Ontogeny of Floral Organs in Flax 1079

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

O NTOGENY OF FLORAL ORGANS IN FLAX ( L INUM USITATISSIMUM ; LINACEAE)1

Lauren C. Schewe, Vipen K. Sawhney, and Arthur R. Davis 2

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 fl ower development of this species is lacking. Here we describe the pattern of initiation and a program of key developmental events in fl ax fl ower ontogeny. This study provides important fundamental information for future research in various aspects of fl ax 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 fi ve sepals originated in a helical pattern, followed evidently by simultaneous initiation of fi ve stamens and fi ve petals, the former opposite of the sepals and the latter alternate to them. The gynoecium, with fi ve carpels, was produced from the remaining, central region of the fl oral apex. Stamens at early stages were dominated by anther growth but fi laments 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 fl oral 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 fi lament bases. • Conclusions: This is the fi rst detailed study on fl ax fl oral organ development and has established a key of 12 developmental stages, which should be useful to fl ax researchers.

Key words: ﬂ ax; ﬂ ower development; Linum usitatissimum ; Linaceae; scanning electron microscopy.

Flax ( Linum usitatissimum L.) is an important crop worldwide, Each fl ower has fi ve sepals, fi ve petals, and fi ve stamens (Figs. with Canada leading the world in production and export of its oil 1, 2 ; see also Lay and Dybing, 1985 ). Petals are typically blue, ( FAO, 2010 ). Flax has been grown for at least 8000 yr both for oil but may vary from white to pink or pale to dark blue (Lay and (also called linseed oil) from seed and for fi ber for use in fabrics, Dybing, 1985). Petals and stamens alternate, and the bases of ropes, and other products (Simpson and Ogorzaly, 2001; Vaisey- stamens are widened to form a fused ring around the base of the Genser and Morris, 2003 ). In North America, fl ax is primarily gynoecium (Fig. 2; see also Williams, 1988). The petals are used in the production of oil, which is most commonly used narrow at their bases and insert into this ring. Small, fl at necta- in coating products such as paints and varnishes ( Levetin and ries are present at the widened bases of the stamens (Williams, McMahon, 1999; Dean, 2003). Flax oil is also used in the manu- 1988). The gynoecium has fi ve carpels, each divided by a false facturing of linoleum fl ooring. In addition, interest in fl ax oil and septum and producing up to two ovules (Williams, 1988; seeds as food products has increased due to their health benefi ts. Diederichsen and Richards, 2003 ). In most fl owers of L. Flax seeds contain relatively large amounts of α -linolenic acid, usitatissimum, the anthers encircle and reach over top of the an omega-3 fatty acid, which is considered essential for human stigmas, but in some varieties, the stigmas extend beyond the health (Vaisey-Genser and Morris, 2003). Despite the importance anthers slightly ( Dillman, 1938 ). of this crop, little is known about the early stages of fl ower devel- The anthers in L. usitatissimum fl owers dehisce before the opment in fl ax. An understanding of the pattern and growth of bud fully opens, but the fl ower does not self-pollinate immedi- fl oral organs should be benefi cial to researchers interested in ately because the anthers face outwards and are slightly dis- breeding, biotechnology, and molecular biology of fl ax. tanced from the stigmas until shortly after the fl ower opens Flowers of L. usitatissimum are terminal, complete and per- (Kadam and Patel, 1938; Williams et al., 1990; Williams, 1991). fect, and borne in panicles ( Lay and Dybing, 1985 ; Diederichsen Anthers open longitudinally while they are facing away from, and Richards, 2003) or in mixed sympodia (Weberling, 1989). and level with, the stigmas. As the fl ower opens, the stamens begin to twist, pushing the anthers together to form a cap over the stigmas ( Kadam and Patel, 1938 ). 1 Manuscript received 29 October 2010; revision accepted 31 March 2011. In two previous studies, keys of developmental stages for L. us- The authors thank Dr. G. Rowland, Crop Development Centre, for seeds itatissimum exist, but their focus on bud and fl ower development is of the fl ax cultivar CDC Bethune, Ms. J. Smith for help with plant growth, limited. Freer (1991) included fi ve stages of bud and infl orescence and Dr. G. Liu for assistance with SEM. They appreciated the helpful development: the appearance of buds in the leaf axils, the exten- comments of Dr. S. Tucker and an anonymous referee, which improved the sion of the pedicels, the development of the infl orescence, the revision. emergence of petals, and the opening of fl owers. In the key de- This study was supported by a Natural Sciences and Engineering Research Council (NSERC) of Canada Undergraduate Student Research scribed by Smith and Froment (1998) , no further characteristics Award to L.C.S., and NSERC Discovery Grants to V.K.S. and A.R.D. about fl ax fl ower development are available. On the other hand, 2 Author for correspondence (e-mail: [email protected] ) morphological features and detailed stages of fl ower development have been described for several important agricultural crops in- doi:10.3732/ajb.1000431 cluding brassicas ( Polowick and Sawhney, 1986 ; Huang et al.,

