Bull. Natl. Mus. Nat. Sci., Ser. B, 36(2), pp. 33–37, May 22, 2010

Dark Material Accumulation and Sclerotization During Seed CoatFormation in Vanilla planifolia Jacks. ex Andrews (Orchidaceae)
Goro Nishimura1,2* and Tomohisa Yukawa321
Keisen Jogakuen University, 2–10–1 Minamino, Tama, Tokyo, 206–8586 Japan
Research Associate, Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki, 305–0005 Japan3
Department of Botany, National Museum of Nature and Science,
Amakubo 4–1–1, Tsukuba, Ibaraki, 305–0005 Japan* E-mail: nishimu@keisen.ac.jp(Received 17 February 2010; accepted 24 March 2010)

Abstract Vanilla planifolia Jacks. ex Andrews has the sclerotic seed coat, an exceptional character state of seed coat in the Orchidaceae. We observed seed coat formation of the species in every
10 days after pollination. The ovule develops into a nucellar filament with 6 to 8 nucellar cells at
the time of anthesis prior to artificial pollination. The inner integument differentiates at 20 days
after pollination. The outer integument differentiates at 30 days after pollination. The cells of theoutermost layer of the outer integument start to thicken at 40 days after pollination. Dark material
starts to accumulate in the outer and lateral cell walls of the outermost layer of the outer integument at 50 days after pollination. The dark material accumulates further at 60 to 80 days, while theinner integument has degenerated at 80 days. The thickened cell walls with dark material occupy
the whole cell cavity and the cells become sclerotic at 120 days after pollination. The inner layercells of the outer integument have degenerated and only the outermost layer of the outer integument remains as the seed coat at 180 days after pollination when the seed matures.
Key words : integument, Orchidaceae, ovule, sclerotization, seed coat, Vanilla.

Introduction
In most orchid species the seed coat usuallyconsists of a transparent, single cell layer (Wirthand Withner, 1959). Exceptionally, the seed coatthickens and dark material accumulates as observed in Apostasia nipponica Masam.
(Nishimura and Tamura, 1993), Selenipedium
chica Rchb.f. (Garay, 1960), Vanilla planifolia(Swamy, 1947), and Galeola (Burgeff, 1936).
Developmental process of the opaque and sclerotic seed coat of orchids has been scarcely
known. However, the following two observations
indicate that the structure of the sclerotic seedcoat in orchids is diversified. Nishimura andTamura (1993) clarified that this type of seedcoat in A. nipponica consists of two layers of theouter integument. The outer layer is thin andtransparent, and the inner layer thickens andturns opaque. On the other hand, Swamy (1947)
reported in V. planifolia that the inner and outer
integument and even nucellus tissue take part in
the seed coat formation and the dark material
accumulates in the outermost layer of the outer
integument. However, he did not show accumulating process of the dark material in detail andthe observation did not incorporate time scale
during the ovule development.
We therefore observed the seed coat formationof Vanilla planifolia every 10 days after the pollination with emphasis on the accumulatingprocess of the dark material in the integument.
Materials and Methods
The materials of Vanilla planifolia were culti-

34

Goro Nishimura and Tomohisa YukawaFigs. 1–9. Development of ovule in Vanilla planifoloia I. 1. Development of the ovule primordium at anthesis
prior to artificial pollination (longitudinal section). Note two nucellar cells covered with the epidermis. 2. The
nucellar cells arrange in line at anthesis prior to artificial pollination (longitudinal section). The nucellar filament begins to bend. 3. Development of the inner integument at 20 days after pollination (longitudinal section). 4. Development of the outer integument at 30 days after pollination. The embryo sac is surrounded by
the inner integument (longitudinal section). 5. Thickened outermost cell layer of the outer integument at 40days after pollination (longitudinal section). The ovule and the suspensor are connected. 6. Thickened outermost layer of the outer integument at 40 days after pollination. Note that the ovule is detached from the placental tissue (longitudinal section). 7. Early stage of dark material accumulation in the outer and lateral cellwalls of the outermost layer of the outer integument at 50 days after pollination (transverse section). 8. Further accumulation of the dark material at 60 days after pollination (transverse section). 9. The ovule at 60
days after pollination (longitudinal section). i, inner integument; n, nucellar cells; o, outer integument; oi,inner cell layers of the outer integument; oo, outermost cell layer of the outer integument; p, placental ridge;s, suspensor. Scale bars are 20 m m in 1–3, 50 m m in 4–6, and 100 m m in 7–9.

