ANATOMY OF THE STIGMA AND STYLE OF SWEET POTATO

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1 New PhytoL (1967) 66, ANATOMY OF THE STIGMA AND STYLE OF SWEET POTATO BY F. W. MARTIN AND SONIA ORTIZ Federal Experiment Station, Crops Research Division, Agricultural Research Service, U.S. Department of Agriculture, MayagUez, Puerto Rico {Received 2T, Atigust 1966) SUMMARY The stigma and style are the sites where self-incompatibihty and hybrid sterihty reactions occur in the sweet potato. The stigma is a highly branched structure covered with a specialized papillate epidermis which exudes a sticky fluid. The cells of the body of the stigma are arranged in gradually narrowing columns which conduct the pollen tubes from stigma to style. The style contains a central, uniform core of pollen-tube conducting tissue. The junction of style and ovary provides a continuous but contorted pathway of conducting tissue which pollen tubes follow. The incompatibility reaction, which occurs at the stigmatic surface, is hypothesized to be due to interactions of substances in the pollen and in the stigmatic exudate. Sterility, on the other hand, may be due to pollen tube disorientation or failure at the stigma-style junction and the style-ovary junction, where passage may be mechanically more difficult. INTRODUCTION A complex of sterility barriers and a system of self-incompatibility impede seed set after self- and cross-pollination in the sweet potato. The incompatibility reaction occurs at the surface of the stigma and results in failure of pollen to germinate (Togari and Kawahara, 1942). Varieties may be divided into three to eight inter-compatible, intra-incompatible groups. In contrast, sterility occurs after pollen germination and is manifest in failure of pollen tubes to pass from stigma to style; failure of tubes which have passed through the style to effect fertilization; and in abortion of embryos and failure of small or weak seeds (Martin and Cabanillas, 1966). Sterility is of general occurrence, and exists to some degree in all crosses. The physiological mechanisms associated with these incompatibility and sterility systems have not been adequately studied. As a base for future investigations, we have studied the morphology and anatomy of stigmas and styles of sweet potato with especial reference to pollen germination and the pollen tube pathway. Both stigma and the style are specialized structures with related but separate functions. Whereas the role of the former appears to be to catch and germinate pollen, the role of the latter appears to be conduction of the pollen tubes to the ovary. Whitehouse (1950) has emphasized the importance of the style in the development of the flowering plants. He suggests that the long, narrow style serves as a sieve where incompatible tubes, through inhibition, are screened from compatible tubes. However, morphology of stigma and style must, necessarily, be related to mode of pollination, and thus it would seem equally probable that long styles facilitate insect pollination in some species. Self-incompatibility systems involving failure of pollen germination may be associated with failure of an enzyme system to break down cutin covering the stigma (Linskens, 109

