1962 151 The Abnormal Stomata on the Diploid and the Tetraploid Bracken Ferns Chihiro Takahashi Biological Laboratory, Department of General Education, Nagoya University, Mizuho-ku, Nagoya, Japan Received September 25, 1961 There were many deviations from the normal morphology and physiology on the tetraploid sporophyte of the bracken fern, Pteridium aquilinum var. latiusculum (Takahashi 1962). The stoma formation was no exception to this. Reports of the various kinds of the abnormal stomata occurring naturally or pathologically, or induced experimentally, have occasionally ap peared in the literature. The present writer intends to add some new know ledge to this field. Material and method While the writer has been engaged in the study on induced apospory of ferns these years, he obtained in large numbers both the diploid and the tetraploid sporophytes of Pteridium aquilinum (L.) Kuhn var. latiusculum (Desv.) Und. in the laboratory (Takahashi 1961). The diploid sporophyte and the tetraploid sporophyte were sexually produced respectively on the haploid prothallium developed from a spore and on the diploid prothallium developed aposporously from the sporophytic tissue. The stoma observation was made under the microscope without any treatment of fixing or staining agent with the exception of special mention. The term stoma is defined here as a structure which is normally con structed out of paired guard cells and a pore between them, or as a structure which is abnormally constructed but corresponds ontogenetically to the normal stoma. Observation The normal course of the stoma formation on the leaf of the present material is as follows (also see Fig. 3): a stomatal initial appears differentiating in the dermatogen of the young leaf; a stomatal initial divides once or twice, giving rise to a stomatal mother cell which is somewhat circular or elliptical; then a cell division occurs once again in a stomatal mother cell, giving rise to paired semicircular cells; they become differentially-thick-walled guard cells and form a pore between them, accomplishing the stoma formation. The sequence is identical with the description by Kondo and Toda (1956).
152 C. Takahashi Cytologia 27 The stoma is raised above the level of the epidermis. The epidermis cells have the wave-like shape entering into each other and many large chloroplasts. Fig. 1. Photographs of two-year-old sporophytes. Both two pots have the same size of six centimeters in diameter. Left, the normal diploid plant. Right, the abnormal tetraploid plant with the poreless multicellular stoma. It was found that there were three types of the anomalous stomata on the leaves. The first type is designated here as a persistent stomatal mother Fig. 2. The leaf epidermis of the tetraploid sporophytes. ~ 100. Left, the normally constructed stomata. Right, the poreless multicellular stomata and the epidermized stoma (the guard cell is marked by ~). cell. It remains undi vided into a pair of guard cells on the fully matured leaf. It has somewhat circular or elliptical cell shape, chloroplasts and not differentially thick wall. The persistent stomatal mother cell may be very rarely observable on all plants, but more on the tetraploid plants, especially on three of them mentioned later in this paper, than on the diploid plants. The stoma of the tetraploid plant is larger than that of the diploid plant, but it is normally constructed (Takahashi 1962). In addition to such
1962 The Abnormal Stomata on the Diploid and the Tetraploid Bracken Fer ns 153 large but normally constructed stoma and the persistent stomatal mother cell, there are other types of the anomalous stomata on the tetraploid plants. The second type is designated here as an epidermized stoma. Although the stomatal mother cell divides into two cells in this type, they do not develop normally into the guard cells but do erratically into the epidermis -like cells. They have the wave-like shape, chloroplasts, no thick wall and no pore between them. They are not raised above the level of the epidermis. They are hardly distinguished from the usual epidermis cells apart from paired cells and the pres ence of the subsidiary cell. The epidermized stoma is observable rarely but more on the later mentioned three tetraploid plants than on the other tetraploid plants. The third type is des ignated here as a poreless multicellular stoma. It has unique features as in the following: it is the stoma which is constructed out of many guard cells (three to more than ten) caused by the further divisions of two cells originating from a stomatal mother cell; it is the stoma whose guard cells are not differentially thick-walled; it is the stoma with no pore for gas exchange and evaporation even after the full growth Fig. 3. The successive stages of the stoma formation. ~ 250. Left, the normal diploid stomata. Right, the poreless multicellular stomata. Lowest, the cross section of the mature stomata of the leaf. The guard cells of this stoma are somewhat spindle-like shaped. Most of them stand side by side with each other. There are also some cells which divide perpendicularly to the existing cells. Sometimes the outer most guard cell assumes somewhat epidermized shape. There can be seen such a stoma as is constructed out of two guard cells which remain undivided
