Indian Journal of Experimental Biology
Vol. 46, January 2008, pp. 71-78
Abnormal anther development and high sporopollenin synthesis in benzotriazole
treated male sterile Helianthus annuus L.
S M Tripathi & K P Singh
Department of Botany, R.B.S. College, Agra 282 002, India
Received 6 July 2007; revised 9 October 2007
Foliar application of 1.5% benzotriazole induced 100% pollen sterility in H. annuus. Pollen abortion in treated plants was
mainly associated with abnormal behaviour of tapetum. A limited number of anther locule showed early degeneration of
tapetum followed by disintegration of sporogenous tissues. On the other hand, some locules showed normal development of
tapetum at initial stages. However, this tapetum exhibited degenerated and non-functional cell organelles. In both these
situations tapetum failed to provide proper nourishment to developing microspores. The ultrastructure of both tapetum and
microspores is different from that of control material with irregularities of exine deposition, endopolyploidy of tapetal nuclei
and an alteration of organelle composition being correlated with sterility. Pollen grains thus developed were devoid of
nucleus and cell organelles and were complete sterile.
Keywords: Helianthus annuus L., Benzotriazole, Induced male sterility, Tapetum development.
Chemical induction of male sterility is a unique
phenomenon to achieve hybrid seeds in F1
generation1. In this system the affected organ and
tissues are stamens and pollen grains. The stamen
plays an integral role in crop production because it is
responsible for carrying out the male reproductive
process. Development of pollen grains within the
anther is a precisely timed, high metabolic demand
process. The tapetal layer of anther provides enzymes,
hormones, nutritive materials, nucleosides and
nucleotides for microsporogenesis to proceed.
Therefore, the tapetum assumes a vital nutritive role
especially during and after microsporogenesis. Many
indirect and circumstantial evidences indicate that
abnormal tapetal behaviour is the root cause of male
sterility in higher plants. It appears that male sterility
is the result of multitude effects, which are more or
less related to insufficient or mistimed supply of
necessary resources for developing microspores.
Some ultra-structural investigations on cytoplasmic
male sterility have been performed in order to find out
the causes of male sterility2-4. But, literature related to
ultra-structural studies of anther development in
chemical hybridizing agent (CHAs) treated plants is
insufficient. There are only a few reports dealing with
___________
*Correspondent author
Phone: +919411651734
e-mail: [email protected];
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the effects of CHAs on anther development and
microsporogenesis at light- and electron-microscopic
level5,6. Ultra-structural studies revealed that tapetum
exhibited premature degeneration of mitochondria and
plastids in ethrel and benzotriazole treated plants of
Vicia faba5 and Brassica juncea6. Tapetal cells of
benzotriazole treated plants of B. juncea secreted
large amount of sporopollenin which accumulated at
several places in anther locule. Pollen grains of
treated plants possessed degenerated protoplast5,6.
Thus this investigation has been carried out to
study the effect of benzotriazole on anther
development and microsporogenesis in Helianthus
annuus L. at light and electron microscopic level.
Materials and Methods
Plant materials—Seeds of Helianthus annuus L.
cv. MSFH-17 were obtained from IARI, New Delhi
and were sown at the Botanic garden, School of Life
Sciences, Dr. B.R. Ambedkar University, Agra in
randomized row design.
Treatments—Twenty-five plants were treated once
with an aqueous solution of 1.5% (w/v) benzotriazole
(C6H5N3). The foliar sprays were done a week before
floral bud initiation. Another group of 25 plants was
sprayed with distilled water to serve as control.
Pollen fertility and sampling—Pollen viability was
tested at regular intervals with the Flurochromatic
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INDIAN J EXP BIOL, JANUARY 2008
reaction (FCR) test7 and by Alexander’s stain8.
Anthers at different stages of development were
collected from inflorescences of male fertile control
plants and benzotriazole treated 100% male sterile
plants.