Seed Coat Formation in Vanilla planifolia

vated at the greenhouse of Tsukuba Botanical
Garden, National Museum of Nature and Science. The ovaries were collected at anthesis andevery 10 days after artificial pollination fromAugust 4 to December 24, 2005 and from March
21 to July 2, 2006. The materials were fixed in
FAA solution containing 2.5% formaldehyde,
2.5% glacial acetic acid and 50% ethanol for 3
days at room temperature. The materials werethen dehydrated in n-butyl alcohol series, andembedded in paraffin (melting point: 56–58°C).
Serial sections were cut at 6 µm thick with a steel
knife and stained with Delafield’s haematoxylin(Nishimura and Tamura, 1993).
Results
The ovule primordium, which consists of nucellar cells and a single layer of epidermis, differentiates from the placental ridge (Fig. 1), andbecomes a nucellar filament which contains 6 to
8 linear nucellar cells at anthesis. At this stage,some filaments begin to bend (Fig. 2). The inner35

integument differentiates at 20 days after pollination (Fig. 3). The outer integument differentiates
and grows into 4–5 cell layers thick at 30 days
after pollination. At this stage, the inner integument surrounds the embryo sac (Fig. 4). At 40
days after pollination, the cells of the outermost
layer of the outer integument thicken (Fig. 5) andthe ovule is detached from the placental tissue(Fig. 6). The dark material starts to accumulate in
the outer and lateral cell walls of the outermost
layer of the outer integument at 50 days after pollination (Fig. 7). The cell walls thicken further at
60 days after pollination (Figs. 8, 9). As the embryo develops into globular stage at 80 days afterpollination, the cells of the inner integument havedegenerated (Fig. 10). The thickened cell walls
occupy the cavity of the outer integument cells,and they become sclerotic at 120 days after pollination (Fig. 11). At 180 days after pollination,
the inner cell layers of the outer integument havedegenerated and the thickened outermost layer ofthe outer integument only persists as the seedcoat (Fig. 12).

Figs. 10–12. Development of ovule in Vanilla planifolia II. 10. Dark material occupies most of the cell cavity ofthe outermost cell layer of the outer integument at 80 days after pollination. The cell layers of the innerintegument have degenerated (longitudinal section). 11. Dark material occupies the whole cell cavity of theoutermost cell layer of the outer integument at 120 days after pollination (longitudinal section). 12. The innercell layers of the outer integument are degenerated and compressed into a thin layer enclosing the embryo
proper, while the thickened outermost layer of the outer integument only persists as the seed coat at 180 days
after pollination. Some cells of the outermost layer of the outer integument are broken by the edge of microtome (longitudinal section). e, embryo; i, inner integument; oi, inner cell layers of the outer integument; oo,outermost cell layer of the outer integument;. Scale bars are 100 m m in 10–12.

