A Study on Development of the ]Embryo Sac in Impatiens balsamina. by Shizuyo TAKAO*

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1 Bot. Mag. Tokyo 79: (September 25, 1966) Received July 30, 1966 A Study on Development of the ]Embryo Sac in Impatiens balsamina by Shizuyo TAKAO* On the embryological field in the family Balsaminaceae, there have already been published several investigations. Among them, a few studied, 2, 3) have dealt with such problems relating to development of the female gametophyte, as the cell formation in the coenocytic embryo sac, fusion of the polar nuclei, structure of the egg apparatus, stored starch grains in the embryo sac and others. But, as to Impatiens balsamina there have still been few earlier works4 5'. Thus, the present study deals with development of the embryo sac in I. balsamina in relation to the problems mentioned above. Material and Methods The material is a garden balsam, Impatiens balsamina. The buds of various size were collected and fixed with sublimate alcohol (8 g of mercuric chloride and 5 ml of glacial acetic acid in 100 ml of 50 per cent ethanol) and Telyesniczky's fluid for 24 hours. The fixed samples were imbedded in paraffin in routine manner and sectioned at 10, i thick. To observe the morphological changes in the course of development the sections were stained with Heidenhain's iron-hematoxylin. Lillie's method for polysaccharides and iodine reaction for starch were applied to the grains appearing in the embryo sac. Observations Ovule and megasporogenesis: The ovules emerge as protuberances of the placenta, when the pollen mother cells are formed through the last mitotic division in the anthers. The ovary is five carpellary with an axial placentation. Each carpel holds usually three ovules but occasionally two or four. The ovule is of so-called anatropous type, i.e., it is gradually bent downwards with growth. One of the subepidermal cells lying near the apex of this protuberance differentiates into an archesporial cell, which differs from adjacent cells by its larger size, denser cytoplasm and fine chromonematous structure of the nucleus, where as the other cells have chromocenter nuclei (Fig. 1). On the other hand the inner integument grows to cover the nucellus and finally forms a micropyle. By the time when the micropyle is completed, orientation of the ovule also becomes entirely anatropous. The outer integument differentiates from the inner one. The most part of the former is in a fused state with the latter (Fig. 2). At this stage, a yellow-green pigment appears in the subepidermal cells at the base of the funicle. The region containing the pigment then spreads towards the ovule, and then into the epidermal cells of the ovule from its base to apex. The pigment seems to be chlorophyll judging from the results of paper-chromatography. Without cutting off a parietal cell, the archesporial cell * Biological Laboratory, Aichi Prefectural University, Nagoya, Japan.

2 438 Bot. Mag. Tokyo Vol. 79 functions directly as a megaspore mother cell, which is elongated later (Fig. 3). At the same time the nucellar cells sustaining the megaspore mother cell also elongate along the ovular axis. Then, the megaspore mother cell undergoes the first meiotic division as usual to form two dyad cells (Fig. 4), and by the second division a linear tetrad is produced (Fig. 5). The megaspores are different in size from each other, and the chalazal one which develops a functioning megaspore is the largest. Megagametogenesis: The development of the embryo sac follows the Polygonumtype. The functioning megaspore enlarges, while the upper three cells degenerate (Fig. 6). The latters are shoved off by elongating megaspore, so that they are crushed under the nucellar epidermis and disappear by the time when the female gametophyte attains to the four-nucleate stage. These degenerating cells show characteristically densely granulated cytoplasm and small and deeply stainable nuclei. The nucleus of functioning megaspore divides thrice to give rise to eight nuclei, i.e., one pair of quartets, the micropylar quartet and the chalazal one (Figs. 7, 8, 9). Subsequently, cell formation takes place on the coenocytic embryo sac, and an eightuncleate seven-celled embryo sac is produced (Fig. 10). The antipodes are ephemeral so they do not exist in the mature embryo sac. Cell formation : In the course of a series of divisions by which the eight-nucleate embryo sac has been formed, only the nuclear divisions progress and the cell plate may not be formed successively. This coenocytic embryo sac, however, is gradually bordered by the septa. The cell formation proceeds in a short time successively. First, the initial antipodal cell with three nuclei, which originate in chalazal quartet, is cut off (Fig. 11-a). A cell wall is laid down between this initial antipodal cell and the cell which comes to the central cell later. The wall takes a rose colour with Lillie's staining for polysaccharides. Thereafter, the cell formation of the antipodes takes place on this trinucleate cell; one antipode, whose nucleus is a sister of chalazal polar one, is cut off and then the remaining binucleate cell is cut in two. Thus, the three antipodes are completed (Figs. 11-b, c). The antipodes are observed usually in cellular form. But occasionally they are composed of one massive cytoplasm with three nuclei only, or of two cells, the chalazal one of which is binucleate. At micropylar part the cell formation for the egg apparatus starts soon after beginning of the antipodal cell formation. At first, the binucleate initial synergid cell, whose nuclei are the sisters, is cut off (Fig. 11-a). Then, the egg cell separates from the remaining binucleate cell, and finally the initial synergid cell is cut in two synergids. Thus, the binucleate central cell is situated in the center of the embryo sac (Fig. 10). In this manner, the eight-nucleate seven-celled embryo sac is formed. At this stage each nucleus takes its origin as follows; the synergid nuclei which are the sister ones are derived from the uppermost or micropylar terminal nucleus at four-nucleate stage, the egg nucleus and the upper polar nucleus from the second nucleus, the lower polar nucleus and one antipodal nucleus from the third, and the other two antipodal ones from the fourth or the most chalazal nucleus, respectively. Egg apparatus : After the cell formation, the upper three cells constitute the egg apparatus. In the beginning these cells have little differences from each other in their nuclear structure and cytoplasmic conditions. These cells elongate to organize the egg apparatus. a) Synergid The upper ends of the initial synergids elongate towards the micropylar portion (Fig. 12); at an early stage of elongation the cells vacuolate extremely in the tip part, thereafter the cells begin to elongate (Fig. 13-a). Their

