Ultrastructure of the stigma and style of Mangifera indica L.

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Ultrastructure of the stigma and style of Mangifera indica L. Esme de Wet*, P.J. Robbertse and J. Coetzee 1 Margaretha Mes Institute for Seed Research, University of Pretoria, Pretoria, 0002 Republic of South Africa and 1Electron Microscopy, University of Pretoria, Pretoria, 0002 Republic of South Africa

Accepted 7 November 1989 The stigma of Mangifera indica L. is non-papillate and wet, with the style semi-solid. The exudate of the stigma and style is of the mixed type and is transported from vesicles in the secretory cells, via plasmatubules, to the surrounding cell walls. Cell walls of the transmitting tissue of the stigma and style are bilayered. The outer wall layer is typically reticulate and the exudate accumulates between the loosely woven wall fibrils. At the base of the style a specialized obturator, the ponticulus, facilitates the direct passage of the growing pollen tubes towards the embryo sac. Die stempel van Mangifera indica L. is papilagtig en nat, en die sty I is semi-solied. Die sekreet van die stempel en styl is van die gemengde tipe en word vanaf vesikels in die sekreterende selle via plasmatubules na die omliggende selwande vervoer. Die selwande van die geleidingsweefsel van die stempel en styl is twee-Iagig. Die buitenste selwandlaag is tipies geretikuleerd en die sekreet versamel tussen die los fibrille van hierdie selwandlaag. 'n Gespesialiseerde obturator, die pontikulus, kom aan die basis van die styl voor en bevorder die deurgang van die stuifmeelbuise na die embriosak. Keywords: Cuticle, Mangifera indica, mango, obturator, ponticulus *To whom correspondence should be addressed

Introduction Some mango cultivars of economic importance are universally characterized by persistent low fruit production. An extensive project addressing possible problems concerning the reproduction biology of Mangifera indica L. has been in progress for 5 years. The structures associated with reproduction, the reproductive processes, initial fruit set and boron application to the trees in an effort to improve fruit set , are being investigated . Some of these results have already been published (de Wet & Robbertse 1986a, b; de Wet et al. 1986; Robbertse et al. 1986; Robbertse et al. 1988 ; de Wet et al. 1989). According to John et al. (1987) reduced fruit set in the mango is the result of the uncommonly thick and ornamented cuticle covering the receptive stigmatic cells. The presence of this cuticle would drastically curtail pollen tube entry , resulting in most ovules remaining unfertilized. In the present investigation of the stigma and style , ultrastructural information was obtained which is at variance with the results and conclusions of these authors. The ultrastructure of the mango stigma and style , with its specialized obturator is described in the present paper. Materials and Methods Material was collected from trees of the cultivars Haden and Sensation growing on the Lisbon Estate , Gazankulu (28°E , 25°S). Flowers at anthesis were collected between 9:00 and 10:00 and the pistils were removed for processing for light (LM) and electron microscopy (EM). For LM, material was fixed in 5% formaldehyde (Collins & MacNichol 1978) , dehydrated in ethanol and

infiltrated with GMA (Feder & O'Brien 1968). The PAS-reaction was carried out on 4 J-lm thick sections, counterstained with toluidine blue 0 in benzoate buffer (Sidman et al. 1961) . Sections were also stained with 0.05% aniline blue in 0.067 mol dm· 3 phosphate buffer (pH 8.5), and the pollen tubes identified with epifluorescence microscopy. For EM, fresh material was fixed for 2 h in 2.5% glutaraldehyde in 0.1 mol dm· 3 phosphate buffer (pH 7.4), post-fixed for 1 h in 1% aqueous osmium tetroxide, dehydrated in acetone and infiltrated with Quetol (Kushida 1974) . Gold interference colour sections were cut using a Reichert Om U-3 ultramicrotome and a diamond knife. The sections were contrasted with uranyl acetate (Watson 1958) and lead citrate (Reynolds 1963). The Thiery test for polysaccharides was performed with the necessary control procedures (Thiery 1967; Courtoy & Simar 1974) . Micrographs were taken with a Philips EM 301 transmission electron microscope . For the morphological study of the stigma and style, the material was studied with a Jeol JSM840 Scanning Electron Microscope (SEM) fitted with a cryostage . Fresh material was plunge-frozen in liquid nitrogen slush and sputter coated with gold before examination at -170°C. Results External morphology of the stigma and style (Figure 1) The stigma is relatively small; non-papillate and of the wet type (type IV) according to the classification of Heslop-Harrison & Shivanna (1977) . The cuticle of the stigmatic cells in the distal part of the style groove is relatively smooth and these cells probably represent the receptive area of the stigma . The remaining stigmatic

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cells with distinctly ornamented cuticle represent the non-receptive area of the stigma.

