American Journal of Botany 98(7): 1077–1085. 2011.

ONTOGENY OF FLORAL ORGANS IN FLAX (LINUM USITATISSIMUM; LINACEAE)1 Lauren C. Schewe, Vipen K. Sawhney, and Arthur R. Davis2 Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, Saskatchewan S7N 5E2, Canada • Premise of the study: Flax (Linum usitatissimum) is an important crop worldwide; however, a detailed study on flower development of this species is lacking. Here we describe the pattern of initiation and a program of key developmental events in flax flower ontogeny. This study provides important fundamental information for future research in various aspects of flax biology and biotechnology. • Methods: Floral buds and organs were measured throughout development and examined using scanning electron microscopy. • Key results: Floral organs were initiated in the following sequence: sepals, stamens and petals, gynoecium, and nectaries. The five sepals originated in a helical pattern, followed evidently by simultaneous initiation of five stamens and five petals, the former opposite of the sepals and the latter alternate to them. The gynoecium, with five carpels, was produced from the remaining, central region of the floral apex. Stamens at early stages were dominated by anther growth but filaments elongated rapidly shortly before anthesis. Early gynoecium development occurred predominantly in the ovary, and ovule initiation began prior to enclosure of carpels. A characteristic feature was the twisted growth of styles, accompanied by the differentiation of papillate stigmas. Petal growth lagged behind that of other floral organs, but petals eventually grew rapidly to enclose the inner whorls after style elongation. Flask-shaped nectaries bearing stomata developed on the external surface of the filament bases. • Conclusions: This is the first detailed study on flax floral organ development and has established a key of 12 developmental stages, which should be useful to flax researchers. Key words: flax; flower development; Linum usitatissimum; Linaceae; scanning electron microscopy.

Flax (Linum usitatissimum L.) is an important crop worldwide, with Canada leading the world in production and export of its oil (FAO, 2010). Flax has been grown for at least 8000 yr both for oil (also called linseed oil) from seed and for fiber for use in fabrics, ropes, and other products (Simpson and Ogorzaly, 2001; VaiseyGenser and Morris, 2003). In North America, flax is primarily used in the production of oil, which is most commonly used in coating products such as paints and varnishes (Levetin and McMahon, 1999; Dean, 2003). Flax oil is also used in the manufacturing of linoleum flooring. In addition, interest in flax oil and seeds as food products has increased due to their health benefits. Flax seeds contain relatively large amounts of α-linolenic acid, an omega-3 fatty acid, which is considered essential for human health (Vaisey-Genser and Morris, 2003). Despite the importance of this crop, little is known about the early stages of flower development in flax. An understanding of the pattern and growth of floral organs should be beneficial to researchers interested in breeding, biotechnology, and molecular biology of flax. Flowers of L. usitatissimum are terminal, complete and perfect, and borne in panicles (Lay and Dybing, 1985; Diederichsen and Richards, 2003) or in mixed sympodia (Weberling, 1989). 1 Manuscript

received 29 October 2010; revision accepted 31 March 2011.

The authors thank Dr. G. Rowland, Crop Development Centre, for seeds of the flax cultivar CDC Bethune, Ms. J. Smith for help with plant growth, and Dr. G. Liu for assistance with SEM. They appreciated the helpful comments of Dr. S. Tucker and an anonymous referee, which improved the revision. This study was supported by a Natural Sciences and Engineering Research Council (NSERC) of Canada Undergraduate Student Research Award to L.C.S., and NSERC Discovery Grants to V.K.S. and A.R.D. 2 Author for correspondence (e-mail: [email protected]) doi:10.3732/ajb.1000431