Stage 1 was characterized by the initiation of sepals, which occurred in a helical pattern, in either a clockwise or counter- clockwise fashion (Figs. 3, 4). All fi ve sepals were initiated before any other fl oral organs. The remaining portion of the fl oral 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 fi ve stamens had been established opposite the sepals, and all fi ve 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 fl oral apex, whereas petals were borne laterally ( Figs. 5 – 7 ). The remaining portion of the fl oral apex appeared fl attened ( 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 fl attened, fi ve- Figs. 1 – 2. Flowers of Linum usitatissimum cv. CDC Bethune. 1. Top view, showing fi ve-part whorls of fl ower at anthesis (stage 12). 2. Lateral lobed buttress occupying the center of the fl oral apex (Figs. view of open fl ower with sepals and petals removed to show fi lament bases 8– 10 ), and throughout this stage, it became more pronounced fused onto a staminal ring, dehisced anthers and relative heights of stamens and began to grow upward ( Fig. 9 ). Five distinct indentations and gynoecium. Bars = 1 mm. were formed between the developing septa of the gynoecium

2010 ), legumes ( Tucker, 1987 , 2003 ), tobacco ( Koltunow et al., Table 1. Average bud lengths and corresponding stages of fl oral 1990 ), corn ( Cheng et al., 1983 ), and the model species Arabidop- development of fl ax (Linum usitatissimum L. cv. CDC Bethune). The sis thaliana (Brassicaceae) ( Smyth et al., 1990 ). corresponding fi gures for each stage are indicated. The objectives of this study were to examine the pattern of Bud length initiation and growth of fl oral organs of fl ax by using scanning ± electron microscopy and to establish a key of developmental Stage (mm) SE N Structural developmental features Figures stages in relation to fl oral bud growth. 1 0.14 ± 0.01 8 Sepals initiated 3, 4 2 0.28 ± 0.03 11 Stamens and petals initiated 5 – 7 3 1.13 ± 0.27 7 Gynoecium initiated, bilobed 8 – 11 MATERIALS AND METHODS anthers and fi laments differentiated. Outer two sepals enclose bud. ± Plant material and growth conditions— Seeds of Linum usitatissimum L. 4 2.50 0.17 9 Anthers distinctly tetralobed. 12 – 14 (cv. CDC Bethune), obtained from Dr. G. Rowland of the Crop Development Ovules initiated and style growth begins. Centre, University of Saskatchewan, were germinated and grown throughout ± the year in Sunshine mix 1 soil (Sun Gro Horticulture Canada Ltd., Vancou- 5 4.36 0.30 7 Style growth encloses carpel 15 – 18 ver, British Columbia, Canada) in individual 5-in pots in a greenhouse with a bases, styles begin to grow upward. photoperiod of 16-h light and temperatures between 20° – 27° C. Gynoecium does not extend past the height of fi laments; petals approximately half the height of Measurement of buds and fl oral organs— Floral buds of varying sizes were stamens. excised and measured, and 12 developmental stages were designated based on 6 5.83 ± 0.19 15 Extensive petal development; 19, 20 bud size and key stages of fl oral development. Whole bud and fl oral organ petals more than half the height of lengths were measured for each bud, with at least seven buds per stage. Buds stamens. Style extension; stigmas and organs less than 1 mm were measured using scale bars on scanning electron reaching beyond height of fi laments. micrographs. Larger buds and dissected fl oral organs were measured using an 7 7.35 ± 0.23 13 Stigmas approaching the height 21, 25 Olympus SZ40 stereo microscope, and supplementary color images were pro- of stamens. Pigmentation of petals duced using a Dino-Eye Digital Eye Piece Camera. begins, petals level with stamens. Nectaries initiated. Scanning electron microscopy (SEM)— SEM was performed as previously 8 8.39 ± 0.14 9 Styles extend past height of 22 – 24, 26, 27 described by Wist and Davis (2008) . Flower buds were fi xed in 2% glutaralde- stamens, styles twist. Petals hyde in 25 mmol/L sodium phosphate buffer overnight, postfi xed with 1% os- enclose inner whorls. Nectaries mium tetroxide in the same buffer, and dehydrated using a graded acetone series. expand, fl ask-shaped. Buds were critical point dried in liquid carbon dioxide, secured to SEM stubs, and 9 8.50 ± 0.15 7 Filaments equal or greater in 28, 29 coated with gold using an Edwards S150B Sputter-Coater. Buds were examined length than anthers. Petals wound using a Philips 505 scanning electron microscope at 29 kV and the images photo- tightly around inner whorls. graphed on Fuji FP-100B B/W fi lm. All images were edited using Adobe (San Nectaries with mature stomata. Jose, California, USA) Photoshop CS5 and labeled using Adobe Illustrator CS5. 10 9.86 ± 0.21 14 Sepals separated slightly, petals 30, 31 protruding past the ends of sepals. Nectar present. RESULTS 11 11.91 ± 0.22 16 Anthers dehisce, petals extended 32 – 34 well past the sepals, wound Floral development — Floral development was arbitrarily loosely around inner whorls. Nectar present. separated into 12 developmental stages based on fl oral bud size 12 13.45 ± 0.24 11 Anthesis, petals refl exed. 1, 2, 35 – 37 and key ontogenetic events. July 2011] Schewe et al. — Ontogeny of floral organs in flax 1079