2 no F. W. MARTIN AND SONIA ORTIZ 1964). Such cutin enzyme systems have been found only in pollen of plants that have stigmas covered with cutin. After incompatible pollination, the enzyme system is not activated, or is inhibited (Christ, 1949). Mutilation of the stigma by physical or chemical means may permit pollen tube penetration in incompatible crosses (Crowe, 1964), but such treatment has not been effective in the sweet potato (Fujise, 1964). A second common incompatibility reaction is inhibition of pollen tube growth in the style. Finally, non-fusion of gametes within the ovule after penetration by male nuclei, and consequent flower abortion, is yet a third incompatibility mechanism (Cope, 1962). Sterility mechanisms, on the other hand, are so numerous, complex, and little known physiologically, that a review does not seem appropriate here (see Stebbins, 1958). In practice, considerable difficulty may be encountered in distinguishing sterility from incompatibility reactions. MATERIALS AND METHODS The sweet potatoes used were commercial varieties from the United States. For anatomical observations, stigmas and styles were harvested early in the morning, whereas for observations of the pollen tube pathway, stigmas were pollinated in the morning and harvested 8 hours later. Materials were fixed in FAA, embedded in paraffin through a ;«/.-butanol series of solutions, sectioned to various thicknesses, and stained with iron haematoxylin. Dilute I-KI solution, and 1% Sudan IV in 70 ^ ethanol or ethylene glycol were used to stain cellular particles and cutin. RESULTS External morphology of stigma, style, and pollen. The stigma consists of two adjacent spherical bodies, each about i mm in diameter, mounted on a slender tapering style, mm in diameter and ^'^^ long (^^^^^ ^' ^ - ^)- ^^^^ spherical lobe actually consists of fifty to seventy-five radiating branches from a central core. The branches are covered with papillate cells which exude a sticky substance. In our trials, pollen grains adhered to the surface of the stigma by mechanical entrapment among the branches of the stigma and by surface tension of the viscous exudate. In addition, the exine of the large (176 /i) pollen grain is itself minutely echinate (Plate 8, No. 2). Stigma and anthers are located in the centre of the flower where pollen is easily deposited on the backs of visiting insects. Internal stritcture of stigma. The main body of the stigma is composed of large (20-50 /( in diameter) spherical or ovoid cells, arranged in columns radiating from the stigma-style juncture to the branches. Traces of vessel elements pass from two bundles coming from the style, to the various branches. The stigma branches completely cover the tissue core. Each branch consists of tv.'enty-five to seventy-five files of parenchymatous cells (Plate 8, No. 3). The branches are about 0.35 mm long and o.i mm in diameter, excluding the papillae. The files of cells making up the branches appear to be continuous with the files in the body of the stigma and these, in turn, are continuous with the files of cells in the central core of the style. In favourable oblique sections (Plate 8, No. 4) files of cells may be traced from stigma branches, through the stigma body, to the central core of the style. However, the cells of the style core are much smaller in diameter (4-12 /O than the cells of the stigma. Thus the stigma-style junction is characterized by a series of intergrading cell sizes where

3 Stylar anatomy in Ipomoea 111 the files of cells from the branches pass to the style. Rough counts of cell numbers in stigma versus style suggest that in addition to increased cell size, more files of cells oecur in the stigma than in the style. The boundary between the parenchymous cells of the stigma, and the papillae of the epidermis is strongly defined by heavier cell walls (Plate 8, No. 3). Characteristics of the epidermis. The epidermis of the stigma is composed entirely of dumb-bell-shaped papillate cells (Plate 8, No. 5). These cells are large ( /< in length), and typically consist of a definite base, a constricted region, a broadened region, and a narrow nipple (Plate 8, No. 6). The nucleus is usually in the broadened portion of the papilla. A distinctive feature of these cells is the presenee of numerous spherical particles. Larger particles (4-6 /< in diameter) are often grouped around the nucleus (Plate 9, No. 7), whereas smaller particles (i /<) are dispersed through the cytoplasm. The two particle types do not constitute an intergrading series. The refractive inde.x of both types of particles is high, and they may be detected in unstained preparations. Sueh particles appear yellowish or greenish. Haematoxylin staining suggests that larger particles are plastids, perhaps chloroplasts, whereas the smaller particles may be mitochondria. Larger particles may contain starch, as they stain a faint blue with iodine. In contrast, particles of the parenchyma cells do not stain well with haematoxylin, but stain dark blue with iodine. The outermost walls of the papillae did not take up Sudan IV stain from alcohol or ethylene glycol, and thus do not appear to be cutinized. Internal structure of the style. The central part of the style consists of a pollen tube conducting tissue of long, narrow cells (4-6 /< in diameter; /(long). This column of cells is of uniform size ( /<) throughout the length of the style. The surface of the style is covered with a uniform epidermis. Between the epidermis and the conducting tissue is a layer of collenchyma which tapers in thickness from about /< at the style-ovary junction to /< at the junction of stigma and style. Two bundles of xylem and phloem are embedded in the collenchyma. The innermost layer of collenchyma is somewhat more uniform and with thicker cell walls than the rest of the tissue (Plate 9, No. 8). At the stigma-style juncture, the collenchyma ends abruptly, whereas the conducting tissue and epidermis intergrade with respective tissues of the stigma. Juncture of style and ovary. The juncture of the style and ovary can only be traced satisfactorily through serial sections. As the style enters the ovary, it becomes continuous with the septum dividing the two loculi of the capsule. The central core of conducting tissue begins to spread laterally at the base of the septum and is widest at the point of juncture with the ovules. Each ovule is attached to the placenta by a short funicular strip, which also delimits the micropyle (Plate 9, No. 9). Part of the tissue of the funicular strip appears similar to, and is continuous with, the pollen tube conducting tissue of the style. This cell type can be traced from the style to funiculus, to the base of the ovule, through the integument of the ovule, upwards and over the upper part of the ovule and down again to the region of the egg. Pollen tube pathway. Pollen grains germinate only in compatible matings. The tubes pass between cells of the epidermis, through the region of thickened cell walls, and between the cells of the parenchyma, crushing cells to the side as they pass through (Plate 9, No. 10). Old pollen tubes in the stigma are completely filled with callose. As the tubes pass into the style they are confined to the central core of conducting tissue. The passage of five to ten tubes through this tissue results in an obliteration of cell outlines, possibly through enzymatic breakdown of cells walls and also by lateral pressure from the tube itself. The amount of callose within the tube varies from little or none, to irregular H NP