154 C. Takahashi Cytologia 27 further, but has not a pore between them. In the guard cells of the poreless multicellular stoma the chloroplasts are present as in those of the normal stoma. But the chloroplasts are very variable in number and sometimes very few. With Lugol solution the normal guard cells can be made to stand out conspicuously from the epidermis cells, probably because the starch grains formed in the chloroplasts of the former are larger than those of the latter and more numerous per volume, while the guard cells of this anomalous stoma do not stand out so much, probably because of the smaller starch grains and the smaller number per volume in comparison with those of the normal guard cells. Although there is no passage for gas exchange and evaporation in three plants, probably the thinner outer cell wall of the anomalous stoma than that of the epidermis cells is of use. Under each dome of the anomalous stoma as under the normal stoma there is a respiratory cavity which leads to the intercellular space in the mesophyll. This interest ing stoma was found only on three of the tetraploid plants. Not a stoma like this was observed on the diploid plants or on the tetraploid plants except three so far. Most surprisingly the normal stoma in structure and function was never found on these three tetraploid plants. All the stomata except those of above mentioned two types which are rarely observable, are of the poreless multicellular type. Namely these three tetraploid plants are com pactly covered with the poreless epidermis. It appears that in spite of the unfavourable structure two of those plants are growing fairly well, not so inferior to the plants having the normal stomata. They exhibit the irregularly pinnated leaf, the thick leaf, the uneven leaf surface, the dark green and the hairiness. The other plant is a feeble-looking dwarf and bears small leaves with the poor laminae. Such characters are, however, not specific to three plants, but appear in various combinations on the other tetraploid plants. Discussion The stoma is known as one of the most important and stable characters specific to the sporophyte of the vascular plant. The present writer found three types of the anomalous stomata on the tetraploid leaf of Pteridium aquilinum var. latiusculum. The persistent stomatal mother cell is caused by the failure of further development beyond the formation of the stomatal mother cell. The abnor mality of this type is of a low degree. It seems that the occurrence of such stomata is not related with any environmental conditions. In fact the per sistent stomatal mother cell is observable even on the normal sporophyte thriving in the habitat. Many investigators also reported such a structure on various angiosperms. For example, formerly Gertz (1919) and recently Dehnel (1961) reported its occurrence on Paeonia gall of fungal and insect etiology, and on the normal leaf of Begonia, respectively. This type was
1962 The Abnormal Stomata on the Diploid and the Tetraploid Bracken Ferns 155 also observed on colchicine treated leaf (Weber 1943). In the normal course of the stoma formation a pair of cells into which a stomatal mother cell divided, develop into a pair of guard cells. On the contrary it happens that a pair of cells do not develop into the normal guard cells but into the epidermis-like cells by mistake. This is the case of the epidermized stoma on the tetraploid plants. Cholodny (1924) also reported the abnormal stomata on Lysimachia leaf caused by the submergence, which had a similar nature to the present writer's case. In the present study it was observed not rarely that the leaf, especially the first leaf, grew diving into the culture solution or the agar, or contacting with the agar surface. But such culture conditions did not result in the occurrence of any anomalous stomata. From the consideration of these facts it seems that a pair of just divided cells are not finally destined to be the specially differentiated guard cells but they are able to become the usual epidermis cells under certain physiological aberrations caused by the doubling of the chromosome number or by the abnormal environment. The occurrence of the persistent stomatal mother cell and the epidermized stoma is the exceptional and partial phenomenon on a given leaf. On the contrary the occurrence of the poreless multicellular stoma is the general phenomenon on the leaf of three tetraploid plants. These plants never form a stoma normal in structure and function. Therefore, they are entirely covered with the poreless epidermis. The interesting questions to be solved in future arise. How will they become? To what extent will they grow? Will they produce spores? If they do, will spores be viable? Into what prothallia will spores develop? What sporophytes will such prothallia pro duce? Furthermore, there is another important problem. What factor is responsible for the stomatal ontogenetic aberration exhibited by three plants? From the fact that all the stomata are of the poreless multicellular type on the exceptional tetraploid plants, it is likely that the nuclear aberration with relation to the tetraploidy is causally involved in the formation of such anomalous stomata. There have been reports of the multicellular stomata, such as twin, triplet or quadruplet stomata, or a stoma with three or four guard cells, which derived from a stomatal mother cell as result of its excessive division. They occurred naturally (Haberlandt 1886, Reuter 1955), pathologically by the fungal infection (Kuster 1925) or by the virus infection (Weber and Kenda 1953), or on the hybrid plant (Fukasawa 1958). Never theless there has been no report on such multicellular stomata as were observed by the present writer, which were constructed out of so many guard cells and had no pore; there has been no report on such plants as had none of the normal stoma and, what is more, none of the pore for gas exchange and evaporation; there has been no report on such multicellular stomata as were of the polyploid origin.
