INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
ISSN Print: 1560–8530; ISSN Online: 1814–9596
10–698/AWB/2011/13–4–603–606
http://www.fspublishers.org
Full Length Article
Male and Female Gametophyte Development in Cichorium
intybus
ABDOLKARIM CHEHREGANI1, FARIBA MOHSENZADEH AND MONA GHANAD†
Department of Biology, Bu-Ali Sina University, Hamedan, Iran
†Department of Biology, Faculty of Science, Islamic Azad University, Broujerd Branch, Iran
1
Corresponding author’s e-mail: [email protected]; [email protected]
ABSTRACT
Gametophyte development and embryogenesis of Cichorium intybus L. were studied. The flowers and buds, in different
developmental stages, were removed, fixed in FAA70, embedded in paraffin and sliced at 7-10 µm. Staining was carried out
with PAS and contrasted with Hematoxylin. Results showed that anther is ovoid-shaped and tetrasporangiated. Development
of anther wall follows the dicotyledonous type, which is composed of an epidermal layer, an endothecium layer, one middle
layer and tapetum. The tapetum is secretory type at the beginning and plasmodial type at the end of anther development.
Microspore tetrads are tetragonal. Pollen grains are spherical, tricolpate and bi-cellular at the mature form. Ovule is
anatropous, unitegmic and tenuinucellate. Development of ovule starts with the formation of primordium. In this primordium,
an archeosporial cell produces a megaspore mother cell, which undergoes meiosis, forming a linear tetrad. The micropylar cell
is functional megaspore that survives and will function in megagametophyte development. Embryo sac development is of the
Polygonum type. The mature embryo sac is composed of 7 cells, one central cell contained polar nuclei or secondary nucleus,
two synergids and one egg cell that formed egg apparatus and three antipodal cells that are degenerate in the mature embryo
sac before fertilization. © 2011 Friends Science Publishers
Key Words: Asteraceae; Male gametophyte; Female gametophyte; Cichorium intybus
INTRODUCTION
Asteraceae is the largest plant family. This family
contained about 1600 genera and 23000 species (Kadereit &
Jeffrey, 2007; Funk et al., 2009). Its abundant members, its
global distribution and the fact that it comprises many
medicinal species have made it the subject of many
researches (Martin et al., 2009). The circumscription of the
genera is often problematic and some of these have been
frequently divided into minor subgroups (Hind et al., 1995).
The principal taxonomic problems within the family are
interrelationships amongst the genera, the circumscription of
sub-tribal taxa and polymorphic species (Torrel et al., 1999;
Inceer & Beyazoglu, 2004; Chehregani & Mehanfar, 2007;
Arabaci & Yildiz, 2009).
Essential oils medicinally important compounds have
been isolated from Asteracean species (Beerentrup &
Robbelen, 1987; Heywood & Humphries, 1997). Many
researchers have studied the karyological properties of the
Asteracean species (Valles et al., 2005), but there are few
embryological studies, so new studies are necessary to
improve the embryological knowledge of the family (Valles
et al., 2005). Based on present embryological studies, many
exceptional events were reported in the members of this
family, including Nemec phenomenon (Davis, 1968;
Batygina, 1987), increasing of synergids (Cichan & Palser,
1982), increasing of antipodal cells (Richards, 1997;
Pandey, 2001), four-celled female gametophyte (Harling,
1951) and apomixis (Davis, 1968; Chaudhury et al., 2001).
The aim of this study was to study of gametophyte and
embryo development in Cichorium intybus. Although there
are some reports about other members of Asteraceae
(Lakshmi & Pullaiah, 1979; Pullaiah, 1979; Rangaswamy &
Pullaiah, 1986; Hiscock et al., 2003) but this is the first
embryological investigation in C. intybus.