Each flower has five sepals, five petals, and five stamens (Figs. 1, 2; see also Lay and Dybing, 1985). Petals are typically blue, but may vary from white to pink or pale to dark blue (Lay and Dybing, 1985). Petals and stamens alternate, and the bases of stamens are widened to form a fused ring around the base of the gynoecium (Fig. 2; see also Williams, 1988). The petals are narrow at their bases and insert into this ring. Small, flat nectaries are present at the widened bases of the stamens (Williams, 1988). The gynoecium has five carpels, each divided by a false septum and producing up to two ovules (Williams, 1988; Diederichsen and Richards, 2003). In most flowers of L. usitatissimum, the anthers encircle and reach over top of the stigmas, but in some varieties, the stigmas extend beyond the anthers slightly (Dillman, 1938). The anthers in L. usitatissimum flowers dehisce before the bud fully opens, but the flower does not self-pollinate immediately because the anthers face outwards and are slightly distanced from the stigmas until shortly after the flower opens (Kadam and Patel, 1938; Williams et al., 1990; Williams, 1991). Anthers open longitudinally while they are facing away from, and level with, the stigmas. As the flower opens, the stamens begin to twist, pushing the anthers together to form a cap over the stigmas (Kadam and Patel, 1938). In two previous studies, keys of developmental stages for L. usitatissimum exist, but their focus on bud and flower development is limited. Freer (1991) included five stages of bud and inflorescence development: the appearance of buds in the leaf axils, the extension of the pedicels, the development of the inflorescence, the emergence of petals, and the opening of flowers. In the key described by Smith and Froment (1998), no further characteristics about flax flower development are available. On the other hand, morphological features and detailed stages of flower development have been described for several important agricultural crops including brassicas (Polowick and Sawhney, 1986; Huang et al.,

American Journal of Botany 98(7): 1077–1085, 2011; http://www.amjbot.org/ © 2011 Botanical Society of America

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Figs. 1–2. Flowers of Linum usitatissimum cv. CDC Bethune. 1. Top view, showing five-part whorls of flower at anthesis (stage 12). 2. Lateral view of open flower with sepals and petals removed to show filament bases fused onto a staminal ring, dehisced anthers and relative heights of stamens and gynoecium. Bars = 1 mm.

2010), legumes (Tucker, 1987, 2003), tobacco (Koltunow et al., 1990), corn (Cheng et al., 1983), and the model species Arabidopsis thaliana (Brassicaceae) (Smyth et al., 1990). The objectives of this study were to examine the pattern of initiation and growth of floral organs of flax by using scanning electron microscopy and to establish a key of developmental stages in relation to floral bud growth.

Stage 1 was characterized by the initiation of sepals, which occurred in a helical pattern, in either a clockwise or counterclockwise fashion (Figs. 3, 4). All five sepals were initiated before any other floral organs. The remaining portion of the floral apex was dome-shaped (Figs. 3, 4), and at this stage, buds were on average 0.14 mm long (Table 1, Fig. 38). At stage 2, all five stamens had been established opposite the sepals, and all five petals had been initiated simultaneously in positions alternate to sepals (Figs. 5, 7). Apices bearing stamen primordia but lacking petal protuberances were never seen. However, based on a differential primordial size between the two adjacent whorls, it is possible that stamens were initiated sooner or concurrently to petals (Fig. 5). Stamens were originated in a more vertical position on the floral apex, whereas petals were borne laterally (Figs. 5–7). The remaining portion of the floral apex appeared flattened (Figs. 6, 7) and buds at this stage averaged 0.28 mm in length (Table 1, Fig. 38). At stage 3, the gynoecium was initiated as a flattened, fivelobed buttress occupying the center of the floral apex (Figs. 8–10), and throughout this stage, it became more pronounced and began to grow upward (Fig. 9). Five distinct indentations were formed between the developing septa of the gynoecium Table 1.

Average bud lengths and corresponding stages of floral development of flax (Linum usitatissimum L. cv. CDC Bethune). The corresponding figures for each stage are indicated.

Stage 1 2 3

MATERIALS AND METHODS Plant material and growth conditions—Seeds of Linum usitatissimum L. (cv. CDC Bethune), obtained from Dr. G. Rowland of the Crop Development Centre, University of Saskatchewan, were germinated and grown throughout the year in Sunshine mix 1 soil (Sun Gro Horticulture Canada Ltd., Vancouver, British Columbia, Canada) in individual 5-in pots in a greenhouse with a photoperiod of 16-h light and temperatures between 20°–27°C. Measurement of buds and floral organs—Floral buds of varying sizes were excised and measured, and 12 developmental stages were designated based on bud size and key stages of floral development. Whole bud and floral organ lengths were measured for each bud, with at least seven buds per stage. Buds and organs less than 1 mm were measured using scale bars on scanning electron micrographs. Larger buds and dissected floral organs were measured using an Olympus SZ40 stereo microscope, and supplementary color images were produced using a Dino-Eye Digital Eye Piece Camera.