156 C. Takahashi Cytologia 27 Summary The normal and the abnormal stomata on the leaf of Pteridium aquilinum var. latiusculum were studied. The normal stoma on the diploid or the tetraploid plant is formed as follows: a stomatal initial appears in the dermatogen; dividing once or twice it gives rise to a stomatal mother cell; a stomatal mother cell divides into a pair of cells; they develop into a pair of guard cells differentiated typically. The persistent stomatal mother cell is rarely observable on all diploid and tetraploid plants. It remains undivided, circularly or elliptically shaped, and not thick-walled. The epidermized stoma is rarely observable on the tetraploid plants. In this type a stomatal mother cell divides into a pair of cells but they develop erratically into the epidermis-like cells. They are not raised above the level of the epidermis. Each cell of the epidermized stoma has the wave like shape, no thick wall and no pore between two cells. The existence of the poreless multicellular stoma is restricted to three of the tetraploid plants. This interesting stoma is characterized by the follow ing features: it is constructed out of many cells (three to more than ten) caused by the further divisions of two cells originating from a stomatal mother cell; guard cells are not differentially thick-walled; no pore is formed; guard cells are stained not so well with Lugol solution; the chloroplast number is variable and sometimes very few; not a normal stoma in structure and function is formed on these three plants. The tetraploid plants bearing this stoma exhibit many other deviations from the normal morphology and physiology. However, such deviations are not specific to three plants but appear on the other tetraploid plants. Also the causes of the occurrence of these anomalous stomata were discussed. The writer wishes to express his cordial thanks to Profs. Drs. M. Kumazawa and I. Harada of Nagoya University for their guidance and encouragement. References Cholodny, N. 1924. Zur Frage nach der Wirkung des Wassers auf den anatomischen Bau der Landpflanzen. Biol. Zentralb. 44: 138-144. Dehnel, G. S. 1961. Abnormal stomatal development in foliage leaves of Begonia aridicaulis. Amer. J. Bot. 48: 129-133. Fukasawa, H. 1958. Studies on restoration and substitution of nucleus (genome) in Aegilotricum, VII. Curious abnormalities of stomata appearing in the leaves of male-sterile durum plants. Cytologia 23: 128-142. Gertz, O. 1919. Ueber einen neuen Typus stomatarer Thyllenbildung nebst anderen Beo bachtungen zur pathologischen Anatomie des Spaltoffnungsapparates bei Paeonia
1962 The Abnormal Stomata on the Diploid and the Tetraploid Bracken Ferns 157 paradoxa. Ber. Deut. Bot. Ges. 37: 237-244. Haberlandt, G. 1886. Beitrage zur Anatomie und Physiologie der Laubmoose. Jahrb. Wiss. Bot. 19: 359-498. Kondo, T. and Toda, H. 1956. A contribution to the study of fern stomata (I), with special references to their development and structure. (In Japanese). Res. Bull., Hamama tsu Branch, Fac. Educ., Shizuoka Univ. 5: 60-80. Mister, E. 1925. Pathologische Pflanzenanatomie. 3 Aufl. Jena. Takahashi, C. 1961. Chromosome study on induced apospory in the bracken fern. Kromo somo (Tokyo) 48: 1602-1605.- 1962. Cytological study on induced apospory in ferns. Cytologia 27: 79-96. Weber, F. 1943. Spaltoffnungsapparat-Anomalien cholchicinierter Tradescantia-Blatter. Pro toplasma 37: 556-565.- and Kenda, G. 1953. Stomata-anomalie von Opuntia-Virustragern. Oest. Bot. Zeit. 100: 153-159.