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A groove, forming the distal part of the stylar canal, begins at the stigmatic region and gradually closes

Figures 1-5 1. Electron micrograph of the stigma and style. s = stigma; st = style. Bar = 100 f.Lm. 2. Ultrastructure of the receptive and non-receptive stigmatic cells. ns = non-receptive stigmatic cells; rs = receptive stigmatic cells. Bar = 1 fLm. 3. Receptive papilla cell with external cuticle rupturing in places and releasing the exudate. Cell wall consists of two layers: a dense inner layer and an outer reticulated layer of loosely woven fibrilae. c = cuticle; e = exudate ; i = inner wall layer; 0 = outer wall layer. Bar = 0.5 fLm. 4. Convoluted cuticle from non-receptive area of stigma. c = cuticle. Bar = 0.5 fLm. 5. Transection of stigmatic cell . Note active dictyosomes, numerous polysomes, rough endoplasmic reticulum and mitochondria. Bar = 0.5 fLm.

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towards the proximal end of the style (see also Scholefield 1982), forming a semi-solid type of style (Figure 1). Internal structure of the stigma and style Stigma

The receptive area of the stigma is characterized by a smooth and thin discontinuous cuticle (Figures 2 & 3) and the non-receptive area, by a thick continuous cuticle (Figures 2 & 4). The latter has a convoluted topography which is a reflection of the uneven thickness of the distal parts of the epidermal cell walls (Figures 2 & 4). Optimum stigma receptivity is found to occur at full anthesis, when a very small amount of superficial exudate is secreted by the receptive cells. The cytoplasm of the stigmatic cells is dense and restricted to the periphery of the cell. Rough endoplasmic reticulum (RER), polysomes and actively vesiculating dictyosomes are abundant (Figure 5) . Mitochondria, lipid bodies , microbodies and plastids are numerous and the nucleus is lobed. Each cell contains a large central vacuole with multivesicular bodies and ergastic compounds. The plasmalemma is convoluted and is often differentiated into plasmatubules (Figure 6). Cells of the receptive area have bilayered walls and each layer may be of varying thickness. The outer layer is reticulate and the wall widens due to the formation of interstitial openings that are filled with exudate (Figure 7). This exudate or secretion within the wall stains positively for polysaccharides (Thiery 1967) as well as for lipids (Bahr 1954). The inner layer is more electron dense and does not exhibit this loose fibrillar structure (Figure 7). Style The rugose outer surface of the external stylar epidermal cell walls is covered by a thick cuticle, similar to that occurring on the non-receptive area of the stigma. The adjacent cortical tissue is traversed by many small vascular bundles. Some of the parenchymatous cortical cells are tanniniferous . The structure of the transmitting cells in the stylar canal corresponds to that of the receptive stigmatic cells. The inner epidermal cell layer (or endothelial cells) of the transmitting tissue that lines the stylar canal is covered by a discontinuous cuticle. In places the outer reticulate wall layer of these epidermal cells is ruptured (Figure 8), facilitating the release of exudate into the stylar canal. Polysomes, RER and mitochondria are abundant in these cells while very few dictyosomes and microbodies are present. All the plastids contain starch and few lipid bodies occur. Ultrastructurally the secretory product in the stylar canal appears to be heterogeneous and stains positively for polysaccharides (Thiery 1967; Figure 9) and for lipids (Bahr 1954). The accumulation of exudate beneath the cuticle causes it to rupture. Eventually only remnants of the cuticle are to be found in the stylar canal (Figure 9). The plasmalemma is convoluted and differentiated into plasmatubules , similar to that of the stigmatic cells.

