Embryology of Gentiana angulosa and G. pontica (Gentianaceae).

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Proc. Geor. Acad. Sci., Biol. Ser. B, 2, 1-2 : 29-34; 2004

Embryology of Gentiana angulosa and G. pontica (Gentianaceae)

Botany

M. Akhalkatsi, G. Gvaladze, M. Gachechiladze, N. Taralashvili
Institute of Botany, Georgian Academy of Sciences
Abstract
Embryology of two gentians - Gentiana angulosa and G. pontica, has been studied for the
first time. They show similar morphology and are considered to be relative species.
Embryological study has revealed considerable similarity in structure of reproductive organs
of G. angulosa and G. pontica. The common characters are: four-loculate anther; 2-celled
mature pollen; superior, cenocarpous and paracarpous ovary; unitegmic, tenuinucellate and
anatropous ovule; hypodermal unicellular archesporium; linear tetrad of megaspores;
Polygonum type embryo sac; pre-mitotic type of fertilization; nuclear endosperm and Solanad
type of embryogenesis. The investigated species differ mainly by two quantitative features –
numbers of cell layers in the integument (5-8 in G. angulosa, 9-10 in G. pontica) and the
antipodals (3-6 in G. angulosa, 3 in G. pontica). The structural difference is only slightly
expressed in seed coat structure, which has taxonomic significance.

reziume
reziumereziume
reziume
embriologiurad pirvelad iqnen Seswavlili Gentiana angulosa da G.
pontica. isini sistematikosTa azriT axlo naTesaur kavSirs amJRavneben.
maT axasiaTebT rigi saerTo niSnebi: oTxbudiani mtvriana; 2-ujrediani
mwife mtvris marcvali; zeda, cenokarpuli, parakarpuli naskvi;
erTsafarveliani, tenuinucelaruli da anatropuli Teslkvirti;
hipodermuli, erTujrediani arqesporiumi;
tetrada;
Polygonum-tipis Canasaxis
ganayofiereba; birTvuli endospermi; Solanad-tipis Canasaxi. Seswavlili
saxeobebi erTmaneTisagan gansxvavdebian ZiriTadad ori raodenobrivi
niSniT, rogoricaa integumentis Sreebis raodenoba (5-8, G. angulosa-Si, 8-
10, G. pontica-Si) da antipodebis ricxvi (3-6, G. angulosa-Si, 3, G. pontica-Si).
mcireodeni struqturuli sxvaobaa nanaxi Teslis garsis zedapiris
agebulebaSi, romelsac taqsonomiuri mniSvneloba aqvs.

Key Words: Embryology, ovule, seed, Gentiana.

Introduction
Embryology of two species of the genus Gentiana - G. angulosa and G. pontica, has
been studied for the first time. They show similar morphology and are considered to be
relative species. Often they are identified as subspecies or varieties of one species (Gagnidze,
1985; Halbmayr, 1990). In “Flora of Europe” (Tutin et al., 1972) one of them is identified as
variety (G. verna var. angulosa Kusn.) and the other as subspecies (G. verna ssp. pontica
(Soltok) Hayek) of G. verna. All three species are perennial herbs. Vegetative parts and
flowers of G. verna are smaller than that of G. angulosa and G. pontica. The main difference,
however, is a form of calyx. Teeth and tube of calyx in G. angulosa and G. pontica are with
clearly visible wings, which are absent in G. verna. G. pontica, in addition has more wide,
ovate and obtuse leaves. These data show that morphological characteristics are not sufficient
criteria for their identification. It is necessary to conduct further study of these species using
embryological and molecular systematic approaches.
megasporebis
premitozuri
xazuri
tipis parki;

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Shamrov, 1987, 1988, 1991, 1996; Akhalkatsi, Wagner, 1997) of the family Gentianaceae
have shown that reproductive organs reveal number of variable features on species and genus
level having a taxonomic value. For example, ovule in most species is anatropous -Gentiana
spp., Gentianopsis spp., but sometimes might be orthotropous - Halenia elliptica (Stolt,
1921), Cotylanthera tenuis, Voyriella parviflora, Voyria spp. (Öhler, 1927), hemitropous -
Comastoma tenellum, Gentianella spp. (Stolt, 1921), or ana-campilotropous - Swertia spp.
(Shamrov, 1991, 1996). Ovules in all species are tenuinucellate and unitegmic. The number of
layers of the cells in a single integument varies among species from 2 to 20.
Megagametophyte develops according to Polygonum type in all Gentianaceae. However,
mature embryo sacs differ among species in number of antipodals. There are three groups of
species in the family Gentianaceae differing in antipodal structure (Shamrov, 1987, 1988): 1.
antipodals uninucleate, ephemeral, degenerate before fertilization. They might be three
(Gentiana prostrata), or 3-9 (Gentiana asclepiadea, G. lutea, G. tibetica); 2. antipodals
always three, degenerate after fertilization (Enicostema littorale, Exacum affine, Hoppea
dichotoma); 3. antipodals multinucleate, or with polyploid nuclei, hypertrophic, 3 (Halenia
elliptica), 6 (Comastoma tenellum), or more than 6 (Gentianella amarella, G. caucasea, G.
germanica, G. uliginosa) .
On the basis of these suggestions we have decided to conduct embryological
investigation of two gentians, which have not been studied before. The determination of
embryological features will contribute to the further taxonomic evaluation of these species.