Processing for microscopy—Anthers were fixed for
24 hr in 3% glutaraldehyde in 0.1 M PO4 buffer at pH
6.8. Then the samples were rinsed twice in the same
phosphate buffer for 5 min each. Post fixation was
done with 1% osmium tetra oxide in the same buffer
for 2 hr. Samples were dehydrated in an ethyl
propylene oxide series, embedded in spurr’s low
viscosity embedding media and polymerized at 60°C
for overnight. For light microscopy, sections were cut
at 1 μm with Reichert OMU-2 ultramicrotome and
mounted on a glass slide. For electron microscopy
they were cut at 60-80 nm and picked up on goldcoated copper grids.
Sections for light microscopy were stained with the
solution of 0.5% (w/v) toluidine blue in 1% (w/v)
sodium borate and for electron microscopy with
uranyl acetate and alkaline lead citrate. The light
microscopic sections were viewed and photographed
with an oil immersion microscope and a CCD
Camera. Subsequent electron microscopic observations on chosen anther locules were made on a Philips
Cryo CM 10 electron microscope at the All India
Institute for Medical Sciences (AIIMS), New Delhi.
Results
The male fertile control plants possessed dehiscent
anthers with only 5.25% sterile pollen. The anther
wall at sporogenous tissue stage composed of
epidermis and endothecium to the outside and a
flattened middle cell layer and uniseriated tapetum
surrounding the sporogenous mass. Sporogenous cells
were connected to each other and to the tapetum by
plasmodesmotal connections. The tapetal cells were
cytoplasmically denser at this stage (Fig. 1).
Sporogenous tissue acquire callose wall and became
pollen mother cell. Pollen mother cells undergo
meiosis and produce dyads and tetrads. At tetrad stage
tapetum started to separate from other wall layers and
become periplasmodial in nature (Fig. 2). Tapetum is
binucleated at tetrad stage. Tetrad has shown
microspores, which possessed well-developed nucleus
and functional cell organelles. Each microspore was
surrounded by primexine (Fig. 3). This tapetal
cytoplasm was rich in mitochondria, plastids,
ribosomes, ER, microtubules, dictyosome, lipid
inclusions etc. Mitochondria present in the tapetum
were oval with mitochondrial cristae (Fig. 4). The
metabolically active nature of tapetum was
represented by the presence of functional cell
organelles. Microspores in a tetrad separated from
each other by disappearance of callose and released in
anther locule. Periplasmodial mass of tapetum came
in to anther locule and mixed with developing pollen
grains (Fig. 5). The plasmodial tapetum disintegrated
and concomitantly the pollen grains began to fill with
food reserves (Fig. 6). Cell organelles particularly
mitochondria still remained functional at young
pollen grain stage (Fig. 7). Mature pollen grains
exhibited spiny ektexine with thick endexine and thin
intine. Pollen cytoplasm possessed well-developed
nucleus and cell organelles, showing physiologically
active nature of pollen. (Fig. 8).
On the other hand, single spray with 1.5%
benzotriazole induced 100% pollen sterility. The
leaves became slightly scorched at their margins after
the treatment, however, it was failed to cause any
other phytotoxic effects. The male sterility in
benzotriazole treated plants mainly occurs due to
abnormal behaviour of tapetum. The tapetal cells,
normally the supplier of nutrients and precursor of
sporopollenin, were deeply stained, slightly expanded
and contained irregular endopolyploid nuclei at
sporogenous tissue stage in sterile anthers (Fig. 9).
The remarkable feature is that a couple of anther
locules showed early degeneration of tapetum which
was followed by disintegration of sporogenous tissue
due to lack of nutrition and thus leaving a degenerated
mass in the anther cavity (Fig. 10). Whereas,
remaining two anther locules showed normal
development of tapetum at primary stage of
development particularly up to pollen mother cell
stage (Fig. 9).
On further development, at tetrad stage, the
protoplasmic content of tapetum became disintegrated
due to presence of deformed and non-functional
organelles (Fig. 11). At this stage, tapetum also
exhibited a large number of vacuoles (Fig. 11) which
became enlarged with further development (Fig. 12).
Mitochondria and plastids were smaller and both were
devoid of cristae and lamellae respectively (Figs 11
and 12).
