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Caryologia: International
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Microsporogenesis and Pollen

Development in Crocus Sativus
L.
Giuseppe Chichiricc

Dipartimento di Scienze Ambientali, Universit di

L'Aquila, L'Aquila, Italy
Published online: 30 Jan 2014.

To cite this article: Giuseppe Chichiricc (1989) Microsporogenesis and

Pollen Development in Crocus Sativus L., Caryologia: International Journal
of Cytology, Cytosystematics and Cytogenetics, 42:3-4, 249-257, DOI:
10.1080/00087114.1989.10796972
To link to this article: http://dx.doi.org/10.1080/00087114.1989.10796972

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CARYOLOGIA

Vol. 42, n. 3-4: 249-257, 1989

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MICROSPOROGENESIS AND POLLEN DEVELOPMENT IN

CROCUS SATIVUS L.
GIUSEPPE CHICHIRICCO
Dipartimento di Scienze Ambientali, Universita di L'Aquila, L'Aquila, Italy

SUMMARY - Microsporogenesis and pollen development were studied in Crocus

sativus L., using light microscope. During meiosis, microsporocytes often displayed
cytoplasmic degeneration or cellular deformation; consequently, they did'nt complete
meiosis or produced deformed microspores. These anomalies were often associated
with abnormal behaviour of tapetal cells. Both normal and malformed microspores
showed several starch grains in the cytoplasm and a nucleus with one or two prominent
nucleoli. During development, starch grains disappeared in most spores and lipid
globules accumulated as reserve material. Many spores aborted or abnormally developed. Pollen grains were of different size and often malformed too. They were
binucleate and showed a thin inaperturate exine and a variously thickened intine.
Most of them (62%) were densely cytoplasmic and filled with lipid globules. The
remaining (38%) comprised pollen grains with loose or no cytoplasm, frequently filled
with starch granules. A variety of pollen grains showed callosic accumulation.

INTRODUCTION

Crocus sativus L. (lridaceae) is a triploid plant cultivated because it

produces saffron. It fails to set seed and, therefore, multiplies only vegetatively
(MATHEW 1977). This fact prompted studies aimed at investigating the cytological, physiological and structural features correlated with the plant sterility
(CHICHIRICCO 1984, 1987; CmcHIRicco and GRILLI CAIOLA 1982, 1984, 1986;
GRILLI CAIOLA et al. 1985).
The present study revealed some cytological abnormalities during microsporogenesis and pollen development, which may be correlated with low and
abnormal germination of pollen observed in vitro and in vivo (CHICHIRICCO and
GRILLI CAIOLA 1986).

Research supported by grants from Ministry of Public Education (M.P.I. 60%).

250

CHIClilRICCO

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MATERIALS AND METHODS

Bulbs of Crocus sativus L. in cultivation at Navelli Highland, L' Aquila (Italy),

were grown at the Botanical Garden of the University of L' Aquila. Anthers at various
stages of development, including dehiscent ones, were excised from buds and fixed in
acetic-alcohol or 3% glutaraldehyde in 0.05 M sodium cacodylate pH 6.9, dehydrated
in an ethanol series and embedded in LKB historesin. Material for lipid localization
was fixed in formol-calcium (PEARSE 1985) and dehydrated in a resin-water gradient to
absolute LKB resin. Serial longitudinal sections were cut at 3-7 m with Jung blades.
Alternatively, pollen mother cells, tetrads and young individual microspores were
removed from fresh cut anthers; after a brief staining with azure B, they were lightly
squashed on slides and observed at light microscope.
Insoluble polysaccharides were detected by (i) IKI (Lugo!), or (ii) periodic acidSchiff (PAS) reaction, counterstained with toluidine blue (FEDER and O'BRIEN 1968).
Lipids were localized by Sudan IV, Nile blue (SouTHWORTH 1973) and Sudan black B
(PEARSE 1985). Nucleic acids were stained with azure B. Callose was identified by its
fluorescence in decolorized aniline blue (KHO and BAER 1968), using a Zeiss photomicroscope with an HBO 50 mercury vapor lamp and Zeiss 09 or 10 filters (450-490 nm).
Autofluorescence was observed using the same filters.
RESULTS

At premeiotic stages, numerous closely packed microspore mother cells

(MCs) fill the anther locule. The locule is surrounded by a continuous layer of
tapetal cells. Both the sporogenous and tapetal cells are polyhedral in shape,
and densely cytoplasmic each with a nucleus containing three nucleoli.