Materials and Methods
Two species of the family Gentianaceae have been studied – G. angulosa M. Bieb.
and G. pontica Solotk. (Czerepanov, 1995). Both are perennial mountain plants (2n=26)
distributed in Caucasus, Asia Minor and Iran. G. angulosa is widely distributed on the Great
Caucasus. G. pontica is more common in the Minor Caucasus. They grow between 2000-3600
m. above see level. Depending on altitude they are flowering in May-June. Fruit matures in
July-August.
Plant material was collected during 1985-1989 in Kazbegi and Bakuriani. Buds,
flowers and fruits at different stages of development were fixed in FAA (formalin, acetic acid,
70% ethanol, 5:5:90) and embedded in paraffin. 10-12 µm thick sections were prepared on
microtome Reichert, Austria, and stained in hematoxylin according to known method by
Meier. Examination was carried out during 2001-2003 using light microscope Polivar,
Reichert, Austria. Photographs were taken with a digital camera NikonCoolpix5000.

Results
Comparative embryological investigation has revealed number of features common for
G. angulosa and G. pontica. Anther is four-loculate, opens longitudinally. Anther wall
consists of epidermis, endothecium and 1-3 layers of glandular tapetum. Tetrad of
microspores is tetrahedral. Mature pollen contains two cells.
The gynoecia of the investigated species are superior, unilocular, bicarpellate (very
rare tricarpellate) and paracarpous. They are terminated by a short style and a 2-lobed stigma.
The gynophore is 4-5 mm long. Numerous ovules develop on the parietal placentae along the
fused margins of the carpels. Vascular bundles reach chalaza, but do not enter ovule. The
Ovule is anatropous, tenuinucellar and unitegmic (Fig. 1). Nucellus degenerates during the
extension of the embryo sac. The archesporium is hypodermal, unicellular and functions
directly as megaspore mother cell. Tetrad of megaspores is linear. Polygonum type of embryo
sac develops from chalazal megaspore. The polar nuclei (Fig. 2) fuse before fertilization and
secondary nucleus is located near the egg cell (Fig. 3). The mature embryo sac consists of the
Embryological investigations (Stolt, 1921; Öhler, 1927; Bouman, Schrier, 1979;

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three-celled egg apparatus, the central cell and different number of ephemeral antipodals. The
synergids have a well developed filiform apparatus.
Fertilization is porogamous. The pollen tube enters the embryo sac through the
micropyle and discharges its contents into one of the synergid. Triple fusion precedes
syngamy. Gametic fusion is of the pre-mitotic type. The primary endosperm nucleus moves
towards the middle of the embryo sac and undergoes successive synchronous divisions (Fig.
4). Endosperm is of a nuclear type (Fig. 5). Cell wall formation proceeds centripetally after
128 endosperm nuclei have developed. At the same time, first division of the zygote takes
place. Embryogenesis follows the Solanad type (Fig. 6). Mature seed contains embryo at early
torpedo stage, cellular endosperm and seed coat (Fig. 7). The seed coat consists of a
sculptured outer layer, derived from the epidermis of the ovule, and a transparent
membranous envelope originated from the crushed cells of the inner layers of the integument.
The sculpturing is of the reticulate type (Fig. 8) in both species. However the pit like structure
of periclinal walls is more prominent in G. pontica.
Investigated species differ by some quantitative characters, which usually are variable
within the family Gentianaceae. These features are the number of antipodals and cell layers in
the integument. Integument of G. angulosa consists of 5 layers of cells in the lateral, adjacent
to the embryo sac, region and 8 layers in the micropylar part (Fig. 2). The micropyle is 100
µm long. G. pontica possesses 9-10 layers of cells of the integument (Fig. 1). The micropyle
is 160 µm long. Antipodals are mostly 3 (Fig. 2), rarely 6 (Fig. 3) in G. angulosa. They are
ephemeral and degenerate during fertilization. In G. pontica there are always 3 ephemeral
reduced antipodals degenerating during or before fertilization.
Number of anomalous ovules has been found in both investigated species. Most of the
anomalies cease the developmental process in the megagametophyte and make impossible the
fertilization. The following anomalies have been observed: 1. gynoecium consists of three
instead of two carpels. In consequence the stigma is three lobed, ovules form 6 marginal
layers in the ovary; 2. archesporium is absent in the ovule; 3. megaspore mother cell
degenerates; 4. tetrad of megaspores is formed but degenerates completely; 5. two or more
megaspores begin to develop but degenerate at later stages; 6. polarization 0-2 is observed in
two-nucleate embryo sac; 7. polarization 1-3 is found in four-nucleate embryo sac; 8. twin
embryo sacs are observed containing 16, or 12 nuclei randomly located into one cenocyte;
and, 9. polar nuclei do not fuse before fertilization and are located in the center of the embryo
sac.