Figs. 3 –11. Scanning electron micrographs of Linum usitatissimum fl oral 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, fl oral apex; an, anther; b, bract; f, fi lament; g, gynoecium; p, petal; s, sepal; st, stamen.

(Fig. 10). Sepals grew rapidly during stages 2 and 3, with the (0.04 mm), and the buds averaged 1.13 mm in length ( Table two outermost sepals enclosing the bud by the beginning of 1, Fig. 38). stage 3. Stamen development was rapid in early stages, and Anthers had a distinctly tetra-lobed form in stage 4 ( Figs. 12, in stage 3, anthers and fi laments had differentiated ( Fig. 11 ) 13), with an average length of 0.26 mm, about double that of and anthers had become bilobed (Fig. 10). At stage 3, sta- the fi laments (0.12 mm) ( Figs. 12, 13, 38 ). The petals (0.17 mens (0.13 mm) were approximately twice the height of pet- mm) were located between the fi laments, and petal growth con- als (0.07 mm) and over 3-fold longer than the gynoecium tinued to lag behind that of stamens ( Fig. 12 ). The formation of 1080 American Journal of Botany [Vol. 98

Figs. 12 – 20. Scanning electron micrographs of Linum usitatissimum fl oral buds at stages 4– 6. Figs. 12– 14 . Stage 4. 12. Sepals removed to show anther and fi lament 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 magnifi cation, 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 fi laments. Bars = 0.1 mm. Figure abbreviations: an, anther; c, carpel; f, fi lament; g, gynoecium; o, ovule; p, petal; ps, socket into which the petal attaches; sa, possible staminode; se, septum; sl, style; sp, stigmatic papillae. July 2011] Schewe et al. — Ontogeny of floral organs in flax 1081 possible staminodes (rudimentary sterile stamens) sometimes Whereas mean bud length in stage 9 (8.50 mm) barely ex- began in stage 4 ( Fig. 14 ), although these small antipetalous ceeded that of stage 8 ( Table 1 , Fig. 38 ), indicating almost a outgrowths were not always present even by stage 5 (Fig. 15). cessation in sepal growth (Fig. 38), there were noteworthy The development of 10 ovules per ovary began in stage 4, as changes in other fl oral organs. In stage 9, fi lament elongation they grew laterally from each side of the fi ve developing septa was prominent; their average length (2.25 mm) now exceeded (Fig. 14). The tips of the fi ve carpels began to grow between that of anthers (2.03 mm; Figs. 28, 38). Growth of the gynoe- each septum and were the precursors to the styles and stigmas cium continued gradually, with the styles and stigmas (2.56 mm) ( Figs. 13, 14 ). On average, stage 4 gynoecia were 0.12 mm long accounting for the majority of the length of the gynoecium and fl oral buds were 2.5 mm in length ( Table 1 , Fig. 38 ). (4.94 mm; Fig. 28). Mature stomata (with pores now visible Stage 5 was characterized by the enclosure of the carpel between the two guard cells) were apparent on the nectaries at bases by the growth of the overarching styles (Figs. 15, 16). this stage ( Fig. 29 ) and petals (5.56 mm; Fig. 38 ) tightly wound The styles fi rst enclosed the center of the apex and then ex- the inner whorls of the bud, as seen in stage 8 ( Fig. 24 ). Sepals tended upward, often to the same height, but not beyond the had separated slightly so that petals became visible externally, stamen fi laments (Figs. 15, 16), and the bases of stamens had but had not extended past the sepal tips. fused to form a ring of tissue (Fig. 16). Papillate stigmas began Filament extension continued in stage 10, reaching an aver- developing on the tips of the styles at this stage ( Fig. 17 ). The age length of 4.20 mm, whereas the anthers had reached their anthers had elongated to 0.69 mm on average (Figs. 16, 38), and maximum average length of 2.32 mm (Figs. 30, 38). By the end gynoecia were 0.31 mm in length, compared to the fi laments of stage 10, the stamens were just below the tips of the stigmas (0.37 mm) ( Fig. 38 ). Petal growth accelerated in stage 5, with (Fig. 30). At the stamen bases, nectar was present on the nectary an average length of 0.56 mm, appearing to reach approxi- surfaces for the fi rst time, at this stage. Gynoecium growth con- mately half the height of the stamens (0.88 mm) (Fig. 38). Ad- tinued, in both the ovary (3.14 mm) and the styles and stigmas ditionally, petals grew laterally and enclosed the fi laments ( Fig. (3.89 mm) above. The petals (8.39 mm) were enlarged laterally 18). Petal attachment to the fused ring at the base of the fi la- and distally and began to protrude beyond the sepals in a tightly ments became apparent, with the point of attachment appearing wound cone ( Figs. 31, 38 ). Buds averaged 9.86 mm in length at as a socket on the ring between each fi lament ( Fig. 16 ). Buds of stage 10 ( Table 1 , Fig. 38 ). stage 5 were 4.36 mm long ( Table 1 , Fig. 38 ). At the beginning of stage 11, anthers had dehisced longitudi- In stage 6, mean petal length (1.04 mm) had attained more nally, and fi laments were either straight or twisted, with the an- than half the height of, but did not extend beyond the tips of, the thers reaching just below the tips of the stigmas (Fig. 32). During stamens (Figs. 19, 20, 38). Staminodes of variable size were this stage, fi laments extended farther and twisted around the gy- detectable along the staminal ring, in between the fertile sta- noecium, repositioning the anthers around the stigmas (Figs. 32, mens ( Figs. 19, 20 ). Style and stigma growth continued at this 33). The average stamen length was 7.5 mm and that of the gy- stage, with the tips of stigmas extending beyond the fi laments noecium was 7.08 mm (Fig. 38). Average anther length decreased (0.55 mm) below the enlarged tetrasporangiate anthers (Figs. from the previous stage to 1.69 mm ( Fig. 38 ) because they had 19, 20, 38). Gynoecia reached an average height of 0.71 mm, shrunken upon desiccation and dehiscence. The petals loosened and buds were 5.83 mm long ( Table 1 , Fig. 38 ). and extended well beyond the tip of the sepals (Fig. 34). Average Style elongation was prominent in stage 7, but was still short bud length in stage 11 was 11.91 mm (Table 1, Fig. 38). of the anther tips (Fig. 21). The average gynoecium length was In stage 12, the fl ower opened, and fi laments continued to 1.64 mm and that of stamens was 1.98 mm ( Fig. 38 ). Petals extend slightly and twist tightly around the gynoecium, placing began to develop blue pigment near their bases, with this pig- the anthers closer to, and in most cases covering, the stigmas mentation extending upward as the stage progressed, and with (Figs. 2, 35). Pollen grains, which were round and tricolpate an average height of 2.29 mm, were curled over the stamens (Fig. 36), had an average diameter of 50 µ m (± 0.2 µ m SE, ( Figs. 21, 38 ). The average bud length at stage 7 was 7.35 mm N = 10). Anthers had fully dehisced, and pollen grains were pre- ( Table 1 , Fig. 38 ). sent on both the anthers and the papillate stigmas (Figs. 2, 35), In stage 8, styles had extended past the tips of the anthers including some with germinated pollen tubes on the stigmas ( Figs. 22, 23 ) for the fi rst time ( Fig. 38 ), and were twisted to ( Fig. 37 ). Eventually petals and anthers abscised from the ma- varying degrees, although in some fl owers they remained ture fl ower. Average length of the mature fl ower at anthesis in straight ( Fig. 22 ). At this point, the combined style and stigma stage 12 was 13.45 mm ( Table 1 , Fig. 38 ). length matched that of the ovary (average 2.00 mm), thus con- tributing equally to the total length of the gynoecium ( Fig. 38 ). Petals had developed dark purple or blue pigmentation and had DISCUSSION continued to enlarge both laterally and distally (5.19 mm) so as to tightly enclose the inner whorls of the bud (Fig. 24). The This study examined the pattern of initiation and growth of fl o- versatile nature of the anthers became apparent at this stage, as ral organs of fl ax and established a key of developmental stages in they began to pivot at their attachment point to the fi lament, relation to fl oral bud growth. The fl oral development of several approximately halfway up the anther ( Fig. 23 ). Total stamen other agricultural crops has been studied in detail (Cheng et al., length was on average 3.22 mm. In stage 7, nectaries were ini- 1983 ; Polowick and Sawhney, 1986 ; Tucker, 1987 , 2003 ; tiated on the outer surface at the bases of each of the fi laments, Koltunow et al., 1990 ; Huang et al., 2010 ), and for the model spe- fi rst appearing as a curved, elongate bulge ( Fig. 25 ), later ex- cies Arabidopsis thaliana ( Smyth et al., 1990 ), and some of these panding and taking on a fl ask-like shape in stage 8 ( Fig. 26 ). have established a developmental program for use in genetic and Immature stomata were evident on the external surfaces of molecular studies. No detailed study on fl oral development in the ledge-like bulge at the bases of the nectaries by stage 8 fl ax has been conducted, and since it is an important crop grown (Fig. 27). Average bud length at stage 8 was 8.39 mm ( Table 1 , worldwide, an understanding of the initiation and development of Fig. 38 ). fl oral organs will be valuable for researchers in fl ax biology. 1082 American Journal of Botany [Vol. 98