4 5

6

7

Scanning electron microscopy (SEM)—SEM was performed as previously described by Wist and Davis (2008). Flower buds were fixed in 2% glutaraldehyde in 25 mmol/L sodium phosphate buffer overnight, postfixed with 1% osmium tetroxide in the same buffer, and dehydrated using a graded acetone series. Buds were critical point dried in liquid carbon dioxide, secured to SEM stubs, and coated with gold using an Edwards S150B Sputter-Coater. Buds were examined using a Philips 505 scanning electron microscope at 29 kV and the images photographed on Fuji FP-100B B/W film. All images were edited using Adobe (San Jose, California, USA) Photoshop CS5 and labeled using Adobe Illustrator CS5.

8

RESULTS

11

Floral development— Floral development was arbitrarily separated into 12 developmental stages based on floral bud size and key ontogenetic events.

9

10

12

Bud length (mm) ± SE

N

Structural developmental features

Figures

0.14 ± 0.01 8 Sepals initiated 3, 4 0.28 ± 0.03 11 Stamens and petals initiated 5–7 1.13 ± 0.27 7 Gynoecium initiated, bilobed 8–11 anthers and filaments differentiated. Outer two sepals enclose bud. 2.50 ± 0.17 9 Anthers distinctly tetralobed. 12–14 Ovules initiated and style growth begins. 4.36 ± 0.30 7 Style growth encloses carpel 15–18 bases, styles begin to grow upward. Gynoecium does not extend past the height of filaments; petals approximately half the height of stamens. 5.83 ± 0.19 15 Extensive petal development; 19, 20 petals more than half the height of stamens. Style extension; stigmas reaching beyond height of filaments. 7.35 ± 0.23 13 Stigmas approaching the height 21, 25 of stamens. Pigmentation of petals begins, petals level with stamens. Nectaries initiated. 8.39 ± 0.14 9 Styles extend past height of 22–24, 26, 27 stamens, styles twist. Petals enclose inner whorls. Nectaries expand, flask-shaped. 8.50 ± 0.15 7 Filaments equal or greater in 28, 29 length than anthers. Petals wound tightly around inner whorls. Nectaries with mature stomata. 9.86 ± 0.21 14 Sepals separated slightly, petals 30, 31 protruding past the ends of sepals. Nectar present. 11.91 ± 0.22 16 Anthers dehisce, petals extended 32–34 well past the sepals, wound loosely around inner whorls. Nectar present. 13.45 ± 0.24 11 Anthesis, petals reflexed. 1, 2, 35–37

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Figs. 3–11. Scanning electron micrographs of Linum usitatissimum floral buds at stages 1–3. 3. Stage 1 (early) showing sepal initiation. 4. Stage 1 (late). Numbering indicates helical initiation of sepals. 5. Early stage 2 with both petal and stamen primordia. 6. Stage 2 showing stamen initiation opposite sepals. 7. Stage 2; sepals detached to expose whorled initiation of stamens and petals and disparity in their growth. 8. Early stage 3; two sepals excised to show petal development lagging behind that of stamens and gynoecium initiation. 9. Late stage 3; lateral view to show the initiation of carpels around the central apex. 10. Late stage 3; top view of gynoecium development. 11. Stage 3; sepal removed to show stamen development ahead of carpels. Bars = 0.1 mm. Figure abbreviations: a, floral apex; an, anther; b, bract; f, filament; g, gynoecium; p, petal; s, sepal; st, stamen.

(Fig. 10). Sepals grew rapidly during stages 2 and 3, with the two outermost sepals enclosing the bud by the beginning of stage 3. Stamen development was rapid in early stages, and in stage 3, anthers and filaments had differentiated (Fig. 11) and anthers had become bilobed (Fig. 10). At stage 3, stamens (0.13 mm) were approximately twice the height of petals (0.07 mm) and over 3-fold longer than the gynoecium

(0.04 mm), and the buds averaged 1.13 mm in length (Table 1, Fig. 38). Anthers had a distinctly tetra-lobed form in stage 4 (Figs. 12, 13), with an average length of 0.26 mm, about double that of the filaments (0.12 mm) (Figs. 12, 13, 38). The petals (0.17 mm) were located between the filaments, and petal growth continued to lag behind that of stamens (Fig. 12). The formation of