In sterile anthers the process of formation of tapetal
periplasmodium becomes slightly delayed in
comparison of fertile ones. In fertile anthers tapetum
become periplasmodial at tetrad stage whereas, in
TRIPATHI & SINGH: ABNORMAL ANTHER DEVELOPMENT AND HIGH SPOROPOLLENIN SYNTHESIS
73
Figs 1-4—Light and transmission electron micrographs of anther development in male fertile control plants of Helianthus annuus. 1:
Transmission electron micrograph showing sporogenous tissue (spg) surrounded by well developed tapetum (tp). 1500 × 2: LM
photograph of tapetum (tp), showing its detachment from other wall layers at microspore tetrad stage. Tetrad (t) exhibited well developed
callose wall. 540 × 3: Transmission electron micrograph of tapetum at microspore tetrad stage showing well developed mitochondria
(mt), endoplasmic reticulum (er), golgi bodies (gb), ribosomes (r) and few small vacuoles (v). 4800 × 4: Transmission electron
micrograph of well developed microspore tetrad showing nucleus (n) in microspores, primexine (pex) and callose wall (ca). 4800 ×.
sterile anthers periplasmodial formation occurred at
microspore stage. This disintegrated plasmodial mass
invades into anther cavity and intermingled with
developing pollen grains. This plasmodial mass
exhibited degenerating cell organelles particularly
mitochondria and plastids (Figs 12 and 13). At pollen
grain stage a large number of lipid and sporopollenin
bodies have been observed resulting in the formation
of thick ektexine (Fig. 13).
Pollen grains of anthers of benzotriazole treated
plants showed well differentiated ektexine with the
presence of cavus I and internal foramina (Figs 13 and
14). Endexine was also made up of lamellae and
colpus showed the presence of fibrous material in
these pollen grains. However, the space between
ektexine and endexine or cavus II failed to shrink with
maturity thus both of these layers remain separated
throughout the period (Figs 13 and 14). Spine
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INDIAN J EXP BIOL, JANUARY 2008
Figs 5-8—Light and transmission electron micrographs of anther development in male fertile control plants of Helianthus annuus. 5: LM
photograph showing microspores (m) surrounded by mass of periplasmodial tapetum (pl). 540 × 6: LM photograph of anther locule
showing pollen grains (pg) and absorbing mass of tapetal periplasmodium (pl). 620 × 7: Transmission electron micrograph of tapetum at
pollen grain (pg) stage showing functional mitochondria (mt) and plastids (p). 2400 × 8: Transmission electron micrograph of mature
pollen showing well developed nucleus (n) with nucleolus (nu). Cytoplasm rich with mitochondria (mt), golgi bodies (gb) and lipid
inclusions (l). Pollen wall differentiated into ektexine (ek) and endexine (en) with well organized Cavus (cv- I & II). 3800 ×.
formation was also greatly inhibited in these pollen
grains (Figs 13 and 14).
The cytoplasm of sterile pollen grains was without
nucleus, vacuolated and possessed degenerated cell
organelles with starch and lipid inclusions. These
features made the pollen physiologically inactive and
sterile (Figs 13 and 14). Tapetal periplasmodium try
to migrate, inside the pollen grain through the cavus I
and fill the cavus II and ektexine get disrupted in a
limited number of pollen grains. These pollen grains
were devoid of endexine and protoplasm. In these
pollen grains tapetal debris with sporopollenin like
material invaded inside the cavity and accumulated in
the center (Fig. 14). All these pollen grains were
physiologically inactive and sterile.
Discussion
Anther development in benzotriazole treated plants
has shown remarkable deviation from the fertile once.
TRIPATHI & SINGH: ABNORMAL ANTHER DEVELOPMENT AND HIGH SPOROPOLLENIN SYNTHESIS
75
Figs 9-12—Light and transmission electron micrographs of anther development in benzotriazole treated male sterile plants of Helianthus
annuus. 9: LM photograph showing two anther locules with stages of development viz. sporogenous tissue (spg) and pollen mother cell
(pmc) with dense tapetum (tp). 540 × 10: LM photograph of anther locules showing early degeneration of tapetum followed by
sporogenous tissues. Arrow indicated degenerated mass of tapetum and sporogenous tissues. 540 × 11: Transmission electron micrograph
of tapetum at microspore tetrad stage showing vacuolation (v) with disintegrated endoplasmic reticulum (er), golgi bodies (gb) and
mitochondria (mt). 2400 × 12. Transmission electron micrograph of tapetum at microspore showing large vacuoles (v) and degenerated
cell organelles. 1900 ×.
