BotanicalJournal of the Linnean Society (1995), 179: 35-43. With 12 figures

Reproductive biology of Nymjhaea caflemis Thunb. var. xanxibariensis (Casp.) Verdc. (Nymphaeaceae)

ISABELLE ORBAN AND JULES BOUHARMONT

Department of Plant Science, Cytogenetic laboratory, 5 bte 13 place Croix-du-Sud, B- 1348 Louvain-la-Neuve, Belgium

Received February 1995, accepted for publication May 1995

Anthesis in Nymphaea capensis var. mwibariensis is diurnal with flowers opening and closing for three consecutive days. On the first day of anthesis, the stigmatic papillae secrete fluid and the outermost anthers are dehiscent. On the second day of anthesis the stamens form a cone above the dry stigmatic cup. The middle stamens open and turn outward. On the third day of flowering, all the stamens open and the dry stigmatic cup is exposed. The flowers are homogamous and not protogynous as the other Nymphaea. The gynoecium of the self- compatible N. capemis var. wnzjbariensis, is characterized by a wet papillate stigma, a short hollow style, and secretory cells on the ventral surface of the ovary. The pollen is released on the receptive stigma. Following initial growth in intercellular spaces in the transmitting tract of the stigma, pollen tubes travel through the stylar canal and into the ovary. 0 1995 The Linnean Society of London

ADDITIONAL KEY WORDS:-floral biology - pollen tube - pollination - style structure.

CONTENTS

Introduction . . . . . . . . . Material and methods . . . . . . . Results . . . . . . . . . .

Gynoecial structure and pollen tube growth Discussion . . . . . . . . . Acknowledgements . . . . . . . References . . . . . . . . .

Floral biology . . . . . . . .

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INTRODUCTION

Nymphaea, with about 40 species of waterlilies, is the largest and most widely distributed genus in the Nymphaeaceae. They are distributed on all continents except Antarctica. The classification of Conard (1905), still generally accepted, recognized five subgenera: the north-temperature subgenus Nymphaea, neotropical subgenus Hydrocallis, palaeotropical subgenus Lotos of the syncarpous group, the Australian subgenus Anecphya and the pantropical subgenus Brachywras of the apocarpous group.

This paper discusses the reproductive biology of N. capensis Thunb. var. 35

0024-4074/95/090035+09 $12.00/0 0 1995 The Linnean Society of London

36 I. ORBAN AND J. BOUHARMONT

,vwibariensis (Casp.) Verdc. from the subgenus Brachyceras. Differences within and among the subgenus with regard to floral biology may contribute to understanding of the reproductive biology of Nymphaeu. We describe also the structure of the gynoecial tissues and the growth of the pollen tube to the ovules. Nymphueu has a meagre literature in this area of the structural aspects of the breeding system. In particular, there is little information on the course that the pollen tubes takes in the gynoecium.

For practical reasons, a basic knowledge of the events in the floral biology and progamic phase is critical for successful plant breeding programmes.

MATERIAL AND METHODS

Plants of N. capensis var. zunzibariensis were grown in greenhouses in Louvain-la-Neuve, Belgium. Floral buds of various ages were selected. The flowers were emasculated before anthesis and artificially pollinated on the day of anthesis. Pollinated flowers were removed 1 and 6 h after the artificial pollination. All the samples were fixed by Carnoy's solution (70% Alcohol, glacial acetic acid, 3 : l v/v). After 12 h of fixation, the samples were stored in 70% alcohol.

For light microscopy, the carpels were isolated from the flowers. The samples were processed through tertiary butyl alcohol series and the usual embedding procedures were followed. Sections were cut at 12 pm thickness on a rotary microtome and were stained with iron alun-haematoxylin and fast green (Jensen, 1962).

The pollen tube entry into the gynoecium was observed by fluorescence microscope using aniline blue staining. After rehydration, sections were stained in a 0.1% solution of water-soluble aniline blue dye (Anilinblau WS, Merck 1275) in 0.1 N K3P04 for 1 h. The styles were mounted in a few drops of the staining medium.

For squashes in aniline blue, the isolated carpels were rinsed in water and treated in a nearly saturated aqueous solution of sodium hydroxide (8 N) for 2 h. Staining was accomplished in 0.1% solution of water soluble aniline blue dye in 0.1 N K3P04 for 1 h (Martin, 1959). For observation the carpels were mounted in a few drops of the staining medium on clean glass slides and split longitudinally. The ovules were removed with fine forceps and covered with a cover slip.