The helical pattern of sepal initiation followed the known The arrangement of stamens and petals in fl ax was similar to phyllotaxis of fl ax ( Meicenheimer, 2006 ) as is common in ca- that observed in several species of Delea and Malina (Fabaceae) lyx formation in many other species ( Greyson, 1994 ). All fi ve such that petals are inserted into sockets between the fi ve fused stamens originated simultaneously and, on the basis of their stamens ( McMahon and Hufford, 2002 ). These sockets have larger relative size, may have preceded petal initiation, whereas been described as pedestals, which provide a location for the petal development certainly lagged behind that of stamens, sim- attachment of petals in both Linum and Hesperolinon (Linaceae) ilar to the pattern observed in many other fl owers, including and may indicate that the fused ring at the base of the androe- those of brassicas (Sattler, 1973; Polowick and Sawhney, 1986; cium may in fact originate from the receptacle rather than the Huang et al., 2010). In Arabidopsis thaliana (Smyth et al., stamens themselves ( Sharsmith, 1961 ). In L. usitatissimum, 1990 ) and other species ( Sattler, 1973 ), however, petals and petal abscission occurred by the day after anthesis, following a stamens are initiated simultaneously. The gynoecium was initi- similar pattern as in L. lewisii (Addicott, 1977). Petals abscised ated later with a fi ve-lobed ridge forming in the center of the readily with little mechanical disturbance, even at early stages, apex similar to the pattern of development as in Pelargonium indicating a weak attachment to the bud. zonale (Geraniaceae) (Sattler, 1973) and tomato (Chandra Initiation of stamens and petals in fl ax occurred prior to any Sekhar and Sawhney, 1984) . The initiation of the gynoecium fusion within or between whorls. Several previous studies after stamens is common in other species, with individual car- have identifi ed antipetalous staminodes in L. usitatissimum pels becoming apparent soon after initiation ( Greyson, 1994 ). ( Narayana and Rao, 1976 ) and other members of the Linaceae As in Solanum lycopersicum (Solanaceae), distinct locules are ( Narayana, 1964 ; Kumar, 1976 ; Narayana and Rao, 1977 ). formed as indentations on the raised fl oral apex (Chandra These short appendages have been described as both teeth- Sekhar and Sawhney, 1984 ). like and stub-like, both of which were observed in this study.