4 112 F. W. MARTIN AND SONIA ORTIZ masses or complete plugs. Pollen tubes are not necessarily circular in cross-section. Evidently, tbe first tubes to pass through tbe style are crushed or distorted by the passage of later tubes (Plate 9, No. 11). Within the ovary, tbe tubes follow tbe circuitous path of the conducting tissue, as previously described. We have seen very few tubes in this region and have not been able to trace individual tubes from the style to the egg. But we have found tubes only in the conducting tissue and not in tbe lumen of the micropyle itself. DISCUSSION The stigma and style of the sweet potato probably differ in no fundamental way from those of related fertile species of Ipomoea. Yet within these organs, pollen tube growth restriction occurs between stigma and style, and between style and ovary (Martin and Cabanillas, 1966). These restrictions are probably not related to the self-incompatibility system in the species, for the latter is known to be controlled by a system of pollengermination inhibition (Togari and Kawahara, 1942; Martin and Ortiz, 1966). In addition, although not documented here, these restrictions occur in almost if not all fertile sweet potato crosses. What is the cause and physiological control of these barriers? The answer to this question probably lies in the nature of the sweet potato itself. It is a hexaploid with ninety chromosomes. Although chromosomes pair normally, considerable secondary association occurs, indicating that partial homology exists among the genomes constituting the sweet potato (Jones, 1965). Thus gametes may not always carry a well-balanced set of chromosomal material. Poor germination of seeds (Martin and Cabanillas, 1966), and irregular growth of seedlings (Thompson, 1925) have been attributed to such imbalances. Although much more difficult to document, it is highly probable that a large portion of gametes produced by any sweet potato variety are weak or imbalanced. Such weakness could be expressed durijig critical growth phases of the pollen tube. In the path of the pollen tube are three critical areas. The first is the surface of the stigma and many pollen grains fail to pass this obstacle even in fertile matings (Martin and Cabanillas, 1966). The second obstacle is the stigma-style juncture where the pollen tube pathway is suddenly and drastically restricted. A change in pollen-tube physiology probably occurs at this point (Martin and Ortiz, 1966). The third obstacle is the styleovary juncture where the pollen tube pathway becomes irregular and less well-defined. We venture the hypothesis that the sterility barriers of the sweet potato are no more than the sites or processes where weak or inadequate gametes are eliminated. Elimination may occur for mechanical or physiological reasons, either before or after pollination. The specialized structure of the papillate cells of the stigma is indicative of the important role these cells play in the incompatibility reaction. High concentrations of mitochondria- and plastid-like particles suggest that such cells could be rich in enzymes and inhibitors effecting germination. Exudates on the stigma surface come from these cells and could affect pollen before germination begins. The nature of the particles requires further study by electron microscopy, whereas the exudates could be studied with histochemical techniques. At present there is no evidence of a cuticular barrier to pollen tube penetration. The causes and control of the sterility is hypothesized above but the physiological mechanism of the self-incompatibility needs much further study.