3 September, 1966 TAKAO, S. 439 Figs Developing ovule with differentiated archesporial cell. x Ovule with two integuments, megaspore mother cell completed (p, layer containing yellow-green pigment). x Megaspore mother cell. x Megaspore dyad. x Megaspore tetrad. x Functioning megaspore and three degenerating ones. x Two-nucleate stage of the embryo sac. x Four-nucleate stage. x 650.

4 440 Bot. Mag. Tokyo Vol. 79 Figs Eight-nucleate stage. 10. Early eight-nucleate seven-celled stage, cell formation finished. 11. Process of cell formation, a, Beginning of cell formation. Formation of antipodes precedes to that of egg apparatus. b and c, Cell formation of antipodes. 12. Later eightnucleate seven-celled stage. Early stage of egg apparatus formation, antipodes degenerating and polar nuclei fusing. 13. Elongating embryo sac. a, Egg apparatus formation and appearance of starch grains in each cell of the embryo sac. Antipodes degenerated. b, More advanced stage. Figs a. x700, Fig. 13-b. x590.

5 September, 1966 TAKAO, S. 441 nuclei are either near the rounded bottom or in the middle of the cells and a vacuole usually appears at the bottom of each cell. Small grains increase numerously in the elongating synergids; though a few grains have been contained even in the initial synergid cell, they increase in number also somewhat in size to reach the maximum at the elongating stage. These grains are considered to be starch grains, because they are stained in a blue-purple colour after iodine reaction and in a rose-red colour with Lillie's method for polysaccharides. With growth of the ovule, the synergids elongate filiform (Fig. 13-b), attaining to 80 to 105p in length, and 135p in an extreme case. Their heads, the synergid caps, are a nib-like or more swollen in shape. The synergid cap is so vacuolated that its fine structure can hardly be observed with hematoxylin staining. With Lillie's method, however, the cap is stained entirely in a bright rose colour and shows to involve a netlike structure stained more deeply than the cytoplasm (Fig. 15). At the final stage of elongation, the synergids are situated in the long narrow canal of the micropyle. The nuclei are oviform with one nucleolus. Starch grains are also observed in the mature synergids but decreased in number. b) Egg cell The third cell next to the synergids elongates upwards in parallel with the tatters and then is completed as the egg cell, showing a filiform shape similar to the synergids (usually 90 to 115~c in length, 145~c in an extreme case) (Figs. 12, 13). In an early stage of elongation the upper part of the cell vacuolates, so that the cytoplasm locates only in the bottom of the cell. Small starch grains appear and increase in number in the egg cell, as in the synergids, at an early stage of the development, then the cytoplasm is filled up with numerous starch grains (Fig. 13-a). Afterwards most of them, however, begin to disappear, and only a few grains remain in the egg cell matured. Polar nuclei: In the beginning of the egg apparatus formation, the nuclei of the central binucleate cell, the polar nuclei, which have been located separately at each side of the cell, move towards the center and come to contact with their nuclear membranes (Figs. 10, 12). During this process the nuclei increase in size, becoming larger than the other nuclei in the embryo sac (Figs. 14-a, b). By the time when the elongation of the egg apparatus comes almost to its maximum and the starch grains in the embryo sac cavity begin to dissolve, the polar nuclei finish their fusion; the contacting membranes disappear, then the large nucleoli approach to each other until they contact closely to form one large nucleolus (Figs. 14-c f). The fused polar nucleus, the secondary nucleus, has one large nucleolus containing frequently a large vacuole. In the mature embryo sac the secondary nucleus lies next to the egg cell, in many cases contacting with each other. Antipodes : Soon after the cell formation is completed, the antipodes begin to degenerate in the embryo sac (Fig. 11). At this stage their nuclei become smaller and the cytoplasm are deeply stained by hematoxylin. Finally they are shoved down into the nucellar tissue. Thus, the antipodes cannot be observed in the embryo sac matured. Pear-shaped mature embryo sac: After the cell formation, the embryo sac elongates towards both the micropylar and the chalazal parts, especially towards the latter. To start with, many small starch grains are accumulated in the dense cytopltasm of the central cell, though some of them have been stored since the megaspore mother cell stage. Then, they increase rapidly in number and in size to fill up the elongating embryo sac. The chalazal end of the embryo sac penetrates