36Goro Nishimura and Tomohisa Yukawa

Discussion
We found that the ovule of Vanilla planifolia
develops into a nucellar filament with 6 to 8 nucellar cells at anthesis prior to pollination. The
results did not support Swamy (1947)’s observation in which the ovule starts to differentiate only
after pollination. Vij and Sharma (1986) classified the ovule development of orchids at anthesis
into three groups. In the first group the ovule development does not occur at anthesis and takes
place only after pollination. This group includes
genera such as Cymbidium (Swamy, 1942), Phalaenopsis (Duncan and Curtis, 1942a; Poddubnaya-Arnoldi, 1960; Nimoto and Sagawa, 1961,1962), Cottonia, Dendrobium (Swamy, 1943),
Cattleya (Poddubnaya-Arnoldi, 1967) and Epidendrum (Cocucci and Jensen, 1969; Yeung andLaw, 1989). In the second group, the ovule isweakly developed at anthesis. Apostasia nipponica (Nishimura and Tamura, 1993), Cypripedium(Duncan and Curtis, 1942b; Poddubnaya-Arnoldi, 1960), and Paphiopedilum (Duncan and Curtis, 1942b) belong to this group. In the thirdgroup, the ovule differentiates and ready for fertilization at anthesis. Epipogium aphyllum Sw.
(Afzelius, 1954), Gastrodia elata Blume (Kusano, 1915), Epipactis papillosa Franch. & Sav.
(Sato, 1974), and Chamaegastrodia shikokiana
Makino & Maek. (Tohda, 1967) constitute thisgroup. Our observation showed that V. planifolia
belongs to the second group. Members of thisgroup, viz., Apostasia, Vanilla, Cypripedium, andPaphiopedilum, belong to the earliest divergent
clades in orchids (Cameron et al., 1999). By contrast, members of the other groups belong tomore derived clades. The weakly developed
ovule at anthesis thus may represent a plesiomorphic character state in the Orchidaceae.
We found that the cells of the inner integument
and the inner cell layers of the outer integument
have degenerated when the seed maturates, andthat the sclerified outermost layer of the outer integument only persists as the seed coat. We confirmed that Swamy (1947)’s observation in which
he noted the outer and inner integuments, andeven the nucellar cells take part in the seed coatis due to misinterpretation. In comparison withthe structure of another sclerotic seed coatspecies, Apostasia nipponica, in which the innercell layer of the outer integument only sclerifies
and deposits the dark material, it is clear thatsclerified, opaque seed coat in orchids does notrepresent a single character state. So far asknown, the dark material accumulates in the outermost cell layer of the outer integument in theorder Asparagales, the sister group of the Orchidaceae (Wunderlich, 1937; Dahlgren and Clifford, 1982; Dahlgren et al., 1985; Wittich andGraven, 1995). These observations indicate thatsclerotization in the outermost cell layer of theouter integument may represent a plesiomorphic
character state in the Orchidaceae.
Chemical composition of the dark material of
the sclerified seed coat in orchids has not beenstudied yet. Similar dark material, called phytomelan, characterizes the seed coat of Asparagales (Dahlgren and Clifford, 1982). The darkmaterial of the orchid seed coat may be homologous with phytomelan if taking the phylogenetic
position of the Orchidaceae and Asparagales into
consideration. Wittich and Graven (1995) studied
chemical composition of phytomelan in Gasteria
verrucosa (Asphodelaceae) using histochemical
technique and the results indicated that phytomelan cannot be stained for cellulose, pectin, lignin,carbohydrates, proteins or fatty substances, but
the presence of phenolics is indicated. This suggests that the phytomelan is a type of melanin,because phenolics play role in the melanin
synthesis. Dahlgren and Clifford (1982) also reported that phytomelan is a charcoal-like substance which is very rich in carbon and has ahydrogen : oxygen proportion of ca. 2 : 1 suggesting dehydration of carbohydrates. Furtheranatomical and histological analyses of the otherorchid and Asparagales species exhibiting thesclerified, opaque seed coat are needed to elucidate the diversity and evolution of this character
state.