Microsporogenesis.
As meiosis progressed, the microsporocytes enlarged, becoming spherical.
At the same time, each became surrounded by a layer of callose developing
interior to primary wall. However, some meiocytes appeared collapsed or
deformed (Fig. 1); other meiocytes showed symptoms of cytoplasmic degeneration as revealed by dark-colored cytoplasm (Fig. 1). These abnormalities
affected a few to many MCs, often within different locules of the same anther.
MCs degeneration generally occurred at any meiotic stage following Profase I,
tetrad stage included (Fig. 2). Deformed MCs produced abnormal tetrads
including one or more collapsed microspores which, in section, appeared
semilunar or lenticular (Fig. 4). However, collapsed microspores might be
observed also into undeformed MCs. Both normal and deformed tetrads
showed a variable number of nucleoli (as revealed by MCs squashing); thus,
they may be grouped into three main groups, viz: (i) tetrads including spores
each one containing one nucleolus, (ii) tetrads including three spores each one
containing one nucleolus, and one spore with two nucleoli, (iii) tetrads includ-

MICROSPOROGENESIS IN CROCUS SATIVUS

251

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ing two spores each one containing two nucleoli, and two spores each one with
one nucleolus. The first type occurred most frequently (51.9% of 79 tested
tetrads), followed by the second (32.9%) and the third (15.2%).
Meiosis was of simultaneous type and, generally, synchronous until Metaphase I.
After meiosis, microspores were released from the tetrad callose into the
locule. They were rather dissimilar both in size and in shape, and in section
appeared: roundish, oval, lenticular or starlike (Fig. 6). In spite of shape,
microspores showed several starch grains in the cytoplasm and, as revealed by
squashing, a nucleus which included one (89.5% of 502 tested spores) or two
(10.5%) prominent nucleoli.

Tapetal development during microsporogenesis.

T apetal cells underwent nuclear division during meiosis, becoming binucleate or, sometimes, multinucleate; at the same time, they grew in volume and
projected into the locule. However, often a variable number of tapetal cells
were degenerating, appearing dark-stained (Fig. 1) or without cytoplasm (Fig.
3). Moreover, sometimes, a number of tapetal cells abnormally proliferated up
to partially occlude the locule (Fig. 5). The above abnormalities were often
associated with those of the MCs.

Pollen development.
After dissolution of tetrad callose, the thin exine wall enveloping microspores, rapidly tickened. Thereafter, intine wall deposited interior to the exine,
showing a strong PAS stainability; during its development lipid globules began
accumulating in the cytoplasm of most spores (Fig. 15). Meanwhile, microspores vacuolated and their nucleus underwent mitosis producing vegetative
and generative nuclei, the generative one developing into spindle-shaped cell
(Fig. 16). A variety of spores developed into scantly cytoplasmic pollen grains
which did'nt accumulate lipid globules. Pollen grains frequently grew deformed; in this regard, the anther locules may be divided in two groups: i)
those containing pollen grains variously shaped (Fig. 15), and ii) those containing only collapsed pollen (Fig. 7). Sometimes, both the types of locules
occurred in the same anther. At anther dehiscence, pollen grains were of
different size, and in section they appeared roundish, oval, semilunar, lenticular or irregularly shaped (Fig. 15). In spite of shape, pollen grains showed a thin
layer of sporopollenin wall which gave positive reaction with either basic dyes
or Sudan black B (Table 1; Figs. 16, 17). The underlying intine was generally
thicker than exine (Fig. 16) and variable among grains, ranging from about 7.5