Discussion
The structure of reproductive organs of G. angulosa and G. pontica revealed
considerable similarity. Most of these common characters usually occur in all species of the
family Gentianaceae. These are: four-loculate anther; 2-celled mature pollen; superior,
cenocarpous and paracarpous ovary; unitegmic and tenuinucellate ovule; hypodermal
unicellular archesporium; linear tetrad of megaspores; Polygonum type embryo sac; pre-
mitotic type of fertilization; nuclear endosperm and Solanad type of embryogenesis.
There are several features, which are variable within the Gentian family, such as ovule
type, number of layers of cells of the integument, number and structure of antipodals and
synchronization of embryogenesis and endosperm development. Only two of them are
different in the investigated species – thickness of the integument and number of the
antipodals. The ovule is anatropous in both species. First division of the zygote takes place
similarly in the investigated species after 128 endosperm nuclei are formed.
Integument thickness varies within the family Gentianaceae from 2-3 (Gentianopsis
ciliata) to 20 cells (Gentiana lutea, Shamrov, 1987, 1988). The investigated species differ by
this character. G. pontica has 9-10 layers of cells, G. angulosa – 5-8 layers. The number of

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the layers of integumentary cells and length of micropyle determine seed coat texture. It is
known (Miège, Wüest, 1984; Yuan, 1993) that shape of seeds and seed coat structure are
variable within the genus Gentiana and have taxonomic significance. The studied species
possess oblong seeds with reticulate structure of the seed coat. The texture differs
insignificantly among these species.
Ovule with a single integument is common feature within the family Gentianaceae.
Unitegmic ovules occur in other sympetalous taxons, such as Asteranae and Lamianae
(Netolitzky, 1926). Single integument is usually formed by both epidermal and subepidermal
cells of a placenta. (Bouman, Schrier, 1979). Unitegmy is originated from bitegmy. The
studies of ovules showing transitional stages between bi- and unitegmy make it apparent that
there are three possible ways in which the change-over from bitegmy to unitegmy took place:
1. the reduction of one of the two integuments; 2. the fusion of integument primordia; and, 3.
the process of integument shafting, when subepidermal cells are dividing more intensively
and overlappe the epidermal layer resulting in their fusion (Bouman, 1977).
The investigated species differ only slightly by number and structure of antipodals,
which is a variable feature within the family Gentianaceae. Both species might have 3
ephemeral antipodals, or 6 antipodal cells can be observed in some ovules of G. angulosa. It
was shown (Akhalkatsi, Wagner, 1996, 1997) that there is definite relations between number
and structure of antipodals and the life history of a species. The short-lived monocarpic
Gentianella species with proliferated antipodal tissue show a high plasticity in habitat
colonization and flowering time. In G. caucasea the time span for seed development amounts
to 16-20 days (Akhalkatsi, Wagner, 1996), in G. germanica to 20-25 days (Wagner, et al.,
1995). It was supposed that such rapid development of a seed might be promoted by
hypertrophous antipodals, an apparently nutritive tissue, which substitute the endosperm in
the early stages and accelerate embryogenesis. The first division of a zygote in these species
takes place at 8-nuclate stage of endosperm development. For comparison, in the perennial
species G. pyrenaica, with ephemeral antipodals, the first division of a zygote occurs only
after formation of 128 nuclei in the endosperm. The total duration of seed formation in this
species exceed 30 days (Wagner, et al., 1995). The investigated perennial polycarpic G.
angulosa and G. pontica show similar synchronization of embryogenesis and endosperm
development as it is described for G. pyrenaica. These data confirm earlier suggestion
concerning the role of proliferated antipodals in acceleration of seed development
(Akhalkatsi, Wagner, 1996, 1997).
Thus, the investigated species differ mainly by two quantitative features – the number
of cell layers in the integument and that of antipodals. The structural difference is only
slightly expressed in seed coat structure, which has taxonomic significance.