6 442 Bot. M ag. Tokyo Vol. 79 Figs. e, Vanishing Process of fusion of polar nuclei, a of contacting membranes and fusion and b, Contacting polar nuclei. c, of nucleoli. f, Fused polar nucleus. d and x650.

7 September, 1966 TAKAO, S. 443 into the nucellus, and elongates more and more so as to reach the chalaza (Fig. 13). After that, dissolution of the starch grains takes place in the chalazal portion of the embryo sac to come up to upper portions in a short time. Subsequently, the embryo sac vacuolates and its chalazal half expands at the portions where the integumentary tapetum and the adjacent tissue have degenerated, so that the embryo sac develops into a " pear shape " (Fig. 17). But, a small number of starch grains still remain scattered in the embryo sac mainly around the fused polar nucleus. Stored starch grains: As mentioned above, the stored starch grains are observed in the course of embrys sac development. They first appear in the megaspore mother cell and then in each cell of megaspore tetrad, also in each cell at eightnucleate seven-celled stage as well. They increase rapidly in the elongating embryo sac. Though some starch grains are also contained in degenerating cells, the abortive megaspores and the antipodes, their further increase is not observed. Nuclear features in the embryo sac : In the mature stage the generative nuclei, the nuclei in the egg apparatus and the fused polar nucleus, exhibit a fine chromonemat ous structure whereas the nuclei of the somatic tissues surrounding the embryo sac have the chromocenters in them. The generative nuclei are somewhat different in size from each other. Compared each nucleus with its original one in volume, it develops as follows; the egg nucleus is large as 10 to 20 times as the latter, the synergid nuclei 2 to 5 times and the fused polar nucleus 15 to 24 times, respectively. The fused polar nucleus is the largest, and its original nuclei are a little larger than the others, among the quartets at eight-nucleate stage. Integumentary tapetum : The innermost layer of the inner integument forms an integumentary tapetum. Though being not well developed and not regularly arranged, cells of the tapetum differ from the others of integumentary tissues. In the cross sections of mature embryo sac at the levels of the egg nucleus and the fused polar nucleus, these cells are arranged radially (Figs. 16-b, c). With growth of the ovule, the collenchymatous change of cell walls progress in the cells surrounding the micropyle as well as in the adjacent part of the integumentary tapetum. On the other hand, in the chalazal half of the tapetum the cell walls suffer no change as mentioned above; the cells of these portions and adjacent tissues break down themselves, when the embryo sac develops into the "pear shape" (Fig. 16-d). The wall of the embryo sac : In the present species, the embryo sac is entirely enveloped with a thin wall of polysaccharides stainable with Lillie's method (Figs. 16, 17). Moreover, on the inner surface of this wall there are numerous minute granules stainable more deeply than the wall. In the portions which envelope the micropylar half of the embryo sac, they are observed abundantly. Discussion The megasporangium of Impatiens balsamina consists of a nucellus and two integuments. The mature ovule is anatropous with a dorsal raphe, and the nucellus which elongates temporarily at first is absorbed up by elongating embryo sac. The 15. An oblique section containing synergid caps and collenchymatous cells around the micropyle. x Cross sections of mature embryo sac. a, Section at the egg apparatus level. b. Section at the egg nucleus level. c, Section at the fused polar nucleus level. d. Section at the vacuolated embryo sac cavity. Integumentary tapetum degenerated. x Pearshaped mature embryo sac (w, wall of the embryo sac). x280.