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Figs. 12–20. Scanning electron micrographs of Linum usitatissimum floral buds at stages 4–6. Figs. 12–14. Stage 4. 12. Sepals removed to show anther and filament growth ahead of petal development and lobing of anthers. 13. Sepals and anthers detached to reveal gynoecium development and tetralobed anthers. 14. Gynoecium of Fig. 13 at higher magnification, showing carpel development and ovule initiation. Possible staminodes present. Figs. 15–18. Stage 5. 15. Sepals, petals, and stamens removed to show style growth enclosing the carpel bases (early). No staminodes evident. 16. Sepals plus two petals and one stamen removed to reveal upward growth of styles (late). 17. Styles, and stigmas with papillae forming. 18. Sepals removed to show petal growth reaching about half the height of stamens. 19. Stage 6 (early); sepals removed to show petals extending past half the height of the stamens. Petals and stamens dissected to reveal gynoecium growth. 20. Stage 6 (late); sepals detached to show petal growth nearly reaching the tips of anthers, petals, and portions of some stamens removed to show gynoecium growth past the height of filaments. Bars = 0.1 mm. Figure abbreviations: an, anther; c, carpel; f, filament; g, gynoecium; o, ovule; p, petal; ps, socket into which the petal attaches; sa, possible staminode; se, septum; sl, style; sp, stigmatic papillae.

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possible staminodes (rudimentary sterile stamens) sometimes began in stage 4 (Fig. 14), although these small antipetalous outgrowths were not always present even by stage 5 (Fig. 15). The development of 10 ovules per ovary began in stage 4, as they grew laterally from each side of the five developing septa (Fig. 14). The tips of the five carpels began to grow between each septum and were the precursors to the styles and stigmas (Figs. 13, 14). On average, stage 4 gynoecia were 0.12 mm long and floral buds were 2.5 mm in length (Table 1, Fig. 38). Stage 5 was characterized by the enclosure of the carpel bases by the growth of the overarching styles (Figs. 15, 16). The styles first enclosed the center of the apex and then extended upward, often to the same height, but not beyond the stamen filaments (Figs. 15, 16), and the bases of stamens had fused to form a ring of tissue (Fig. 16). Papillate stigmas began developing on the tips of the styles at this stage (Fig. 17). The anthers had elongated to 0.69 mm on average (Figs. 16, 38), and gynoecia were 0.31 mm in length, compared to the filaments (0.37 mm) (Fig. 38). Petal growth accelerated in stage 5, with an average length of 0.56 mm, appearing to reach approximately half the height of the stamens (0.88 mm) (Fig. 38). Additionally, petals grew laterally and enclosed the filaments (Fig. 18). Petal attachment to the fused ring at the base of the filaments became apparent, with the point of attachment appearing as a socket on the ring between each filament (Fig. 16). Buds of stage 5 were 4.36 mm long (Table 1, Fig. 38). In stage 6, mean petal length (1.04 mm) had attained more than half the height of, but did not extend beyond the tips of, the stamens (Figs. 19, 20, 38). Staminodes of variable size were detectable along the staminal ring, in between the fertile stamens (Figs. 19, 20). Style and stigma growth continued at this stage, with the tips of stigmas extending beyond the filaments (0.55 mm) below the enlarged tetrasporangiate anthers (Figs. 19, 20, 38). Gynoecia reached an average height of 0.71 mm, and buds were 5.83 mm long (Table 1, Fig. 38). Style elongation was prominent in stage 7, but was still short of the anther tips (Fig. 21). The average gynoecium length was 1.64 mm and that of stamens was 1.98 mm (Fig. 38). Petals began to develop blue pigment near their bases, with this pigmentation extending upward as the stage progressed, and with an average height of 2.29 mm, were curled over the stamens (Figs. 21, 38). The average bud length at stage 7 was 7.35 mm (Table 1, Fig. 38). In stage 8, styles had extended past the tips of the anthers (Figs. 22, 23) for the first time (Fig. 38), and were twisted to varying degrees, although in some flowers they remained straight (Fig. 22). At this point, the combined style and stigma length matched that of the ovary (average 2.00 mm), thus contributing equally to the total length of the gynoecium (Fig. 38). Petals had developed dark purple or blue pigmentation and had continued to enlarge both laterally and distally (5.19 mm) so as to tightly enclose the inner whorls of the bud (Fig. 24). The versatile nature of the anthers became apparent at this stage, as they began to pivot at their attachment point to the filament, approximately halfway up the anther (Fig. 23). Total stamen length was on average 3.22 mm. In stage 7, nectaries were initiated on the outer surface at the bases of each of the filaments, first appearing as a curved, elongate bulge (Fig. 25), later expanding and taking on a flask-like shape in stage 8 (Fig. 26). Immature stomata were evident on the external surfaces of the ledge-like bulge at the bases of the nectaries by stage 8 (Fig. 27). Average bud length at stage 8 was 8.39 mm (Table 1, Fig. 38).