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Figs. 1-24. - Abbreviations: E = exine; GC =generative cell; L =lipid globules; M =micros pores; MCs = microspore
mother cells; S = tapetal cell syncytium; T = tapetum; V =vacuole; VN =vegetative nucleus.
Fig. 1. - Metaphase I. Most of both MCs and tapetal cells are degenerating (dark stained), some MCs are collapsed
(arrows). X 170. Fig. 2. - Degenerating MCs at tetrad stage. X 150. Fig. 3- Degenerated tapetal cells at tetrad
stage; note microspores which have not yet completed meiosis (arrows). X 150. Fig. 4. - Collapsed microspores at
tetrad stage. X 450. Fig. 5. - Anther locule partially obstructed by proliferated tapetal cells; note some degenerated
tapetal cells (arrows). x 130. Fig. 6. - Microspores soon after release from tetrad callose. X235. Fig. 7. - Highly
collapsed microspores; tapetal cells have already developed Ubisch bodies. X 150. Fig. 8. - Tapetal cells surrounded
by Ubisch bodies (arrows). X 245. Fig. 9. - Tapetal cells (arrows) intruding among the microspores. x 260. Fig. 10.
- Tapetal cell syncytium surrounding microspores; note the Ubisch bodies (arrows). x 145. Figs. 11, 12. - Tapetal
cells with a tapering protuberance. x 260. Fig. 13. - Pollen grains and aborted microspore (arrow); note the scantly
cytoplasmic pollen grain filled with starch grains (arrows). X 320. Fig. 14. - Developing microspores before intine
formation, note aborted ones (arrows). X 130. Fig. 15. - Pollen grains stained with Sudan black B, after intine
formation; both cytoplasmic lipids and exine wall, as well as Ubisch bodies (arrows), stain green grey. X 245. Fig. 16.
- Mature pollen grain showing vegetative nucleus and generative cell. x 380. Fig. 17. - Mature pollen grain
stained with Sudan black B, showing numerous lipid globules in the cytoplasm and some spinulae (arrows) on exine;
intine wall does not stain. X 515.

254

CHICHIRICCO

TABLE 1 - Colour reactions and autofluorescence of Crocus sativus sections.

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Exine

In tine

Cytoplasm

Ubisch
bodies

Spore

Pollen

Spore

Pollen

Spore

Pollen

Toluidine
blue

Azure
blue

Azure
blue

Red
violet

Red
violet

Violet

Violet

Azure
blue

Azure B

Green

Green

Violet

Violet

Pale
violet

Light
blue

Green

Schiff's
reagent

Unstained

Unstained

Bright
pink

Pale
pink

Pink
grains

Pink
granules
21%
of pollen

Unstained

Nile blue

Azure

Azure

Violet

Violet

Pale
violet

Blue
grains

Azure

Sudan IV

Unstained

Unstained

Unstained

Unstained

Unstained

Orange
grains

Sudan
black B

Green
grey

Green
grey

Unstained

Unstained

Unstained

Blue
grains

Green
grey

Yellowish

Absent

Absent

Absent

Yellowish

Yellowish

Yellowish
Autofluorescence

m to 1 m; it stained intensely with basic dyes and very faintly with Schiff's
reagent (Table 1). About 62% of pollen grains showed a dense PAS positive
cytoplasm, indicative of dispersed polysaccharides, filled with lipid globules
(Fig. 17); occasionally, they included starch grains. The remaining (about 38%)
comprised pollen grains with loose or no cytoplasm, frequently filled with
starch granules (Fig. 13); occasionally, they included lipid globules. Among
pollen grains a variety of aborted spores might be observed; these appeared as
shapeless and empty grains consisting only of a sporopollenin wall (Fig. 13).

Tapetum behaviour during pollen development.

Tapetal cells attained maximum development after microspore release. At
this time, they might appear hypertrophied, and occasionally some of them
protruded a tapering protuberance into the locule (Figs. 11, 12). Tapetum lost
cell walls shortly after microspore release, and developed Ubisch bodies which
coated the inner protoplast faces (Fig. 8). Tapetal protoplasts generally retained
the initial position around the locule. However, sometimes, they intruded into
the locule (Fig. 9) forming a syncytium around the microspores (Fig. 10). It is
worth noting that also in this case tapetum developed Ubisch bodies following
protoplast fusion (Fig. 10). Tapetum became completely absorbed before pollen
maturation.

MICROSPOROGENESIS IN CROCUS SATIVUS

255

Autofluorescence.

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Autofluorescence manifested itself shortly after microspore release. At

this time it was observed in the whole exine wall, as well as in Ubisch bodies
(Fig. 23; Table 1). However, as development proceeded, it appeared also in the

Fig. 18. - Meiotic tetrads showing fluorescent callosic walls. X 195. Fig. 19. - Mature pollen grain
showing fluorescent callose deposits around intine and within cytoplasm. X 330. Fig. 20. - Callose
dispersed within pollen grain. x 310. Fig. 21. - Callose homogeneously dispersed within pollen grain.
X 370. Fig. 22. - Callose masses scattered within pollen grain. X 325. Fig. 23. - Autofluorescence
of both microspore exine and Ubisch bodies. x 255. Fig. 24. - Autofluorescent mature pollen grains.
x 130.