Figs. 21 – 27. Linum usitatissimum fl oral 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 fi laments. 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 fi lament showing initiation of nectary in stage 7. 26. Magnifi cation of Fig. 22 nectary showing fl ask 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 µ m in Fig. 27 . Figure abbreviations: is, immature stoma; n, nectary. July 2011] Schewe et al. — Ontogeny of floral organs in flax 1083

Stub-like protrusions from the fused staminal ring, also referred Narayana and Rao, 1976 , 1977 ; Weberling, 1989 ). That to as a staminal cup in reference to the closely related genus closely related members of the subfamily Hugonoideae Hesperolinon ( Sharsmith, 1961 ), were observed as early as (Linaceae) have 10 fertile stamens in two whorls (McDill et al., stage 4, but were only consistently present by stage 6. By stage 2009), provides further evidence for this designation as sta- 7, these outgrowths took on a toothed appearance, but varied minodes. However, some studies have referred to these struc- slightly in location and magnitude of invagination between tures as ligular appendages due to their lack of vascularization these staminodal projections. The classiﬁ cation of these ap- (Al-Nowaihi and Khalifa, 1973), and if the fused basal ring of pendages as staminodes is not deﬁ nitive: most prior work the androecium is formed from receptacle tissue, this would has referred to them as such ( Narayana, 1964 ; Kumar, 1976 ; more likely be the case.

Figs. 28– 34. Linum usitatissimum fl oral 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 fi lament 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 fi lament 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, fi laments 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. 1084 American Journal of Botany [Vol. 98

Figs. 35 – 37. Scanning electron micrographs of Linum usitatissimum ﬂ oral 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 µ m in Figs. 36, 37 . Figure abbreviations: an, anther; co, colpus; pg, pollen grain; pt, pollen tube; sp, stigmatic papilla.