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7 Stylar anatomy in Ipomoea 113 REFERENCES CHRIST, B. (1959). Entwicklungs geschichtfiche und physiologische Untcrsucluingcn uhcr die Sebsterilitiit von Cardamine pratensis L. Z. Bot., 47, 88. COPE, F. W. (1962). The mechanism of pollen-incompatibility in Theobioiiia cacan L. Hcieditv, I^ond., 17, 157- CROWE, L. K. (1964). The evolution of outhreeding in plants. I. The angiosperms. Heredilv, Loud., 19, 435- FujiSE, K. (1964). Studies on flowering fruit setting, and self- and cross-incompatibility in sweet potato varieties. Bull. Kyushu agric. Exp. Stn, g, 123. JONES, A. (1965). Cytological observations and fertility measurements of sweet potato (Ipomoea hatiitas (L.) Lam.). Proc. Am. Soc. hort. Set., 86, 527. LiNSKENS, H. F. (1964). Pollen physiology. A.Rei. PL Physiol., 15, 255. MARTIN, F. W. & CABAXILLAS, E. (1966). Post-pollen germination barriers to seed set in sweet potato. Eiiphytica (In press). MARTIN, F. W. & ORTIZ, S. (1966). Germination of sweet potato pollen in relation to incompatibility and sterility. Proc. Am. Soc. hort. Sci., 88, 491. STEBBINS, G. L. (1958). The inviability, weakness, and sterility of interspecific hybrids..4dv. Genet., 9, 147. THOMPSON, T. B. (1925). Production of sweet potato seedlings at the \'irgin Islands Experiment Station. Bidl. Virgin hi. agric. Exp. Stn, 5. ToGARl, Y. & K.^WAHAR.^, V. (1942). Studies on the different grades of self- and cross-incompatibilities in sweet potato. IL Pollen behavior in the incompatible combinations. Bidl. imp. agric. Exp..Stn, Japan, 52, 21. WHITEHOUSE, H. L. K. (1950). Multiple allelomorph incompatibility of pollen and styles in the evolution of the angiosperms. Ann. Bot., x.s., 14, 199. EXPLANATION OF PL.4TES PLATE 8 No. I. The stigma of sweet potato, x 35. No. 2. Pollen grains of sweet potato. Phase contrast, x 180. No. 3. Cross-section of stigmatic branch showing barrier wall and papillae, x 100. No. 4. Oblique section of stigma showing the transition between small cells of pollen tube conducting core of the style, and the large cells of the stigma, x 100. No. 5..^ longitudinal branch of the stigma, showing the two types of cells, x 100. No. 6. A single papilla of the stigma, x 470. PLATE 9 No. 7. Grouping of particles, probably plastids, around the nucleus of a papillate cell, x No. 8. Cross-section of style showing central core of pollen tube-conducting tissue, x 200. No. 9. Cross-section of o\"ary showing pollen-tube conducting tissue continuous with tissue of the style, x 100. No. 10. Callose-fiUed pollen tube in the stigma, x No. II. Pollen tubes in the style, x \mount of callose in tube \aries. Some tubes are partially crushed.

Plant Science 1203L Laboratory 5 - Sexual Reproduction (Pollination and Double Fertilization)

Plant Science 1203L Laboratory 5 - Sexual Reproduction (Pollination and Double Fertilization) Today s lab is about sexual reproduction in plants. As with stem or root structure there are numerous definitions