incidence

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of having two smaller nectar droplets per lateral side (Table2). Again, a detailed examination of Plant 1 is revealing.Only five of 22 flowers exuded a single nectar droplet at eachlateral location, with almost two-thirds of all lateral sidesbearing two smaller nectar droplets. In some examples offlowers from Group 3 (see above), the four droplets could beof different sizes and sugar contents (e.g. 63±9, 20±8 �s. 35±5,40±6 µg per droplet), or very similar (e.g. 32±9, 32±1 �s. 32±5,30±5 µg per droplet). But interestingly, despite the significantdifferences in nectary volume (above), no difference incombined nectar carbohydrate production (from laterallocations) per flower was detected, regardless of whetherflowers bore only two large droplets (Group 1: 172±1³33±6(s.e.) µg, n¯ 3 flowers), one large and two smaller drop-lets (Group 2: 167±0³22±8 µg, n¯ 4), or four small droplets(Group 3: 162±7³21±4 µg, n¯ 10; P" 0±7). This re-lationship also held for the other three plants of 4n B. rapa,and the four plants of B. napus ; the combined, smallerdroplets per nectary location were not significantly dif-ferent in carbohydrate content from the single, largedroplets.

DISCUSSION

The differences in floral morphological features reflect thevariable ploidy levels of the plants studied. Clear differencesin flower dry weights for n, 2n and 4n lines of B. rapa agreewith a photograph depicting flower sizes of B. napus (Kellerand Armstrong, 1978) and the statement of Ockendon(1984) for flowers of n and 2n plants of B. oleracea. Largerpetal size with increasing ploidy in B. rapa also agrees withdata for B. napus (Renard and Dosba, 1980). Significantlylarger pollen grains in 4n than in 2n plants of B. rapa accordwith a study of ploidy in Arabidopsis (Altmann et al., 1994).Moreover, the lack of pollen production in n plants of B.rapa agrees with earlier reports for this species (Baillie et al.,1992) and for B. napus (Keller and Armstrong, 1978;Renard and Dosba, 1980).

In parallel with the trends in flower weight and size offlower parts, average lateral nectary volume in B. rapaincreased with ploidy. However, although floral dimensionswere largest in B. napus, the average size of the lateralnectaries was significantly smaller than that of 4n B. rapa.This relationship of nectaries to other parts is of interestbecause, in floral ontogeny of the Brassicaceae, knowledgeto date (Sattler, 1973; Davis, 1992, 1994) indicates thatdevelopment of the nectaries occurs last. In Brassica, eachof the lateral glands is initiated between the filament basesof a short and two long (tetradynamous) stamens, isbordered by the claws of two petals, and is delimited aboveby the gynoecium. Therefore, the eventual size of the lateralnectaries may be governed strongly by the physicallimitations imposed by the earlier-established, surroundingfloral organs. Where space for the lateral nectary wasseverely restricted, two entirely isolated outgrowths ofnectarial tissue developed at the lateral location, either sideof the short stamen. Less commonly, nectarial tissue wasfound abaxial to the short stamen. That lateral nectaries areof equal sizes in B. napus and 2n plants of B. rapa may

indicate close similarities in the area of floral apicesremaining available to the glands. On the other hand, eachmedian nectary has its inception external to the junctionpoint of the filament bases of two long stamens, such thatthe size attained by median glands apparently is restrictedless.

Indeed, constraint imposed by the earlier-formed organsof the flower seems to be a major factor responsible for theconsiderable variation detected in nectary morphology andsize. The micrographs of sections show clearly the closeproximity between nectaries and bases of either petals orstamens, even to the point where slight interdigitation ofepidermes—one cell yielding to accommodate the other—isevident at the interface between organs (Figs 16–19, 34 and36). It seems clear that at least some forms of nectarymorphology in Brassica are not predetermined entirely, butrather are manifestations of opportunity. The resultingvariation in nectary morphology was evident within thesame gland type between ploidy levels, between plants of thesame ploidy and species, between flowers of the same plant,and even within a flower. Compared to characteristics ofother organ whorls in these flowers, morphological varia-bility among the nectaries ranks highly (see Table 1).

The remarkable functional aspect of Brassica nectariescan be seen to have high ecological significance. Despite theexistence of considerable variation in morphology, nectaryfunction was not impaired because over 99% of all nectarialtissue at the lateral location yielded nectar. Even in extremecases where two separated outgrowths of nectarial tissue(often unequal in size) occurred at the lateral position (e.g.4n B. rapa, B. napus), a direct supply of phloem was receivedby each outgrowth and an isolated, though smaller, nectardroplet was still collectable from each. Quantities of nectarsugar per lateral side of the flower, in turn, usually werecomparable to those on the opposite side, despite starkdifferences in nectary size and form.

