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

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

___________ *Correspondent author Phone: +919411651734 e-mail: singhkprbs@rediffmail.com; tripathismani@rediffmail.com

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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 gold-coated 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 observa-tions 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

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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

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 ×.

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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.

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 ×.

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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 develop-ment and microsporogenesis10. According to this classification, benzotriazole is a copper chelator compound and act as an inhibitor of microspore

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 ×.

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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 Kaul M L H, Male sterility in higher plants (Springer Verlag,

Berline, Germany) 1988. 2 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.

3 Bino R J, Ultrastructural aspects of cytoplasmic male sterility in Petunia hybrida. Protoplasma 127 (1985) 230.

4 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.

5 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.

6 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.

7 Heslop-Harrison J & Heslop-Harrison Y, Evolution of pollen viability by enzymatically induced fluorescence, intercellular hydrolysis of fluoresein diacetate, Stain Technol, 45 (1970) 115.

8 Alexander M P, A versatile stain for pollen, yeast and bacteria, Stain Tech, 55 (1980) 13.

9 McRae D H, Advances in chemical hybridization, Plant Breed Rev, 3 (1985) 169.

10 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.

11 Singh Vandana & Chauhan S V S, Effect of various chemical hybridizing agents on Helianthus annuus L, J Indian Bot Soc, 79 (2000) 71.

12 Singh Vandana & Chauhan S V S, Benzotriazole-a new chemical hybridizing agent for Brassica juncea L, J Cytol Genet, 2 (2001) 81.

13 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.

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 ×.

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14 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.