In sterile anthers of periplasmodial tapetum was
metabolically inactive due to presence of degenerated
cell organelles particularly mitochondria and plastids.
Thus, it failed to provide proper nourishment to
developing microspores. This results into the
development of sterile pollen grains with degenerated
protoplasm.
A large number of gametocides or CHAs have been
identified for hybrid seed production in various
crops9. These CHAs can be categorized into four
classes according to their action on anther development and microsporogenesis10. According to this
classification, benzotriazole is a copper chelator
compound and act as an inhibitor of microspore
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INDIAN J EXP BIOL, JANUARY 2008
TRIPATHI & SINGH: ABNORMAL ANTHER DEVELOPMENT AND HIGH SPOROPOLLENIN SYNTHESIS
development. Benzotriazole put a remarkable effect
on pollen fertility in various plants11-13 and disrupt the
microspore development6. Singh and Chauhan11 have
studied the effect of benzotriazole on plant height,
pollen fertility and yield of H. annuus. According to
them, 1.5% benzotriazole induced 100% pollen
sterility and caused reduction in plant height and total
yield/plant. On the basis of these results, the present
investigation has been carried out to find out the exact
cause of pollen abortion in the anthers of
benzotriazole treated plants by light and electron
microscopy.
Tapetum is the main target site for gametocide
action and which is responsible for abortion of
microspores14. Tapetal cells of fertile and sterile
anthers collapse in different ways. Tapetum
degeneration in the sterile anthers was started with the
commencement of vacuolation which is followed by
degeneration of cell organelles. Vacuolation in tapetal
cytoplasm manifest the first aspect of degeneration
and it frequently precedes pollen sterility3,6.
Mitochondria and plastids were largely affected by
benzotriazole. They were quite smaller with disrupted
cristae and lamellae. The structure of mitochondria
and plastid apparatus was considerably deformed in
various CMS plants15-17. Anther development in
benzotriazole treated Brassica juncea exhibited small
mitochondria with degenerated cristae and plastids
filled with lipids. Alteration in mitochondrial
ultrastructure may be associated with changes in the
energy requirements of the cell18. Degeneration in
mitochondria seems to be responsible for the decrease
in oxygen uptake in the sterile anthers and thus may
be associated with lower metabolic activity of tapetal
cells.
Tapetum of benzotriazole treated anthers of
sunflower release a large amount of sporopollenin due
to which exine become thick. Thick exine and
absence of intine has also been reported in sterile
microspores of Vicia faba19. Jwell et al.20 also
reported irregular pollen development in copper
deficient barley plants due to abnormal behaviour of
tapetum. Similar feature of abnormally high secretion
of sporopollenin was observed in benzotriazole
treated Brassica juncea6. This extra sporopollenin
gets accumulated at several places in anther locules.
An accumulation of unpolymerized sporopollenin
precursors increased the osmotic potential of the
locular fluid and drew water out of the microspores,
causing them to collapse21.
Acknowledgement
The first author is grateful to CSIR for the financial
assistance in the form of JRF and SRF.
References
1
2
3
4
5
6
7
8
9
Figs 13-15—Transmission electron micrographs of anther
development in benzotriazole treated male sterile plants of
Helianthus annuus. 13: Transmission electron micrograph of
pseudoperiplasmodial tapetum at pollen grain stage. Highly
vacuolated pollen grain showing the presence of lipid (l) and
degenerated cell organelles. Ektexine (ek) and endexine (en) are
thick with large Cavus I (cv I) and Cavus II (cv II). Intine is
absent. 1900 × 14: Transmission electron micrograph of pollen
grain is devoid of nucleus showing degenerated plastids (p) and
thick endexine (en). 2400 × 15: Transmission electron micrograph
of completely degenerated pollen grain showing accumulation of
sporopollenin (sp) in centre. Ektexine is thick and endexine and
intine are completely absent. Note the presence of large Cavus I
(cv I) and Cavus II (cv II). 2400 ×.