Observations were made using UV excitation in a Polyvar (Reichert-Jung) fluorescence microscope equipped for epi-illumination. A 200 watt mercury lamp (Osram HBO) with appropriate ultraviolet filter was found to be satisfactory for the purpose. Photomicrographs employed TMX- 100 Kodak 21" DIN/100 ASA film.

To elucidate the reproductive mechanisms of N. cupensis var. wwiburiensis, the following caging experiments were performed:

(1) A control set of five buds was left uncaged. (2) Five preanthesis buds were caged, using containers. These buds were

emasculated and then left undisturbed. (3) To study stigma receptivity, 20 preanthesis buds were caged and

emasculated. The pollen grains of dehiscing anthers removed from second

SEXUAL REPRODUCTION IN NFMPHAEA 37

day flowers were sampled with a brush and deposited on the dry stigma of five preanthesis buds. Five emasculated flowers on first day of anthesis were artificially pollinated. The pollen grains were removed from second day flowers with a brush and submerged in the stigmatic fluid from the five emasculated flowers on the first day of anthesis. Five emasculated flowers on the second day of anthesis and five on the third day of anthesis were also artificially pollinated. The pollen grains were removed from other second day flowers and deposited on the dry stigma from the emasculated flowers on the second and third days of anthesis. The pollinated flowers were then recaged.

RESULTS

Floral biology

N. capensis var. wm'bariensis is a blue flowering species. The calyx is composed of four green sepals with a purple margin and a deep purplish- blue inner surface. The corolla has 18 to 24 light blue petals. The androecium is composed of approximately 50 yellow stamens. The outermost stamens are large, petaloid and oblanceolate with broad, flat filaments and small anthers. Progressing inward, the filaments become thicker in depth, thinner in width and shorter in length while the anthers become larger and more convolute. The gynoecium has 14 carpels. The latter form a ring embedded in cup-shaped receptacular and appendicular tissue to which the appendicular organs are attached laterally. The upper surface of each carpel contributes a ray of stigmatic tissue to the stigmatic disk, which tops the ovary and this ray usually terminates abaxially in a free appendage termed the carpellary appendage or carpellary style (Fig. 1).

Flowers of N. capemis var. wnzibariensis bloom for three consecutive days. On the first day, flowers (Fig. 2) are partially open. The calyx and the corolla together with the stamens are positioned to form a funnel. Examination of 10 opened flowers showed that pollen had already been released from 12 outermost stamens. At the bottom of the funnel is the cup-shaped gynoecium filled with 300 pl of stigmatic secretion. On the second day of anthesis (Fig. 3), the perianth and the outermost stamens reflex to their fullest extent. The indehiscent stamens and carpellary styles slope inward forming a cone over the now dry stigmatic cup. The stamens ripen gradually from the outermost anthers to those nearest the dry stigmatic cup. Twenty-six middle stamens begin to open and bend away from the cone of innermost indehiscent stamens. On the third day (Fig. 4), all the stamens are open and turn outward. On the fourth day, flowers remain closed and begin to submerge due to coiling of the peduncle.

The average number of seeds produced by the pollinated flowers on the first day of anthesis is 1424 per fruit. None of the pollinated buds, the pollinated flowers on the second day of anthesis nor the pollinated flowers on the third day of anthesis set seeds (Table 1).

Gynoecial structure and pollen tube growth

To study gynoecial structure, the isolated carpels were cut longitudinally (Figs 5, 6).

38 I. ORBAN AND J. BOUHARMONT

Figures 1-4. Flowers of h? capensis var. wn&riensk Fig. 1. Schematic drawing of longitudinal section of quartered portion of flower, (sep) sepals, (pet) petals, (s) stamen, (ca) carpellary appendage, (sti) stigma, (fa) floral axis, (c) carpel. Fig. 2. First-day flower. Fig. 3. Second-day flower. Fig. 4. Third-day flower. Scale bars = 20 mm.