References

1. Akhalkatsi M., Wagner J. Reproductive phenology and seed development of Gentianella
caucasea in different habitats in the Central Caucasus. Flora 191, 161-168, 1996.
2. Akhalkatsi M., Wagner J. Comparative embryology of three Gentianaceae species from
the Central Caucasus and the European Alps. Pl. Syst. Evol. 204, 39-48, 1997.
3. Bouman F. Integumentary shafting - a third way to unitegmy. Ber. Deutsch. Bot. Ges. 90,
15-28, 1977.
4. Bouman F., Schrier S. Ovule ontogeny and seed coat in Gentiana with a discussion on the
evolutionary origin of the single integument. Acta Bot. Neerl. 28, 467-478, 1979.
5. Czerepanov S.K. Vascular plants of Russia and adjacent states (The former USSR).
Cambridge, Cambridge Univ. Press, 1995.

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6. Gagnidze R. ojakhi Gentianaceae Juss. In: Florae of Georgia. R. Gagnidze (Ed.) Tbilisi,
Metsniereba, 1985.
7. Halbmayr H. Dem Enzian auf der Spur. Monograph, Wien, 1990.
8. Miège J., Wüest J. Les surfaces tégumentaires des graines de Gentiana et Gentianella
vues au microscope électronique à balayage. Bot. Helv. 94, 41-59, 1984.
9. Netolitzky F. Anatomie der Angiospermen – Samen. In: K. Linsbauer (Ed.) Handbuch der
Pflanzenanatomie. Berlin, Borntraeger, 1926.
10. Öhler E. Entwiklungsgeschichtlich-zytologische Untersuchungen an einigen
saprophytischen Gentianaceen. Planta 3, 641-733, 1927.
11. Shamrov I.I. Semeistvo Gentianaceae. In: Sravnitel’naja embryologia tsvetkovykh. T.B.
Batygina, M.S. Jakovlev (Eds), Leningrad, Nauka, 1987.
12. Shamrov I.I. Razvitje semiapochki i osobennosti stroenija zarodyshevogo meshka u
predstavitelei semeistva Gentianaceae. Bot. Zhurn. 72, 2, 213-222, 1988.
13. Shamrov I.I. The ovule of Swertia iberica (Gentianaceae): structural and functional
aspects. Phytomorphology 41, 213-229, 1991.
14. Shamrov I.I. Ovule development and significance of its features for Gentianaceae
systematics. Opera Bot. Belg. 7, 113-118, 1996.
15. Stolt K.A.H. Zur Embryologie der Gentianaceen und Menyanthaceen. Kgl. Svensk Vet.-
Akad. Handl. 61, 1-56, 1921.
16. Tutin T.G., Heywood V.H., Burges N.A., Moore D.M., Valentine D.H., Walters S.M.,
Webb D.A. Flora Europea 3. Cambridge, Cambridge Univ. Press, 1972.
17. Wagner J., Achalkazi M., Mayr S. Anwendung quantitativ embryologischer Methoden in
Entwicklungsbiologie und Reproduktionsökologie der Pflanzen. Anz. österr. Akad. Wiss.,
Math.-Naturwiss. Kl. 131, 7-18, 1995.
18. Yuan Y-M. Seed coat micromorphology and its systematic implication for Gentianaceae
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Figure 1:
1 – Ovule of Gentiana pontica, x 110;
2 – Ovule and embryo sac of G. angulosa with egg apparatus (EA), polar nuclei (P) in the
center and three antipodals (A). x 200;
3 - Embryo sac of G. angulosa with secondary nucleus (SN) and six antipodals (A). x 180;
4 – Metaphase of second division of the endosperm nuclei in G. pontica, x 400;
5 – Nuclear endosperm (NE) in developing seeds of G. pontica, x 85;
6 – Seed of G. pontica with Solanad type of embryo (E) and cellular endosperm (CE), x 80;
7 – Mature seed of G. angulosa with embryo at torpedo stage and cellular endosperm, x 80;
8 – Seed coat texture in G. angulosa, x 320.

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