For the first time in Brassica spp., the existence of narrowconnections of nectarial tissue between some adjacent lateraland median glands, has been revealed. Although the lateraland median nectarial outgrowths are usually solitary inBrassica, the small gaps between petals and stamens arefilled by merging lateral and median outgrowths that extendby marginal growth, as in Arabidopsis (Davis, 1992, 1994).Fourteen per cent of mature Brassica flowers, but par-ticularly those of 2n B. rapa, had at least one suchconnection. The lower incidence of these connections in nand 4n flowers may have different explanations. In theformer, smaller gland size may account for lateral andmedian glands often not meeting at their edges. In the latter,larger petals and stamen filaments block the merging oflateral and median glands in the zones between corolla andandroecium. Nonetheless, these connections can be com-monplace in mature flowers of other Brassicacean species(von Hayek, 1911; Norris, 1941), where they make itdifficult to interpret whether a flower has only one unitedgland or nectary (Davis, 1994), or whether, in the case ofglandular confluence, the outgrowths should still beregarded as separate nectaries (see Schmid, 1988, for asimilar discussion of nectary trichomes). Based on thedifferences in morphology and quantity of phloem that

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penetrates the gland interior, and ontogenetic disparities atthe two (lateral and median) nectarial locations inArabidopsis (Davis, 1992, 1994), it seems likely that twoseparate glandular systems are in operation in Brassicaceanflowers. Accordingly, we identify each outgrowth as anectary, whether confluence occurs or not. In fact, thisfavours the approach taken by Arber (1931a, b), who, uponfinding a continuous zone of glandular tissue in Capsellabursa-pastoris and Sisymbrium alliaria, such that theboundaries between ‘ individual glands could not be sharplydefined’, still referred to tissue proliferations at variouslocations, as individual ‘glands’.

Apart from the gross variability in their structure, theadded variability in nectarial connections within B. rapa,also has significance for taxonomy of the Brassicaceae. Forexample, even without changes in ploidy, some matureflowers (e.g. 2n B. rapa) fall into Norris’ (1941) category ofthe 4-nectary type, whereas other flowers from the same planthad nectaries of the annular type. If this classificationsystem is to be maintained for Brassicacean nectaries, thenit is recommended that several plants per species should beinvestigated, and proper allowance made for nectaryvariability within an individual species.

Nectary morphology in haploids was simple ; no medianglands were lobed, and very few lateral glands had furrows.On the other hand, multilobed lateral nectaries were detectedcommonly in flowers of 4n B. rapa, and secondarily in B.napus and 2n B. rapa. Only median glands of 4n B. rapa hadmultiple lobes. In Arabidopsis thaliana, lobing of nectarialtissue in young buds was detected almost as soon as suchtissue could be identified, and seemed to indicate more thanone original point of inception at the lateral position (Davis,1992). On balance, the evidence suggests that the prevalenceof furrowed or lobed glands in 4n plants over the 2n and nplants of B. rapa is a manifestation of multiple points ofgland inception, and may be a consequence of additionaltranscriptional signals for nectary development at the timeof gland initiation.

Therefore, in order to determine the basis for the lobingof nectaries, the physical constraints on the glands imposedby earlier-formed floral parts, and the nature of the narrowconnections between nectaries at the lateral and medianpositions, a detailed investigation of the ontogeny of flowersin the Brassicaceae having different ploidy, would appearvaluable. These aspects might be addressed better by alsoincluding species such as Arabidopsis thaliana, where flowersbearing lateral nectarial outgrowths abaxial to the shortstamens would probably experience fewer physicalrestrictions imposed by earlier-formed floral parts. Ac-cessibility of the richly-secreting lateral nectaries, beingconcealed only by the lateral sepals, would allow close,repeated examination of this important glandular tissue,from live material.

ACKNOWLEDGEMENTS

We thank Dr A. M. R. Ferrie for generously allowingaccess to the haploid plants of B. rapa, and Dr P. H.Williams for seed of the rapid-cycling lines. Discussionswith Dr J. King and Prof. Emeritus T. A. Steeves were very

helpful and much appreciated. Mrs S. Stone critical-pointdried most of the tissues, and Mr Y. Yano assisted withtechnical aspects of SEM. Mr D. Dyck and Mr M. Mierauassisted with preparation of most of the figures, and Mrs J.Smith, Mr J. Sullivan and Mrs M. Cowell cared for theplants. We appreciated the suggestions of Dr Jean Gerrathand two other, anonymous, reviewers, which clarified thetext. A.R.D. is very grateful to the Natural Sciences andEngineeringResearch Council of Canada, for a PostdoctoralFellowship.

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floral nectar secretion and ploidy in brassica rapa and b. napus

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