In the mature gynoecium, each stigma is folded longitudinally, thereby forming a groove along the adaxial face. The abaxial epidermis is composed of elongate parenchyma cells, the slightly recurved adaxial surface is covered with stigmatic papillae (Fig. 7). Stigmatic cells form a layer with dense cytoplasm just beneath the stigma surface and lead into a hollow canal (Fig. 8). The canal is lined with a secretory epidermis. The secretory transmitting tract is restricted to the ventral side of the canal. The stylar canal opens into the ovary and the transmitting tract becomes the region where carpel

SEXUAL REPRODUCTION IN NYkfPh54EA 39

TABLE 1. Seed-set of natural and artificial pollinated flowers

Number of Average number of Experimental group flowers seeds per fruit

Control (no caging) Emasculated buds and left undisturbed Pollinated buds 1st day pollinated stigma 2nd day pollinated stigma 3rd day pollinated stigma

613 0 0

1424 0 0

walls meet to form the placenta. All of the carpel is plentifully covered with secretion (Fig. 9).

Pollen is deposited on the stigma of first day flowers. The cup-shaped gynoecium is filled with stigmatic secretion. Then the pollen grains germinate on the stigmatic surface and pollen tubes penetrate through the papillae into the solid transmitting tract of the style. The tubes continue to grow to the short stylar canal. One hour after the pollination, the tubes are at the entrance of the ovary (Fig. 10). Six hours after pollination, the tubes apparently travel through the canal and into the ovary (Fig. 11) until an ovule is encountered (Fig. 12).

DISCUSSION

The sequence of events leading to pollination in N. capensis var. wwihariensis does not closely parallel the events described in other species of Nymphaea subgenus Brachyceras, e.g. N. stellata Willd. (Scott Elliot, 1891 ; Schneider, 1982); N. citrina Peter (Faegri & Van der Pijl, 1979); N. colorata Peter (Schmucker, 1933, 1935), N. capensis Thunb. (Fox-Wilson, 1937). During the first day of anthesis, the flowers are in female stage. All anthers are indehiscent. The stigma secretes a fluid which fills the perigynous cup. During the second and third day of anthesis, the flowers are in male stage. The stamens form a closed cone over the stigma surface which is no longer accessible. Then the stamens develop gradually from the outer part to the dry stigmatic cup. As each stamen ripens, it turns outward. Here the species are not protogynous but as in N. ampla (Salisb.) DC. (Prance & Anderson, 1976), the stamens in the flower of N. capensis var. zanzibariensis liberate pollen at the first day of anthesis.

The period of receptivity lasts the first day of anthesis. It is determinable by the production of seeds when the flowers are artificially pollinated on the first day of anthesis. The receptivity corresponds with the secretion of fluid which covers the stigmatic surface on the first day of flowering.

Although Heslop-Harrison & Shivanna (1977), Cronquist (1981) and Dalgren & Clifford (1982) consider the Nymphaea stigma as the dry type, the stigma bears a copious secretion of exudate. This secretion on the receptive surface of the gynoecium is similar to the secretion of the species with a wet-type stigma.

The stigma of the first day flowers has multicellular uniseriate papillae as described in species with dry stigmas by Heslop-Harrison & Shivanna (1977),

I. ORBAN AND J. BOUHARMONT , stigma

7

papjllae

8

Figures 5-8. Sections of carpels of N. capensir var. taw'bariensii. Fig. 5. Schematic drawing showing one whole carpel. Scale bar = 1 mm. Fig. 6. Longitudinal section of one carpel. Scale bar = 600 pm. Fig. 7. Longitudinal section of the carpel showing the stigma and the upper portion of style. Scale bar = 100 pm. Fig. 8. Longitudinal section through the stylar canal showing the transmitting tissue. Scale bar= 100 pm.

SEXUAL REPRODUCTION IN NYMPHAE.4

Figures 9-12. Carpel, pollinated gynoecia and ovule of N. capemis var. wnzibarimis. Fig. 9. Longitudinal section of one carpel showing the stigma and the stylar canal. Scale bar= 100 pm. Figs 10-11. Fluorescence micrographs of longitudinal sections of pollinated gynoecia. Fig. 10, Pollen tubes in one carpel one hour after pollination. Scale bar = 50 pm. Fig. 11, Pollen tubes in one carpel six hours after pollinafion. Scale bar=50 pm. Fig. 12. Pollen tube penetrating an ovule six hours after pollination. Scale bar = 50 pm.

41

42 I. ORBAN AND J. BOUHARMONT

Helsop-Harrison (198 1) but the stigmatic surface of Nymphaea secretes a copious exudate.