Figure 10 illustrates the structure of the transmitting cells in the proximal part of the style at the level where no canal is present. This transmitting tissue is a continuation of the transmitting cells lining the stylar canal. The contents and walls of the transmitting cells and the structure of the cell walls are similar to those of the cells of the stylar canal and to those of the receptive stigma area. At the base of the style, the ovary wall forms an outgrowth protruding into the ovary locule (Joel & Eisenstein 1980; de Wet & Robbertse 1986b; de Wet et al. 1986) . This outgrowth (or ponticulus) is a proliferation of both dermal and subdermal origin and is therefore a continuation of the transmitting tissue of the style. The ponticulus lies appressed to the raphechalazal region, which would seem to facilitate the direct guidance of the pollen tube (Figure 11) towards the embryo sac. Although relatively more small vacuoles and more dictyosomes are present in the ponticular cells, their ultrastructure does not differ significantly from that of the cells of the rest of the transmitting tissue. The walls of the ponticular cells are generally thinner and the outer cell wall layer is less reticulate than that of the the rest of the transmitting tissue, but an exudate nevertheless does accumulate between the wall fibres (Figure 12). The inner cell wall layer of the ponticulus cells appears to be more organized although no plasmatubules or plasmodesmata can be identified. Ponticular cells are slightly papillate and project into the ovarian locule. Their walls are thinner than those of the rest of the cells of the inner epidermis of the locule. The cuticle of these papillate ponticular cells is discontinuous and a small amount of the secretion is released into the locule (Figure 13). Discussion Stigma

Two areas can clearly be distinguished in the stigma of the mango, namely a receptive area with cells covered by a thin, smooth discontinuous cuticle, and a nonreceptive area with cells covered by a thick, smooth continuous cuticle. The external convo luted appearance of the cuticle is due to the uneven thickness of the distal parts of the epidermal cell walls. In their description of the mango stigma, John et al. (1987) assumed that all stigmatic cells are covered by an uneven, thick, ornamented cuticle. They mention the presence' of a pellicle, which generally is a feature of dry stigmas (Shivanna & Sastri 1981). However, in our opinion this so called pellicle of John et al. (1987) probably represents fragments of the thick cuticle breaking loose and lying over the receptive area. John et al. (1987) concluded that poor fruit set can be attributed to the low percentage of ovules that are fertilized, owing to the inability of most pollen tubes to penetrate the thick cuticle of the stigma. However, the thick cuticle probably has no influence on pollen tube germination and penetration as the stigma has a definite

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receptive area for pollen tube e ntry. Furthermore, a

receptive area of the stigma , through the transmitting

pathway exists for growing pollen tubes from the

tissue of the styl e and ponticulus , towards th e ovule.

Figures 6-9 Stigmatic cell with plasmatubules between plasmalemma and cell wall. Plasmodesmata connect cytoplasm of adjacent cells . p = plasmatubules; pi = plasmodesmata. Bar = 0.25 fLm . 7. Exudate in distended cell wall interstices of receptive sells. e = exudate; i = inner wall layer ; 0 = outer wall layer. Bar = 0.5 fLm. 8. Stylar canal lined by endothelial cells. Exudate is released into stylar canal and remnants of the cuticle are distinguishable. c = cuticle; e = exudate; en = endothelial cells. Bar = 1 fLm. 9. E lectron-dense areas in endothelial cells , corresponding with Thiery positive substances. e = exudate ; i = inner wall layer; o = outer wall layer. Ba r = 0.5 fLm.

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Style

Hanf (1935) described three types of style structures: open styles (characteristic of monocotyledonous plants)

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in which the route for pollen tube growth to the ovary is through a mucilage-filled stylar canal; solid styles (usually found in dicotyledonous plants) in which the

Figures 10-13 10. Transmitting tissue (with distended cell walls) in the solid part of the style. t = transmitting tissue. Bar = f.lm. II. The ponticulus in close contact with the ovule, guiding a pollen tube towards the chalazal region of the ovule. ov = ovule; po = ponticulus; pt = pollen tube. Bar = 100 f.lm. 12. Transection of ponticular cells. Cell walls less reticulate, but exudate still accumulates in distended cell wall interstices. e = exudate. Bar = 0.5 f.lm. 13. Slightly papillate ponticular eell with discontinuous cuticle. c = cuticle; 1 = locule. Bar = 1 f.lm.