256

CHICHIRICCO

cytoplasm of pollen grains (Fig. 24). At maturity, most pollen grains were
brightly autofluorescent both in the exine and cytoplasm.

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Aniline blue induced fluorescence.

Aniline blue induced a yellowish fluorescence in sporocytes (Fig. 18), due

to their callosic wall which persisted till the end of meiosis. Moreover, a similar
fluorescence might be observed in a variety of mature pollen grains. These, in
section, showed one or more fluorescent masses close to intine or widespread
within the grain (Figs. 20, 22), and/or a continuous fluorescent layer around
the intine (Fig. 19); occasionally, fluorescence was homogeneously dispersed
within the pollen grain (Fig. 21).
DISCUSSION

Microsporogenesis, in C. sativus, shows some abnormalities such as meiocyte degeneration or production of deformed microspores. Unsuccessful of
microsporocytes is often associated with degeneration of many tapetal cells,
however a definite correlation between these phenomena cannot be assumed.
Microspores are 1- or 2-nucleolate, according to the following evidences: i)
MCs nucleus contains three prominent nucleoli, ii) the chromosome complement of C. sativus includes three homologous chromosomes with secondary
constriction (CmcHIRicco 1984), iii) the chromosomes associate in trivalents
during Metaphase I and daughter nuclei generally receive either one or two
chromosomes from any trivalent (CHICHIRICCO 1984). However, in this regard,
it would be expected that the percentage of uni-and binucleolate spores were
more or less similar and not different as those observed (89.5% uni-and 10.5%
binucleolate); this discordance could suggest gene inactivation phenomena in
the spore nucleus.
Microspores undergo a different development, generating different sized
and shaped pollen grains which, at maturity, may be distinguished in two main
groups: i) those densely cytoplasmic (62%), and those scantly cytoplasmic
(38%). The former generally undergo amylolysis during development, accumulating lipid globules as reserve material; the latter generally accumulate starch
grains instead of lipids. Among both the above groups, a variety of pollen
grains shows callosic deposits. In this regard, deposition of callose on intine
wall may be related to further pollen germination (pers. unpubl.); anyhow,
massive accumulation within the grain could indicate cytoplasm disorganization (cf. NoHER DE HALAC 1980).
Pollen grains, as previously reported (GRILLI CAIOLA et al. 1985), are
spinulate and lack germinative pores, bacula and tectum. The exine shows a
strong reactivity with: i) basic dyes, due to sporopollenin anionic sites (SOUTHWORTH 1973), and ii) Sudan black B, due to acidic lipids (PEARSE 1985). These
staining properties, as well as the autofluorescence, are observed in Ubisch

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MICROSPOROGENESIS IN

CROCUS SA TIVUS

257

bodies (Table 1), obviously due to chemical similarity between the above
bodies and exine (BHANDARI 1984). Concerning pollen autofluorescence, it has
been observed in several plants (cf. WILLEMSE 1972; AUDRAN and WILLEMSE
1982); in C. sativus pollen, as autofluorescence parallels staining with Sudan
black B (Table 1), it could be ascribed to acidic lipid compounds.
Summarizing, the present study points out that, in C. sativus, both
microsporocytes and tapetal cells, as well as microspores, often show abnormal
development. Variations regarding microspore development, may be correlated
with genie unbalance of microspore itself as consequence of meiotic irregularities (cf. CmcHIRICCO 1984). Otherwise, variations concerning both microsporocyte and tapetal development cannot be at present correlated to any specific
phenomenon. Cytological evidence characterizes the anomalous pollen grains as
displaying scanty cytoplasm, callose accumulation and very thickened intine.
The above considerations may be correlated with low and abnormal pollen
germination which were observed both in vitro and in vivo (CHICHIRICCO and
GRILLI CAIOLA 1986).
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Springer-Verlag, New York.
CmcHIRicco G., 1984. - Karyotype and meiotic behaviour of the triploid Crocus sativus L. Caryologia,
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Received 9 July 1988; revision accepted 4 May 1989