This receptive surface is supported by a secretory tissue. The transmitting tissue opens on a short stylar canal. A layer of cells with dense cytoplasm delimits the canal which is filled with a secretion. This structure of the gynoecium is similar to species with hollow styles. The canal is only delimited by one layer of differentiated cells in Citrus Zimon (L.) Burm. (Ciampolini et aZ., 1981), Ornithogdurn caudatum Ait. (Tilton & Horner, 1980) Gasteria uerrucosa (Mill.) H. Duval (Willemse & Franssen-Verheijen, 1986, 1988) and LiZium longyorum Thunb. (Rosen & Thomas, 1970; Dashek, Thomas & Rosen, 1971). For these species, the substance in the canal is secreted by special cells which line the stylar canal and have useful elements for pollen tube growth (Willemse & Franssen-Verheijen, 1986). For N. capensis var. ,wzzibariensis, the secretion is observed at anthesis. The transmitting tissue continues in the ovary on the surface of the placenta. Stigma, stylar and carpellary secretions most probably have a part in the receptivity of the pollen, provide nutrients for the growth of the pollen tube, are involved in the recognition of the compatible pollen or pollen tube, and the arrival of the pollen tube at the micropyle.

ACKNOWLEDGEMENTS

We are greatly indebted to Prof. L. Waterkeyn and G. Garot for the photographs.

REFERENCES

Ciamplini F, Cresti M, Ssrfatti G, T i e d A. 1981. Ultrastructure of the stylar canal cells of C i t m limon [Rutaceae). Plant Sqstemtic Evolution 138 263-274.

Canard *. 1905.' The Witer-lilies: A monograph of the genus Nymphaea. Publication Carnegie Institute 4: 1-279.

Cronquist A. 1981. An integrated system of clarification offloweringplants. New York Columbia University

Dahlgren NUT, CWord HT. 1982. Monocotykdm: A comparative study. London: Academic Press. Dashek WV, Thomas HR, Rosen WG. 1971. Secretory cells of Lily pistils. 11. Electron microscope

Faegri K, Van der Pijl L. 1979. The principles of pollination ecology. 3rd ed. New York: Pergamon Press

Fox-Wilson G. 1937. Insects captured in flowers of Nymphaea capensis Thunb. Journal of Entomology 12:

Heslop-Harrison Y. 1981. Stigma characteristics and angiosperm taxonomy. Nordic Journal of Botany 1:

Heslop-Harrlson Y, Shivanna KR. 1977. The receptive stigma of the Angiosperm stigma. Annals of

Jensen WA. 1962. Botanical histochistry. Principles and practice. San Francisco: Freeman. Martin FW. 1959. Staining and observing pollen tube in the style by means of fluorescence. Stain

Press.

cytochemistry of canal cells. American Journal of Botany 58: 909-920.

Ltd.

3 1 3 9 .

401-480.

Botany 41: 1233-1258.

Technolorn 34: 125-128. Prance GT; Anderson AB. 1976. Studies of the floral biology of tropical Nymphaeaceae. Acta Amazonica

6: 163-170. Rosen WG, Thomas HR. 1970. Secretory cells of Lily pistils. I. Fine structure and function. American

Schmucker Th. 1933. Zur Blutenbiologie tropischer Nymphaea-Arten. 11. Bor als entscheidender Faktor.

Schmucker Th. 1935. Uber der Einfluss von Borsaure auf Pflanzen, insebesondere keimende

Journal of Botany 57: 1108-1 114.

Phnta 18: 641-650.

pollenkomer. Planta 23: 264-283.

SEXUAL REPRODUCTION IN NYMPHAEA 43

Schneider EL. 1982. Notes on the floral biology of Nymphaea elegans (Nymphaeaceae) in Texas. Aquatic

Scott Elliot GF. 1891. Notes on the fertilization of South Ajican and Madagarcar jlowering plunk Annals

Tilton VR, Horner HT. 1980. Stigma, style and obturator of Ornithogalum caudatum and their function

Willemse MTM, Fransen-Verheijen MAW. 1986. Stylar development in the open flower of Garteria

Willemse MTM, Fransen-Verheijen MAW. 1988. Pollen tube growth and its pathway in Garteria

Botany 12: 197-200.

of Botany 5: 334-335.

in the reproductive process. American Journal of Botany 67: 1113-1131.

uerrucosa (Mill.) H. Duval. Acta Botanica Neerlundica 35: 297-309.

uerrucosa (Mill.) H. Duval. Phytomorphology 38: 127-132.