MATERIALS AND METHODS
The Cichorium intybus L. plants were collected from
the naturally growing area in Broujed. Voucher specimens
are deposited in the local herbarium of the Islamic Azad
University (Broujerd branch) and labeled as follows:
Lorestan province of Iran, 15 km from Broujerd to Malayer,
Alt. 2500 m. The florescence and buds, in different
developmental stages, were removed, fixed in FAA70
(formalin, acetic acid & 70% ethanol, 1:1:17 v/v), stored in
70% ethanol, embedded in paraffin and sliced at 7-10 μm
with a Micro DC 4055 microtome. Staining was carried out
with PAS (Periodic Acid Schiff) according to the protocol
suggested by Yeung (1984) and contrasted with Meyer’s
To cite this paper: Chehregani, A., F. Mohsenzadeh and M. Ghanad, 2011. Male and female gametophyte development in Cichorium intybus. Int. J. Agric.
Biol., 13: 603–606
CHEHREGANI et al. / Int. J. Agric. Biol., Vol. 13, No. 4, 2011
unitegmic (Fig. 2a). During early ovular development, only
a single hypodermal cell was observed. It is larger and
differentiated from the neighboring cells and then become
the megaspore mother cell (mc). The ovule contained small
mass of nucellus (Fig. 2a). Megaspore mother cell (Figs. 2b,
c) continues to growth, enters meiosis, produces dyad (Fig.
2d) and finally results in a linear-shaped tetrad of
megaspores that the chalazal megaspore is functional one.
Mitotic divisions take placed in the functional megaspore
and resulted to form a two nucleated embryo sac (Fig. 2f),
four nucleated embryo sac (Fig. 2g), and finally eight
nucleate embryo sac was produced. Cell formation takes
place in the embryo sac and mature embryo sac was formed
(Fig. 2h). Embryo sac formation and maturation follows as
the Polygonum type.
Egg cells are larger and distinguishable from synergids
by its position (Fig. 2h). The two polar nuclei are fused and
a large secondary nucleus was formed, before the formation
of egg apparatus containing of egg cell and two synergids
(Figs. 2h, i). The polar nuclei are visible in the center of
embryo sac that are fussing just before fertilization and
produced a secondary nucleus (Fig. 2i). Antipodal cells are
degenerated in the studied flowers (Fig. 2i). Secondary
nucleus is migrated toward the egg apparatus (Fig. 2i).
Some gametophyte characters were illustrated in Table I.
Hematoxylin (Chehregani & Sedaghat, 2009). For each
developmental stage, several sections observed under a
Zeiss Axiostar Plus light microscope. Many samples were
studied for each stage and photomicrographs were made
from the best ones.
RESULTS
Male gametophyte development: Results showed that the
anther of Cichorium intybus is tetrasporangiate. Primary
sporogenous cells (Fig. 1a) were developed directly as
microsporocytes (Fig. 1b). Each microsporocyte undergoes
meiosis and resulted in a microspore tetrad. Meiosis
staggers including prophase I, metaphase I (Fig. 1c),
anaphase I and then telophase I (Fig. 1d), followed by
prophase II, metaphase II, anaphase II (Fig. 1e), and
telophase II (Fig. 1f) were observed clearly in the
specimens. Cell wall was not formed between the two
newly formed nuclei in telophase I stage (Fig. 1d). In the all
tetrads, the cytokinesis was done as simultaneous type. The
final tetrads are recognized mostly as tetragonal (Fig. 1f, g).
Callosic wall is formed around the tetrad and between each
monad (Fig. 1g). In the two neighboring pollen sacs,
microspores development is synchronized. The microspores
when released from tetrad are non-vacuolated. They have a
dense cytoplasm with irregular shape and a prominent
centrally placed nucleus (Fig. 1h). Its nucleus takes up a
peripheral position together the central vacuole develops,
i.e., forming a large vacuole squashes the cytoplasm and the
nucleus toward the microspore margin. Nucleus of
microspores then undergoes mitosis and resulted to form
two unequal nuclei, a large vegetative and small generative
one thus to form bi-nucleate pollens (Fig. 1i), further twocell ones. Pollen grains have a considerable thick exine
(Fig. 1i).