77
10
11
12
13
Kaul M L H, Male sterility in higher plants (Springer Verlag,
Berline, Germany) 1988.
Horner H T, A comparative light and electron-microscopic
study of microsporogenesis in male fertile and cytoplasmic
male sterile sunflower (Helianthus annuus), Am J Bot 64 (6)
(1977) 745.
Bino R J, Ultrastructural aspects of cytoplasmic male sterility
in Petunia hybrida. Protoplasma 127 (1985) 230.
Smith M B, Palmer R G & Horner H T, Microscopy of a
cytoplasmic male sterile soybean from an interspecific cross
between Glycine max and G. soja (Leguminosae), Am J Bot,
89 (3) (2002) 417.
Chauhan Surabhi & Chauhan S V S, Ultrastructural studies
on anther development in ethrel induced male sterile Vicia
faba L, J Ind Bot Soc, 84 (2005) 49.
Chauhan S V S & Singh Vandana, Chemical induction of male
sterility in Brassica juncea (L.) Czern & Coss and its
utilization for hybrid seed production, Brassica, 7 (2005) 117.
Heslop-Harrison J & Heslop-Harrison Y, Evolution of pollen
viability by enzymatically induced fluorescence, intercellular
hydrolysis of fluoresein diacetate, Stain Technol, 45 (1970)
115.
Alexander M P, A versatile stain for pollen, yeast and
bacteria, Stain Tech, 55 (1980) 13.
McRae D H, Advances in chemical hybridization, Plant
Breed Rev, 3 (1985) 169.
Cross J W & Schulz P J, Chemical induction of male
sterility, in Pollen biotechnology for crop production and
improvement, edited by K R Shivanna & V K Sawhney
(Cambridge University Press) 1997, 218.
Singh Vandana & Chauhan S V S, Effect of various chemical
hybridizing agents on Helianthus annuus L, J Indian Bot
Soc, 79 (2000) 71.
Singh Vandana & Chauhan S V S, Benzotriazole-a new
chemical hybridizing agent for Brassica juncea L, J Cytol
Genet, 2 (2001) 81.
Chauhan S V S & Chauhan Surabhi, Evaluation of three
chemical hybridizing agents on two varieties of broad bean
(Vicia faba L.), Indian J Genet, 63 (2003) 128.
78
14
INDIAN J EXP BIOL, JANUARY 2008
Chauhan S V S & Kinoshita T, Chemically induced male
sterility in angiosperms (Review), Seiken Ziho, 30 (1982) 54.
15 Atrashenok N V, Lyul’skina E I & IL’chenko V P, The
ultrastructure of anther cells in male-sterile and fertile barley
forms Geterozisi Kolichestern masled stvennost’ minsk
Belorussian SSr Naokai takhika 122-127 From referativnyl
Zhurnal 85511 Cited in Plant Breed Ab 49 (1977) 5813.
16 Warmke H E & Lee S L J, Mitochondrial degeneration in
Taxas cytoplasmic male sterile corn anthers, J Hered, 68
(1977) 213.
17 Nakashima H, Physiological and morphological studies on
the cytoplasmic male sterility of some crops, J Fac Agric
Hokkaido Univ, 59 (1978) 17.
18
Smith R A & Ord M J, Mitochondrial form and function
relationship in vivo: their potential in toxicology and
pathology, Int Rev Cytol, 83 (1983) 63.
19 Audran J M & Willemse T W, Wall development and its auto
fluorescence of sterile and fertile Vicia faba L. pollen,
Protoplasma, 110 (1982) 106.
20 Jwell A W, Murrray B G & Alloway B J, Light and electron
microscopic studies on pollen development in barley
(Hordeum vulgare L.) grown under copper sufficient and
deficient conditions, Plant Cell & Environ, 11 (1988) 273.
21 El-ghazaly G A & Jensen W A, Development of wheat
(Triticum aestivum) pollen wall before and after effect of a
gametocide, Can J Bot, 68 (1990) 2509.