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Whereas mean bud length in stage 9 (8.50 mm) barely exceeded that of stage 8 (Table 1, Fig. 38), indicating almost a cessation in sepal growth (Fig. 38), there were noteworthy changes in other floral organs. In stage 9, filament elongation was prominent; their average length (2.25 mm) now exceeded that of anthers (2.03 mm; Figs. 28, 38). Growth of the gynoecium continued gradually, with the styles and stigmas (2.56 mm) accounting for the majority of the length of the gynoecium (4.94 mm; Fig. 28). Mature stomata (with pores now visible between the two guard cells) were apparent on the nectaries at this stage (Fig. 29) and petals (5.56 mm; Fig. 38) tightly wound the inner whorls of the bud, as seen in stage 8 (Fig. 24). Sepals had separated slightly so that petals became visible externally, but had not extended past the sepal tips. Filament extension continued in stage 10, reaching an average length of 4.20 mm, whereas the anthers had reached their maximum average length of 2.32 mm (Figs. 30, 38). By the end of stage 10, the stamens were just below the tips of the stigmas (Fig. 30). At the stamen bases, nectar was present on the nectary surfaces for the first time, at this stage. Gynoecium growth continued, in both the ovary (3.14 mm) and the styles and stigmas (3.89 mm) above. The petals (8.39 mm) were enlarged laterally and distally and began to protrude beyond the sepals in a tightly wound cone (Figs. 31, 38). Buds averaged 9.86 mm in length at stage 10 (Table 1, Fig. 38). At the beginning of stage 11, anthers had dehisced longitudinally, and filaments were either straight or twisted, with the anthers reaching just below the tips of the stigmas (Fig. 32). During this stage, filaments extended farther and twisted around the gynoecium, repositioning the anthers around the stigmas (Figs. 32, 33). The average stamen length was 7.5 mm and that of the gynoecium was 7.08 mm (Fig. 38). Average anther length decreased from the previous stage to 1.69 mm (Fig. 38) because they had shrunken upon desiccation and dehiscence. The petals loosened and extended well beyond the tip of the sepals (Fig. 34). Average bud length in stage 11 was 11.91 mm (Table 1, Fig. 38). In stage 12, the flower opened, and filaments continued to extend slightly and twist tightly around the gynoecium, placing the anthers closer to, and in most cases covering, the stigmas (Figs. 2, 35). Pollen grains, which were round and tricolpate (Fig. 36), had an average diameter of 50 µm (±0.2 µm SE, N = 10). Anthers had fully dehisced, and pollen grains were present on both the anthers and the papillate stigmas (Figs. 2, 35), including some with germinated pollen tubes on the stigmas (Fig. 37). Eventually petals and anthers abscised from the mature flower. Average length of the mature flower at anthesis in stage 12 was 13.45 mm (Table 1, Fig. 38). DISCUSSION This study examined the pattern of initiation and growth of floral organs of flax and established a key of developmental stages in relation to floral bud growth. The floral development of several other agricultural crops has been studied in detail (Cheng et al., 1983; Polowick and Sawhney, 1986; Tucker, 1987, 2003; Koltunow et al., 1990; Huang et al., 2010), and for the model species Arabidopsis thaliana (Smyth et al., 1990), and some of these have established a developmental program for use in genetic and molecular studies. No detailed study on floral development in flax has been conducted, and since it is an important crop grown worldwide, an understanding of the initiation and development of floral organs will be valuable for researchers in flax biology.