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pollen tubes pass through the transmitting tissue towards the ovary, and semi-solid styles which exhibit intermediate features. Semi-solid styles have been described for a few members of the Cactaceae (Hanf 1935), for Begonia tuberhybrida Voss of the Begoniaceae (Knox 1984) and for Persea americana L. (avocado) belonging to the Lauraceae (Sedgley & Buttrose 1978). Sedgley & Buttrose (1978) speculated that the semi-solid style of the avocado may possibly be an intermediate stage between the primitive carpellary state, as exemplified by Drimys piperita Hook f. (de Boer & Bouman 1974) and the solid style of dicotyledonous plants. The occurrence of a semi-solid style in the mango (belonging to the relatively primitive Anacardiaceae) can similarly be interpreted as a primitive trait. The fact that in the mango, transmitting tissue is found in the central parts of the proximal style, supports the assumption that this type of style developed by longitudinal folding of a carpel. Transmitting tissue The cell wall structure of the transmitting tissue is cHaracteristic, with an organized inner layer and a reticulate outer layer. These structural modifications are present in all tissues concerned with pollen tube growth, and are found in cells of the stigma, style, ponticulus and nucellus. The exudate accumulates within the outer wall layer, through which the pollen tube grows. Plasmatubules It is not clear how the stigmatic and stylar exudate is

translocated from the cell cytoplasm where it is presumably synthesized, to the interstitial openings in the cell walls. This type of transport would normally be expected to be associated with the presence of the well-developed wall ingrowths of transfer cells . However, there is no indication of such cells occurri ng in the transmitting tissues of the pistil. The only structural modifications which may be implicated in the transport of exudate solubles from the symplast to the apoplast, and which are present in these cells, are plasmatubules (Figure 7). Harris et al. (1982) postulated that these structures are present in cells where a large flux of solutes occur across the plasma membrane , especially if this transport is a transient phenomenon (Coetzee & Fineran 1987). These parameters are exactly those which occur in the transmitting tissue of the pistil. In the absence of other structural adaptations which may facilitate this transport, the plasmatubules are probably implicated in the transport of the exudate from the cytoplasm to the cell wall. Ponticulus According to Bor & Bouman (1974), Baillon in 1858 introduced the term 'obturator' for 'a shoulder of conducting tissue' in the ovary. Since then, most authors have used their own criteria in defining this structure. Tilton & Horner (1980) proposed that any portion of the transmitting tissue which occurs in the ovary and which

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facilitates pollen tube growth towards the embryo sac should, irrespective of its anatomical modifications, be considered to be an obturator. Joel & Eisenstein (1980) coined the term 'ponticulus' for protuberances on the dorsal side of the ovule as well as for those on the section of the ovary wall closest to the base of the style. According to these authors the epidermal cells in those areas where the protuberances originate, become meristematic after pollination and form a so-called 'bridge' or ponticulus connecting the ovary wall with the ovule. The pollen tube then grows down the style, through the ponticulus and nucellus, and into the embryo sac. Tn contrast to the description of the development of the ponticulus by Joel & Eisenstein (1980), it was found during this investigation that it was not the epidermal cells of the dorsal part of the ovule that became meristematic after pollination, but the epidermal and the subdermal cells of the ovary wall directly at the base of the style. During subsequent development, the cells of the ponticulus press against the dorsal cells of the chalazal end of the ovule, but no fusion occurs. It is concluded that the ponticulus, located at the stylar base and projecting into the ovary locule, is structurally similar to the transmitting tissue of the style and may guide the pollen tube towards the ovule. The ponticulus is therefore functionally similar to an obturator, in spite of the fact that it is not associated with the micropyle. Tn the mango we prefer the term ponticulus (bridge) for the obturator, as it is a particularly specialized structure ensuring the shortest route for the pollen tube on its way to the ovule. This term also pinpoints its position in the ovary, preventing confusion with other modifications of ovarian transmitting tissues. In the unilocular ovary of the Thymelaeaceae (Wunderlich 1967) a structure similar to the ponticulus of the mango is formed by papillate cells at the base of the stylar canal and the 'roof of the ovary cave'. Bor & Bouman (1974) found a similar transformation of epidermal cells at the base of the style in Euphorbia milii Des Moulins. This epidermal tissue of Euphorbia, is glandular during the early stages of the ontogeny of the ovule, but it loses this function in the mature state. Conclusion As no obvious structural obstacles are present in or on the stigma and in the style which can inhibit pollen tube growth, the low fruit yield of the investigated mango cultivars cannot be attributed to structural defects in the stigma and style. Some of the reasons for the persistent low fruit yield in the mango may be: (i) a possible incompatibility reaction (Dolev 1979; El-Azzouni et al. 1976; Sharma & Singh 1970 & 1972; Singh et al. 1961); (ii) the fact that a low percentage of normal embryo sacs are present (Singh & Sharma 1972; Sturrock 1965; Chadha & Singh 1963; Sharma & Singh 1970, 1972); (iii) possible abortion of the embryo owing to genetic load (Charlesworth 1989a, b; Wiens et al. 1989); (iv) unsynchronized timing of the