8 444 Bot. Mag. Tokyo Vol. 79 hypodermal archesporial cell comes to function directly as a megaspore mother cell and cuts off no parietal cell. These features are similar to those in the other species of Impatiens investigated (Ottleyl', Dahlgren2', Steffen3, Venkateswarlu and Lakshminarayana6', Narayana and Sayeeduddin7', Narayana8 9)). As to the integuments. there have been reported two types in Balsaminaceae, i.e., the free and fused types. I. balsamina belongs to the latter; the two integuments are fused in one in the most parts. In Balsaminaceae there have been reported two types of the embryo sac development, i.e., the Polygonum- and Allium-types. In the present species it conforms to the former as in I. glanduligera (Steffen3j), I. levengii, I. tenella, I. radiata, I. tripetala, I. incospicus (Narayana8'), I. maculata and I. parviflora (Narayana9'). There have been reported some different types in the maturing pattern of embryo sac in the genus Impatiens. The filiform egg apparatus in I. balsamina seems to be similar to those in the other species. But there are some differences when observed in detail; above all in the present species it elongates far longer than in the others. On the netlike structure in the cytoplasm of the synergid cap, we observe it to be a similar structure to what Steffen3' has observed in I. glanduligera. In the present species, the fusion of the polar nuclei has finished by mature stage of the embryo sac. Sometimes, the large nucleolus in the fused nucleus is vacuolated as in I. sultanii (Ottleyl') and I. glanduligera (Steffen3'). The development from coenocytic into cellular embryo sac takes place among the antipodes at first, and a little later in the egg apparatus though overlapping with the former. Also in I. glanduligera Steffen3' has observed a successive change similarly in the present species. There is, however, a difference between them. In I. balsamina the cell formation in the coenocytic embryo sac starts in the pattern of multinucleate initial cells, the trinucleate initial antipodal cell and the binucleate initial synergid cell, which are later cut into cellular form. The cell formation to the synergids and the antipodes takes place successively on each initial cell. On the other hand, in I. glanduligera the cell formation seems to take place in cellular pattern, i.e., the synergids and two antipodes are cut off from both terminals of the coenocytic embryo sac, consequently, there remains four-nucleate cell which can not be observed in I. balsamina. Then, the last antipode and the egg cell are cut off from the four-nucleate cell. In I. balsamina growth of the embryo sac is composed of two steps, i.e., the first is the step in which the embryo sac filled up with numerous starch grains elongates into the nucellus and the second is the step in which the embryo sac develops into its mature state. The enlarged mature embryo sac attains to a pear shape. This shape is likely brought about by the increase in osmotic pressure in the embryo sac; the starch grains are dissolved into soluble sugar so as to result in vacuolation and anatonosis upon the embryo sac. The embryo sac may receive water supply from adjacent cells, integumentary tapetum and integumentary tissue, thus the latters are dehydrated, and finally they are shoved and collapsed by the turgescent embryo sac. The vascular bundles disposed in the chalaza may also supply water necessary for the vacuolation of embryo sac. Then, the lateral expansion of embryo sac has taken place; it takes place only in the chalazal half of the embryo sac, where the adjacent cell walls suffer little collenchymatous change. Thus the mature embryo sac attains the pear shape with long narrow neck. Longo5' has denied the existence of the integumentary tapetum in I. balsamina. According to Maheshwari10', there are some characteristics by which the integumentary