Formation of anther wall: In early stage of development,
several rows of archesporial cells differentiated beneath the
epidermis of the anthers. They have dense cytoplasm and
prominent nuclei. They are divided periclinally, cause to
formation of inner sporogenous cells and outer parietal cells.
Parietal cells are divided and cause to form anther wall that
consists of three layers; the epidermis, endothecia, and
tapetum (Figs. 1a-i). The tapetal cells are uni-nucleate, binucleate or tri-nucleate at the stage of microsporocyte (Figs.
1 d, e). They have rapid mitosis and contained high level
polyploidy (Fig. 1e), indicating their high metabolic activity,
that is similar to the antipodals in the embryo sac.
Extensions of tapetal cells are visible in the anther locule at
the microspores releasing stage (Fig. 1h). The tapetal cells
were degenerated at the stage of uni-cellular or bicellular
pollen grains, and only relics of the tapetum are visible in
the stage (Fig. 1i). In this species, the middle layer is not
developed.
Female gametophyte development: Results showed that in
C. intybus, ovary is composed of a single carpel and a locule
with an ovule (Fig. 2a). The ovule is of anatropous type and
DISCUSSION
Results of this research showed that development of
the three layered anther wall occurred as the
dicotelydonous-type in C. intybus (Davis, 1964).
Archesporial cells are recognized by their prominent nuclei
and compact cytoplasm. They are divided periclinaly
resulted to form outer primary parietal and inner
sporogenous cells that is accordance with the findings of
Xue and Li (2005). The endothelial fibrous thickening is not
as clearly observed as in the most representatives of
Asteraceae (Yurukova-Grancharova et al., 2006). Although
presence of middle layer was reported by Rangaswamy and
Pullaiah (1986). It seems that there is no developed middle
layer. A sharp correlation was observed between the meiotic
division in pollen mother cells (PMCs) and development of
anther’s tapetum (Figs. 1b, g) that was reported for other
Asteraceaen members (Gustafsson, 1946).
Tapetum cells have high level of polyploidy that is
indicating their high metabolic activity (Maheshwari, 1950).
Two basic types of tapetum are recognized in Angiosperms:
secretory and amoeboid type (Pacini et al., 1985). In C.
intybus the tapetum passes at the first a parietal (secretory)
phase with multiplication of the nuclei (2-3 nuclei per cell)
and later it changes in to amoeboidal (periplasmodial) type
so that its periplasmodial extensions are observed toward
the anther locule (Fig. 1h).
In C. intybus, the primary sporogenous cells become
directly pollen mother cells (PMCs) that are as few rows of
PMCs in the pollen sac (Fig. 1b). There are few plant
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Fig. 2: Megasporogenesis and megagametophyte
development in Cichorium intybus, (a) Young ovule
with archesporial cells and nucellus, (b, c) Megaspore
mother cell (m), (d) First meiosis resulted in formation
of dyad cells (arrows), (e) Linear-shaped tetrads
(arrows), (f) Two-nucleate embryo sac (arrows), (g)
Four- nucleate embryo sac, (h) Maturing embryo sac
showing two synergids (sy), an Oospher (os), two polar
nuclei (arrows, PN) that are migrating toward the egg
apparatus, antipodal cells are invisible in this section,
(i) Mature embryo sac that showed well developed egg
apparatus (eg), fused polar nuclei and antipodal cells.