Of all fl oral organs, nectaries were formed last, on the outer In conclusion, this study reports for the fi rst time a detailed surface of each of the fi laments, near their bases, as previously ontogeny of fl ax fl owers and has established a key of major observed ( Williams, 1988 ). The nectaries appeared to form events in the initiation and growth of fl oral organs. This study directly from the tissue of the fi lament. The appearance and structure of fl ax fl oral nectaries were similar to those in the Limnanthaceae ( Link, 1992 ). Stomata were present on the upper surface of the base of the fl ask-shaped gland, and nectar was present on this surface before anthesis. Although the nectaries are active in secretion, they tend not to greatly enhance cross- pollination in fl ax; fl ower-visiting insects tend to position themselves on sepals or pedicels, to collect nectar (Dillman, 1938). Thus, fl ax is primarily self-pollinated (i.e., autogamous), and further research on nectary structure and function in this species is warranted to determine whether fl oral attractiveness to forag- ing insects (potential pollinators) can be stimulated. The styles in most fl ax fl owers were twisted when fully mature and began to take on this form during extension. The de- gree of twisting varied, with some styles remaining completely untwisted, whereas others coiled extensively. The twisting of styles has been reported in Linum grandifl orum, a distylous species, but only after pollination by thrum pollen in a pin morph fl ower (Darwin, 1884; Lewis, 1943). Twisting of styles also oc- curs prior to pollination in Erythroxylum laurifolium (Eryth- roxylaceae), another distylous plant ( Pailler et al., 1998 ). The evolutionary signifi cance of this phenomenon is unknown. In fl ax, as the fl ower 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, fi laments began to twist imme- diately after the anthers dehisced, and pollen grains were observed on the stigmas prior to fl ower opening, although the fi nal position of anthers was not attained until after anthesis. Pollen grains of L. usitatissimum were round and tricolpate as described previ- Fig. 38. Growth of fl oral organs of Linum usitatissimum in relation to ously ( Saad, 1961 , 1962 ; Nair and Sharma, 1980 ). The average fl oral bud growth from organ initiation (stage 1) to anthesis (stage 12), dem- pollen grain diameter (50 µ m) was within the range reported by onstrating relative growth of the entire bud, sepals, petals, stamens, anthers, Nair and Sharma (1980) in L. usitatissimum , with variation be- fi laments, gynoecium, ovary and styles plus stigmas throughout develop- tween varieties. ment. Dimensions are given as mean ± SE with N ≥ 7 buds per stage. July 2011] Schewe et al. — Ontogeny of floral organs in flax 1085 should provide a foundation for research in other areas of fl ax McDill , J. , M. Repplinger , B. B. Simpson , and J. W. Kadereit . 2009 . biology including experimental developmental biology, devel- The phylogeny of Linum and Linaceae subfamily Linoideae, with im- opmental genetics, and molecular biology, and in fl ax breeding plications for their systematics, biogeography, and evolution of het- programs. We presently are examining in detail, the anatomical erostyly. Systematic Botany 34 : 386 – 405 . McMahon , M. , and L. Hufford . 2002 . Developmental morphology and ultrastructural changes during anther and pollen develop- and structural homology of corolla-androecium synorganization in ment in fl ax. the tribe Amorpheae (Fabaceae: Papilionoideae). American Journal of Botany 89 : 1884 – 1898 . LITERATURE CITED Meicenheimer , R. D. 2006 . Stem unit growth analysis of Linum usitatissimum (Linaceae) internode development. American Journal of Addicott , F. T. 1977 . Flower behavior in Linum lewisii: Some eco- Botany 93 : 55 – 63 . logical and physiological factors in opening and abscission of petals. Nair , P. K. K. , and R. K. Sharma . 1980 . Pollen morphology of Linum. American Midland Naturalist 97 : 321 – 332 . Journal of Palynology 16 : 9 – 52 . Al-Nowaihi , A. S. , and S. P. Khalifa . 1973 . Studies on some taxa of Narayana , L. L. 1964 . Contribution to the fl oral anatomy and em- the Geraniales II. Floral morphology of certain Linaceae, Rutaceae bryology of Linaceae. Journal of the Indian Botanical Society 43 : and Geraniaceae with a reference to the consistency of some charac- 343 – 357 . ters. Journal of the Indian Botanical Society 52 : 198 – 206 . Narayana , L. L. , and D. Rao . 1976 . Contributions to the fl oral anatomy Chandra Sekhar , K. N. , and V. K. Sawhney . 1984 . A scanning elec- of Linaceae 6. Journal of Japanese Botany 51 : 92 – 96 . tron microscope study of the development and surface features of fl o- Narayana , L. L. , and D. Rao . 1977 . Contributions to the fl oral anatomy ral organs of tomato (Lycopersicon esculentum ). Canadian Journal of of Linaceae 8. Journal of Japanese Botany 52 : 56 – 59 . Botany 62 : 2403 – 2413 . Pailler , T. , L. Humeau , and J. D. Thompson. 