9 September, 1966 TAKAO, S. 445 tapetum cells are distinguished from the rest, i.e., the radially elongated shape and their contents. In this aspect, in I. balsamina also exists the integumentary tapetum, though not well developed; the innermost layer of the inner integument is distinguished from the rest by their radial arrangement. Moreover, it has another characteristic, i.e., the collenchymatous change of the cell walls. In the integumentary tissue the cell walls scarecely show such a change. In the mature stage, however, its chalazal half has degenerated as a result of the expansion of embryo sac. Longo's figures5' showing no integumentary tapetum in I. balsamina seem to be founded on the preparations in the embryogenic stages. We also observe a great part of the integumentary tapetum to have degenerated in these stages (Takao, unpublished). Thus, the integumentary tapetum exists in early stages of the embryo sac development in L balsamina and its degeneration starts with entering into mature stage. The stored starch grains in the embryo sac have been observed in many plants by many investigators. They appear in various stages of the embryo sac development. For instance, according to Treub", in Loranthus pentandrus they appear even at the megaspore mother cell stage. In I. balsamina also the starch grains are observed since the megaspore mother cell stage. Furthermore, the fact that they are observed even in such cells as degenerating may be considered as follows: the starch grains contained in the original cells, the megaspore mother cell and the multinucleate cell, are distributed into the daughter cells through the embryo sac formation. The changes in the amount of starch grains may be interpreted as follows: in the increase or storage phase nutrient sugar flows actively into the embryo sac through the vascular supply and is utilized for the development, then the remains turn into starch grains: in the decreasing or dissolving phase the starch grains are dissolved into soluble sugar to prepare the embryogeny. The dissolution seems to take place in a short time and then to propagate from chalazal to micropylar part. This might be caused by the release of dissolving enzymes and related to the beginning of a direct contact of the embryo sac with the vascular bundle. Also in Balsaminaceae on the appearance of starch grains in the egg apparatus there have been a few observations. Steffen3' has reported in I. glanduligera that they appear only in the egg cell and disappear before the embryo sac mature. Ottleyl' has described in I. sultanii the appearance of "many stored food" in the embryo sac cavity, but does not refer to their appearance in the egg apparatus. In the present species, the starch grains appear in both the egg cell and the synergids throughout the course of development. At the mature stage they have almost disappeared in the egg cell, remaining only a few, but still present in the synergids. These differences may be due to the specific character of the species. In I. balsamina, the embryo sac is enveloped entirely with a thin wall. This is confirmed in the preparations stained with Lillie's method for polysaccharides. It may be necessary to make further investigations upon this wall. Summary The development of the embryo sac in Impatiens balsamina, and some relating problems are studied. In the present species the development of the embryo sac conforms to the Polygonum-type. The antipodes are ephemeral. The fusion of the polar nuclei has finished just before maturation of the embryo sac. The nuclei of synergids which are the sister ones are derived from the first nucleus of the four-

10 446 Bot. Mag. Tokyo Vol. 79 nucleate embryo sac, and the egg nucleus from the second nucleus. In the course of embryo sac development, many stored starch grains are observed. They appear first in the cytoplasm of the megaspore mother cell, and then in the egg apparatus, furthermore, in the degenerating cells, i.e., the abortive megaspores and the antipodes. Among others they are stored numerously in the elongating embryo sac cavity and dissolved just before maturation. The mature embryo sac attains to a pear shape. This may be caused by the increase in osmotic pressure of the embryo sac which originates in the dissolution of starch grains. The integumentary tapetum exists in early stages of the embryo sac, but the most parts of it degenerate when the embryo sac changes into the pear-shaped one. The author expresses here her deep gratitude to Prof. T. Shimamura for his kind guidance throughout the course of this work and also sincere thanks to Dr. T. Ota for his helpful suggestions. References 1) Ottley, A. M., Bot. Gaz. 66: 289 (1918). 2) Dahlgren, K. V. O., Sv. Bot. Tidskr. 28: 103 (1934). 3) Steffen, K., Planta 39: 175 (1951). 4) Longo, B., Rend. Acc. Lincei Roma 1.6: 591 (1907). 5), Ann. di Bot. (Roma) 8: 65 (1910). 6) Venkateswarlu, J., and Lakshminarayana, L., Phytomorphology 7: 194 (1957). 7) Narayana, L. L., and Sayeeduddin, M., J. Indian Bot. Soc. 38: 391 (1959). 8), ibid. 42: 102 (1963). 9) -, J. Jap. Bot. 40: 104 (1965). 10) Maheshwari, P., An Introduction to the Embryology of Angiosperms, 63 (McGraw Hill Book Comp. Inc., New York, 1950). 3: 184 (1883). 11) Treub, M., Ann. Jard. Bot. Buitenzorg