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release of pollen and stigma receptIvIty (de Wet & Robbertse 1986a, b) and (v) boron deficiency in mango trees (de Wet et al. 1989). Acknowledgements

The authors wish to thank Dr Elzabe Stoffberg for discussions and comments on the manuscript. Financial support for this study was provided by the Department of Agriculture and Water Supply , as well as by the Foundation for Research Development (CSIR) and the University of Pretoria. References BAHR, G .F. 1954. Osmium tctroxidc and ruthenium tetroxide and their reactions with biologically important substances. Exp. Cell Res. 7: 457- 479. BOR, 1. & BOUMAN , F. 1974. Development of ovulc and integuments in Euphorbia milii and Codiaeum variegatum. Phytomorphology Sept/Dec: 280-296. CHADHA , K.L. & SINGH , K.K. 1963. Studies on fruit drop in mango. 1. Fruit set, its retention and factors affecting it. Indian 1. Hort. Sci. 20: 172- 185. CHARLESWORTH, D. 1989a. Why do plants producc so many more ovules than seeds? Nature 383: 21 - 22 . CHARLESWORTH, D. 1989b. Evolution of low femalc fertility in plants: pollcn limitation, resource allocation and genetic load. Tree 4: 289- 292. COETZEE , 1. & FINERAN, B.A. 1987. The apoplastic continuum , nutrient absorption and plasmatubules in the dwarf mistletoc Korthalsella lindsay (Viscaceae). Protoplasmal36: 145- 153. COLLINS, B.A. & MacNICHOL, E.F. jun. 1978. Longterm fixation for histological studies. Microsc. Acta 81: 155- 158. COURTOY, R. & SIMAR, L.l. 1974. Importance of controls for the demonstration of carbohydrates in electron microscopy with the silver methenamine or the thiocarbohydrazide-silver proteinate methods. 1. Microsc. 100: 199- 211. DE BOER , R. & BOUMAN , F. 1974. Integumentary studies in the Polycarpicae. III. Drimys winteri (Winteraceae). Acta bot. neerl. 23: 19- 27. DE WET, E. & ROBBERTSE , P.l. 1986a. A preliminary study of the pollcn of Mangifera indica L. cv. Haden in South Africa. S. Afr. 1. Plant Soil 3: 87- 89. DE WET, E. & ROBBERTSE, P.l. 1986b. Pollen studies and stigma receptivity of Mangifera indica L. cvs Haden and Sensation. S. Afr. Grs. Assoc. Res. Rep. 6: 27- 32. DE WET, E ., ROBBERTSE, P.l. & COETZER, L.A. 1986. Pollination and ponticulus development of Mangifera indica L. cvs Haden and Sensation. S. Afr. 1. Plant Soil 3: 76-79. DE WET, E., ROBBERTSE, P.l. & GROENEVELD, H.T. 1989. The influence of boron and temperature on pollen germination in Mangifera indica L. S. Afr. f. Plant Soil 6: 228-234. DOLEV, M. 1979. Self and cross pollination and their effects on fruit set in some mango varieties. M.Sc. thesis , Fac. of Agric., Hebrew Univ. of Jerusalem. EL-AZZOUNI , M.M. , FOUAD , M.M. , KABEAL , M.T. & EL-KADY, M.1. 1976. The question of low cropping in 'Taimour' mango trees. Egypt. 1. Hort. 3: 29- 35. FEDER, N. & O 'BRIEN, T.P. 1968. Plant microtechnique: some principles and new methods. Am. 1. Bot. 55: 123-142.