1998 . Distyly Cheng , P. C. , R. I. Greyson , and D. B. Walden . 1983 . Organ initiation and heteromorphic incompatibility in oceanic island species of and the development of unisexual fl owers in the tassel and ear of Zea Erythroxylum (Erythroxylaceae). Plant Systematics and Evolution mays. American Journal of Botany 70 : 450 – 462 . 213 : 187 – 198 . Darwin , C. 1884 . The different forms of fl owers on plants of the same Polowick , P. L. , and V. K. Sawhney . 1986 . A scanning electron micro- species. D. Appleton, New York, New York, USA. scope study on the initiation and development of fl oral organs of Brassica Dean , J. R. 2003 . Current market trend and economic importance of oil- napus (cv. Westar). American Journal of Botany 73 : 254 – 263 . seed fl ax. In A. D. Muir and N. D. Westcott [eds.], Flax: The genus Saad , S. I. 1961 . Pollen morphology and sporoderm stratifi cation in Linum , 275 – 291. Taylor and Francis, New York, New York, USA. Linum. Grana Palynologica 3 : 109 – 129 . Diederichsen , A. , and K. Richards . 2003 . Cultivated fl ax and the ge- Saad , S. I. 1962 . Palynologycal studies in the Linaceae. Pollen et Spores nus Linum L.: Taxonomy and germplasm conservation. In A. D. Muir 4 : 66 – 81 . and N. D. Westcott [eds.], Flax: The genus Linum , 22 – 54. Taylor and Sattler , R. 1973 . Organogenesis of fl owers. University of Toronto Francis, New York, New York, USA. Press, Toronto, Ontario, Canada. Dillman , A. C. 1938 . Natural crossing in fl ax. Journal of American Sharsmith , H. K. 1961 . The genus Hesperolinon (Linaceae). University Society of Agronomy 30 : 279 – 286 . of California Publications in Botany 32 : 235 – 314 . FAO [Food and Agriculture Organization]. 2010 . FAOSTAT. Website Simpson , B. B. , and M. C. Ogorzaly. 2001 . Economic botany: http://faostat.fao.org/site/535/DesktopDefault.aspx?PageID=535 [ac- Plants in our world, 3rd ed. McGraw Hill, Boston, Massachusetts, cessed 05 October 2010]. USA. Freer , J. B. S. 1991 . A development stage key for linseed (Linum us- Smith , J. M. , and M. A. Froment . 1998 . A growth stage key for winter itatissimum ). Aspects of Applied Biology 28 : 33 – 40 . linseed (Linum usitatissimum). Annals of Applied Biology 133 : 297 – 306 . Greyson , R. I. 1994 . The development of fl owers. Oxford University Smyth , D. R. , J. L. Bowman , and E. M. Meyerowitz . 1990 . Early Press, New York, New York, USA. development of Arabidopsis. Plant Cell 2 : 755 – 767 . Huang , L. , X. Zhao , T. Liu , H. Dong , and J. Cao . 2010 . Developmental Tucker , S. C. 1987 . Floral initiation and development in legumes. In characteristics of fl oral organs and pollen of Chinese cabbage (Brassica C. H. Stirton [ed.], Advances in legume systematics, part 3, 183– 239. campestris L. ssp. chinensis ). Plant Systematics and Evolution 286 : Royal Botanical Gardens, Kew, UK. 103 – 115 . Tucker , S. C. 2003 . Floral development in legumes. Plant Physiology Kadam , B. S. , and S. M. Patel . 1938 . Anthesis in fl ax. Journal of 131 : 911 – 926 . American Society of Agronomy 30 : 932 – 940 . Vaisey-Genser , M. , and D. H. Morris . 2003 . Introduction: History of Koltunow , A. M. , J. Truettner, K. H. Cox , M. Wallroth , and the cultivation and uses of fl axseed. In A. D. Muir and N. D. Westcott R. B. Goldberg . 1990 . Different temporal and spatial gene ex- [eds.], Flax: The genus Linum , 1 – 21. Taylor and Francis, New York, pression patterns occur during anther development. Plant Cell 2 : New York, USA. 1201 – 1224 . Weberling , F. 1989 . Morphology of fl owers and infl orescences. Kumar , A. 1976 . Studies in Geraniales II. The fl oral anatomy. Journal of Cambridge University Press, Cambridge, UK. the Indian Botanical Society 55 : 233 – 253 . Williams , I. H. 1988 . The pollination of linseed and fl ax. Bee World 69 : Lay , C. , and C. D. Dybing . 1985 . Linum usitatissimum. In A. H. Halevy 145 – 152 . [ed.], CRC handbook of fl owering, vol. III, 302– 305. CRC Press, Williams , I. H. 1991 . Floral phenology, pollination and fertilization in Boca Raton, Florida, USA. linseed. Annals of Applied Biology 28 : 27 – 32 . Levetin , E. , and K. Mcmahon . 1999 . Plants and society, 2nd ed. WCB/ Williams , I. H. , A. P. Martin , and S. J. Clark . 1990 . Pollination re- McGraw Hill, Boston, Massachusetts, USA. quirements of linseed ( Linum usitatissimum ). Journal of Agricultural Lewis , D. 1943 . The physiology of incompatibility in plants: II Linum Science 115 : 347 – 352 . grandifl orum. Annals of Botany 7 : 115 – 122 . Wist , T. J. , and A. R. Davis . 2008 . Floral structure and dynamics of Link , D. A. 1992 . The fl oral nectaries in the Limnanthaceae. Plant nectar production in Echinacea pallida var. angustifolia (Asteraceae). Systematics and Evolution 179 : 235 – 243 . International Journal of Plant Sciences 169 : 708 – 722 .