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The helical pattern of sepal initiation followed the known phyllotaxis of flax (Meicenheimer, 2006) as is common in calyx formation in many other species (Greyson, 1994). All five stamens originated simultaneously and, on the basis of their larger relative size, may have preceded petal initiation, whereas petal development certainly lagged behind that of stamens, similar to the pattern observed in many other flowers, including those of brassicas (Sattler, 1973; Polowick and Sawhney, 1986; Huang et al., 2010). In Arabidopsis thaliana (Smyth et al., 1990) and other species (Sattler, 1973), however, petals and stamens are initiated simultaneously. The gynoecium was initiated later with a five-lobed ridge forming in the center of the apex similar to the pattern of development as in Pelargonium zonale (Geraniaceae) (Sattler, 1973) and tomato (Chandra Sekhar and Sawhney, 1984). The initiation of the gynoecium after stamens is common in other species, with individual carpels becoming apparent soon after initiation (Greyson, 1994). As in Solanum lycopersicum (Solanaceae), distinct locules are formed as indentations on the raised floral apex (Chandra Sekhar and Sawhney, 1984).

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The arrangement of stamens and petals in flax was similar to that observed in several species of Delea and Malina (Fabaceae) such that petals are inserted into sockets between the five fused stamens (McMahon and Hufford, 2002). These sockets have been described as pedestals, which provide a location for the attachment of petals in both Linum and Hesperolinon (Linaceae) and may indicate that the fused ring at the base of the androecium may in fact originate from the receptacle rather than the stamens themselves (Sharsmith, 1961). In L. usitatissimum, petal abscission occurred by the day after anthesis, following a similar pattern as in L. lewisii (Addicott, 1977). Petals abscised readily with little mechanical disturbance, even at early stages, indicating a weak attachment to the bud. Initiation of stamens and petals in flax occurred prior to any fusion within or between whorls. Several previous studies have identified antipetalous staminodes in L. usitatissimum (Narayana and Rao, 1976) and other members of the Linaceae (Narayana, 1964; Kumar, 1976; Narayana and Rao, 1977). These short appendages have been described as both teethlike and stub-like, both of which were observed in this study.

Figs. 21–27. Linum usitatissimum floral buds at stages 7 and 8. Figs. 21–23, 25–27. Scanning electron micrographs. Fig. 24. Photograph at stage 8, most sepals removed. 21. Stage 7 bud with sepals removed to expose enlarged petals curled over tips of anthers, some petals and anthers removed to show gynoecium reaching near the height of the stamens. Figs. 22 and 23. Stage 8 buds with perianth removed to reveal gynoecium and androecium growth. 22. Early in stage; styles barely past stamens, straight. Nectaries on filaments. 23. Late in stage; styles beyond stamens, very twisted. 24. Petals wound tightly around inner whorls of the bud, not extended past the sepal tips. Figs. 25–27. Floral nectaries. 25. Base of filament showing initiation of nectary in stage 7. 26. Magnification of Fig. 22 nectary showing flask shape in stage 8; arrow denotes location of developing stomata. 27. Immature stomata on upper surface of nectary in stage 8. Bars = 1 mm in Figs. 21–24; 0.1 mm in Figs. 25, 26; 10 µm in Fig. 27. Figure abbreviations: is, immature stoma; n, nectary.

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Stub-like protrusions from the fused staminal ring, also referred to as a staminal cup in reference to the closely related genus Hesperolinon (Sharsmith, 1961), were observed as early as stage 4, but were only consistently present by stage 6. By stage 7, these outgrowths took on a toothed appearance, but varied slightly in location and magnitude of invagination between these staminodal projections. The classification of these appendages as staminodes is not definitive: most prior work has referred to them as such (Narayana, 1964; Kumar, 1976;

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Narayana and Rao, 1976, 1977; Weberling, 1989). That closely related members of the subfamily Hugonoideae (Linaceae) have 10 fertile stamens in two whorls (McDill et al., 2009), provides further evidence for this designation as staminodes. However, some studies have referred to these structures as ligular appendages due to their lack of vascularization (Al-Nowaihi and Khalifa, 1973), and if the fused basal ring of the androecium is formed from receptacle tissue, this would more likely be the case.