Antipodal cells were degenerated. ar, archeosporial
cell; nu, nucellus; ne, Nucellar endothelium; m,
megaspore mother cell; in, integument; fme, functional
chalazal megaspore; R, raphe; pn, polar nuclei; ant,
antipodal cells; syn, synergids; Os, oosphere; v,
vacuole. Scale bars = 40µm
Table I: Some gametophyte characters in C. intybus
Characters
Ovule number
Ovule length
Ovule width
Embryo sac length
Ovary hair
Number of nucellus layers in the wide of
embryo-sac
Position of megagametocyte
Form of megaspore tetrads
Fusion time of polar nuclei
Fusion position of polar nuclei
Amyloplast accumulation in embryo sac
Growth pattern of integuments
Number of layers in integument
Number of pollen grains in each anther
Polar length of mature pollen grains
Endothecium thickness
Quantity
1
260 µm
140 µm
180 µm
Present
12-14
Second layer
Linear
Before emigration to
micropylar end
Center of embryo sac
Weak
Grow faster on opposite side of
funicule
4
20-35
60 µm
15 µm
Fig. 1: Microsporogenesis, male gametogenesis and
development of anther wall in Cichorium intybus, (a)
Longitudinal section of anther showing anther wall,
tapetum layer (ta) and archeosporial cells (ar), (b)
Longitudinal section of anther showing Pollen mother
cells (pmc), (c-g) Showing various stages of meiosis in
microsporocytes, (c) Metaphase I, (d) Telophase I, (e)
Telophase II, (f, g) Tetragonal microspore tetrads, (h)
Microspores just released from tetrads, (i) Mature
tricolpate pollen grains. di, dyads, En, endothecium; ip,
immature pollen grains (microspores); pmc, pollen
mother cells; Ta, tapetum layer; te, tetrad; p, mature
pollen grains. Scale bars = 20 µm
1f, g). In two neighboring sporangia, microspores are
synchronized development. The microspores at releasing
time are non-vacuolated. They have compact cytoplasm,
irregular shape, with a prominent and centrally located
nucleus (Fig. 1h). The nucleus is then divided by the mitosis
into two nuclei, a small generative and a large vegetative
nucleus, so called bi-nucleated pollen grain, further two-cell
one that is different from the results of Lakshmi and
Pullaiah (1979) in the other Asteracean member, that
reported that pollen grains are 3-celled when shed.
In C. intybus, the chalazal megaspore of the linearshaped tetrad gives rise to form Polygonum type of embryo
sac as described for more than 70% of angiosperm
(Maheshwari, 1950; Batygina, 1987). The other three
megaspores were degenerated rapidly. Linear tetrads were
reported by prior researchs in other species of Asteraceae
species that have such character (Hu, 1982). The
significance of this type of pollen mother cells development
is not understood in plant phylogeny (Pan et al., 1997).
Each microsporocyte undergoes meiosis and produced
microspore tetrad. The tetrads are mostly tetragonal (Fig.
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(Lakshmi & Pullaiah, 1979; Kapil & Bhatnagar, 1981;
Rangaswamy & Pullaiah, 1986). Remaining megaspore
produced 8-nucleated and then cellulized embryo sac. In
mature embryo sac three cells were differentiated at the
micropylar end that consists of an Oospher and two
Synergids. In this study, both synergid cells were seen in the
embryo sac. It means that C. intybus is an allogamous
species. In the plant, fertilization gives rise to degenerate
one of the synergids, and the other synergid should be
degenerated few days after the flower opening (Chehregani
& Sedaghat, 2009). Two free polar nuclei were located at
the center of embryo sac that came toward the egg
apparatus, fussing took place near the egg apparatus and just
prior to fertilization and secondary nucleus was resulted.
In Polygonum type development of embryo sac,
antipodal cells are placed on the chalazal end of embryo sac.
They are usually three and variable in size and number
(Maheshwari, 1950; Cameron & Prakash, 1994; Xiao &
Yuan, 2006), as reported in Asteraceae (Rangaswamy &
Pullaiah, 1986). Our results indicated that the antipodal cells
are degenerated in the late stages of embryo sac
development. In this species, it seems that antipodal cells
have not any specific role, but their function is importing of
nutrients into the embryo sac at the early developmental
stage (Diboll, 1968).
Acknowledgment: This study was supported by the grant
provided by research and technology council of Islamic
Azad University, Broujerd Branch. The authors wish to
thank Prof. Dr. Shahin Zarre, from the University of Tehran,
for his valuable comments.
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