Seed Coat Formation in Vanilla planifolia

References
Afzelius, K. 1954. Embryo sac development in Epipogium aphyllum. Svensk Botanisk Tidskrift Utgiforen
af Svenska Botaniska Föreningen 48: 513–520.
Burgeff, H. 1936. Samenkeimumg der Orchideen undEntwicklung ihere Keimpflanzen. Gustav Fisher, Jena.
Cameron, K. M., Chase, M. W., Whitten, W. M., Kores, P.
J., Jarell, D. C., Albert, V. A., Yukawa, T., Hills, H. G.
and Goldman, D. H. 1999. A phylogenetic analysis of
the Orchidaceae: evidence from rbcL nucleotide sequences. American Journal of Botany 86: 208–224.
Cocucci, A. E. and Jensen, W. A. 1969. Orchid embryology: The mature megagametophyte of Epidendrum
scutella. Kurtziana 5: 23–38.
Dahlgren, R. M. and Clifford, H. T. 1982. The Monocotyledons. A Comparative Study. Academic Press,London.
Dahlgren, R. M., Clifford, H. T. and Yeo, P. F. 1985. The
Families of the Monocotyledons. Springer, Berlin.
Duncan, R. E. and Curtis, J. T. 1942a. Intermittent growthof fruits of Phalaenopsis. A correlation of the growthphase of an orchid fruits with their internal development. Bulletin of the Torrey Botanical Club 69:
167–183.
Duncan, R. E. and Curtis, J. T. 1942b. Intermittent growthof fruits of Cypripedium and Paphiopedilum. A correlation of the growth of orchid fruits with their internaldevelopment. Bulletin of the Torrey Botanical Club 69:
353–359.
Garay, L. A. 1960. On the origin of the Orchidaceae.
Botanic Museum Leaflet, Harvard University 19:
57–96.
Kusano, S. 1915. Experimental studies on the embryonal
development in an angiosperm. Journal of the Collegeof Agriculture, Imperial University of Tokyo 6: 8–120.
Nimoto, D. H. and Sagawa, Y. 1961. Ovule developmentof Dendrobium. American Orchid Society Bulletin 30:813–819.
Nimoto, D. H. and Sagawa, Y. 1962. Ovule development

37

of Phalaenopsis. Caryologia 15: 89–97.
Nishimura, G. and Tamura, M. 1993. Seed coat formationin Apostasia nipponica. Journal of Japanese Botany 68:
219–223.
Poddubnaya-Arnoldi, V. A. 1960. Study of fertilization inthe living material of some angiosperms. Phytomorphology 10: 185–198.
Poddubnaya-Arnoldi, V. A. 1967. Comparative embryology of the Orchidaceae. Phytomorphology 17: 312–320.
Sato, Y. 1974. Embryological studies in the Japanese Epipactis (Orchidaceae). Scientific Report of the Tohoku
University IV (Biology) 37: 33–45.
Swamy, B. G. L. 1942. Female gametophyte and embryogeny in Cymbidium bicolor Lindl. Proceeding of theIndian Academy of Science, Section B 15: 194–201.
Swamy, B. G. L. 1943. Embryology of Orchidaceae. Current Science 12: 13–17.
Swamy, B. G. L. 1947. On the life-history of Vanilla planifolia. Botanical Gazzette 108: 449–456.
Tohda, H. 1967. An embryological study of Hetaeria
shikokiana, a saprophytic orchid in Japan. Scientific
Report of the Tohoku University IV (Biology) 33:83–95.
Vji, S. P. and Sharma, M. 1986. Embryo sac developmentin Orchidaceae. In : Vji, S. P. (ed.), Biology, Conservation and Culture of Orchids. pp. 31–48. Affiliated EastWest Press, New Dehli.
Wirth, M. and Withner, C. L. 1959. Embryology and development in the Orchidaceae. In: Withner, C. L. (ed.),The Orchids. A Scientific Survey. pp. 155–188, Ronald
Press, New York.
Wittich, P. E. and Graven, P. 1995. Histochemical study ofthe development of the phytomelan layer in the seedcoat of Gasteria verrucosa (Mill.) H. Duval. Protoplasma 187: 72–78.
Wunderlich, R. 1937. Zur vergleichenden Embryologie
der Liliaceae-Scilloideae. Flora 132: 48–90.
Yeung, E. C. and Law, S. K. 1989. Embryology of Epidendrum ibaguense. I. Ovule development. Canadian
Journal of Botany 67: 2219–2226.