Figs. 28–34. Linum usitatissimum floral buds at stages 9–11. Figs. 28–30, 32, and 33. Scanning electron micrographs. Figs. 31 and 34. Photographs of whole buds. 28. Stage 9 bud with perianth removed to show gynoecium and androecium growth, particularly filament extension. 29. Stage 9 nectary, with mature stomata on upper surface. 30. Stage 10 bud with perianth removed to show gynoecium and androecium growth, particularly filament extension. Filaments were straight and anthers were closed upon sampling, but twisted and dehisced, respectively, during critical point drying. 31. Stage 10. Petals remained tightly wound around inner whorls, but protrude beyond the tips of the sepals. 32. Anthers, with their stomia placed near, but not facing, the stigmas. 33. Late in stage 10, with anthers more widely dehisced, filaments further twisted, and some pollen present on stigmas. 34. Stage 11. Petals loosened and extending well past the sepal tips. Bar = 1 mm in Figs. 28, 30–34; Bar = 0.1 mm in Fig. 29. Figure abbreviations: ms, mature stoma; sg, stigma.

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Figs. 35–37. Scanning electron micrographs of Linum usitatissimum floral buds at stage 12. 35. Widely dehisced anther forming cap over stigmas; pollen grains on stigma surfaces. 36. Round, tricolpate pollen grain on surface of stigma. 37. Pollen grains on the stigmas, with one germinated pollen tube. Bar = 1 mm in Fig. 35; = 10 µm in Figs. 36, 37. Figure abbreviations: an, anther; co, colpus; pg, pollen grain; pt, pollen tube; sp, stigmatic papilla.

Of all floral organs, nectaries were formed last, on the outer surface of each of the filaments, near their bases, as previously observed (Williams, 1988). The nectaries appeared to form directly from the tissue of the filament. The appearance and structure of flax floral nectaries were similar to those in the Limnanthaceae (Link, 1992). Stomata were present on the upper surface of the base of the flask-shaped gland, and nectar was present on this surface before anthesis. Although the nectaries are active in secretion, they tend not to greatly enhance crosspollination in flax; flower-visiting insects tend to position themselves on sepals or pedicels, to collect nectar (Dillman, 1938). Thus, flax is primarily self-pollinated (i.e., autogamous), and further research on nectary structure and function in this species is warranted to determine whether floral attractiveness to foraging insects (potential pollinators) can be stimulated. The styles in most flax flowers were twisted when fully mature and began to take on this form during extension. The degree of twisting varied, with some styles remaining completely untwisted, whereas others coiled extensively. The twisting of styles has been reported in Linum grandiflorum, a distylous species, but only after pollination by thrum pollen in a pin morph flower (Darwin, 1884; Lewis, 1943). Twisting of styles also occurs prior to pollination in Erythroxylum laurifolium (Erythroxylaceae), another distylous plant (Pailler et al., 1998). The evolutionary significance of this phenomenon is unknown. In flax, as the flower opens, the twisting of stamens to form a cap over the stigmas and to deposit pollen has been reported (Kadam and Patel, 1938; Williams et al., 1990; Williams, 1991). However, in cv. CDC Bethune, filaments began to twist immediately after the anthers dehisced, and pollen grains were observed on the stigmas prior to flower opening, although the final position of anthers was not attained until after anthesis. Pollen grains of L. usitatissimum were round and tricolpate as described previously (Saad, 1961, 1962; Nair and Sharma, 1980). The average pollen grain diameter (50 µm) was within the range reported by Nair and Sharma (1980) in L. usitatissimum, with variation between varieties.

In conclusion, this study reports for the first time a detailed ontogeny of flax flowers and has established a key of major events in the initiation and growth of floral organs. This study

Fig. 38. Growth of floral organs of Linum usitatissimum in relation to floral bud growth from organ initiation (stage 1) to anthesis (stage 12), demonstrating relative growth of the entire bud, sepals, petals, stamens, anthers, filaments, gynoecium, ovary and styles plus stigmas throughout development. Dimensions are given as mean ± SE with N ≥ 7 buds per stage.

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Schewe et al.—Ontogeny of floral organs in flax

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