S.-Afr.Tydskr. Plantk., 1990,56(2) HANF , M. 1935. Vergleichcnde and cntwicklungsgeschichtliche Untcrsuchungen lIber morphologie und Anatomie der Griffel und Griffellaste. Beihefte zur botanische Zentraalblatt A 54: 99- 141. HARRIS , N., OPARKA, K.J. & WALKER-SMITH , 0.1. 1982. Plasmatubules: an alternative to transfer cells? Planta 156: 461-465. HESLOP-HARRISON , Y. & SHIVANNA , K.P. 1977. The receptive surface of the Angiosperm stigma. Ann Bot. 41: 1233- 1258. lOEL , D.M. & EISENSTEIN, 0.1980. A bridge between the ovule and ovary wall in Mangifera indica L. (Anacardiaceae) . Acta bot. neerl. 29: 202- 206. lOHN , P., RAGHURAM , Y. & NAIR, G.M. 1987. Uncommon cuticular ornamentation of the stigma surface of mango (Mangifera indica L.). Curro Sci. 56: 783- 784. KNOX, R.B. 1984. Pollen- pistil interactions. In: Encyclopedia of Plant Physiology, eds Linskens , H.F. & Heslop-Harrison , J. , Vol. 17, pp. 508- 608 , 743 pp., Springer-Verlag, Berlin. KUSHIDA , H. 1974. A new method for embedding with a low viscosity Epoxy resin 'Quetol 651 ' . 1. Electr. Microsc. 23: 197. REYNOLDS , E.S. 1963. The usc of lead citrate at high pH as an electron opaque stain on electron microscopy. 1. Cell Bioi. 17: 208- 212. ROBBERTSE , P.l. , DE WET, E. & COETZER, L.A. 1988. The influence of temperature and boron on pollen tube growth in mango. S.A. Mango Growers' Assoc. Yrb. 8: 4-6. ROBBERTSE, P.l. , VON TEICHMAN , I. & JANSE VAN RENSBURG , H. 1986. A re-evaluation of the structure of the mango ovule in comparison with those of a few other Anacardiaceae species. S. Afr. 1. Bot. 52: 17-24. SCHOLEFIELD, P.B. 1982. A scanning electron microscope study of the flowers of avocado, litchi, macadamia and mango. Scientae Hort. 16: 262- 272. SEDGLEY, M. & BUTTROSE, M.S. 1978. Structure of the stigma and style of the avocado. Austr. 1. Bot. 26: 663-682. SHARMA , O.K. & SINGH, R.N. 1970. Self-incompatibility in mango (Mangifera indica L.). Hort. Res. 10: 108- 118. SHARMA , D .K . & SINGH, R.N. 1972. Investigations on self-incompatability in Mangifera indica L. Acta Hort. 24: 126- 130. SHIVANNA, K.R. & SASTRI, D.C. 1981. Stigma-surface esterase activity and stigma receptivity in some taxa characterized by wet stigmas. Ann. Bot. 47: 53- 64. SIDMAN, R.L., MOTTLA, P.A. & FEDER, N. 1961. Improved polyester wax embedding for histology. Stain Teclmol. 36: 279- 284. SINGH, R.N. , MAJUMBER , P.K. & SHARMA , O.K. 1961. Self-incompatability in mango (Mangifera indica L.) var. Dashehari. Curro Sci. 31: 209 . SINGH , R.N. & SHARMA, O .K . 1972. Some pollination problems in mango D. Acta Hort. 24: 134-138. STURROCK, T.T. 1965 . Degenerate mango ovaries. Proc. Fla . State Hort. Soc . 82: 321 - 324. THIERY, 1.P. 1967. Mise en evidence des polysacharides sur coupes fines en microscopie electronique. 1. Microscopie 6: 987- 1018. TILTON, V.R. & HORNER , H.T. jun. 1980. Stigma, style , and obturator of Ornithogalum caudatum (Liliaceae) and their function in the reproductive process. Am. 1. Bot. 67: 1113- 1131. WATSON , M.L. 1958. Staining of tissue sections for electron microscopy with heavy metals. 1. Biophys. Biochem. Cytol. 4: 475-478.

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S.Afr.J. Bot., 1990,56(2) WIENS, D., NICKRENT, D.L. , DAVERN , c.r., CALVIN, c.L. & VIVRETTE, N.J. 1989. Developmental failure and loss of reproductive capacity in the rare palaeoendemic

shrub Dedeckera eurekensis. Nature 338: 65-67. WUNDERLICH, R. 1967. Some remarks on the taxonomic significance of the seed coat. Phytomorph. 17: 301-311.