A PRELIMINARY NOTE ON THE EMBRYOLOGY OF CASUARINA EQUISETIFOLIA, FORST BY B. G. L. SWAMs (Bangalore) Received June 27, 1944 (Communicated by Prof. L. S. S. Kumar, r.A.SC.) THE remarkable discovery of Chalazogamy in Casuarina by Treub in 1891 evoked very keen interest and initiated further studies of Casuarinaceae and Amentifera~ fi'om both morphological and anatomical points of view. Certain aspects of the megasporogenesis of Casuarhza stricta was subsequently studied by Frye in 1903 and Juel (1903) recorded his observations on the origin and development of the female archesporium in Casuarina quadrivalvis. In spite of these contributions our present knowledge regarding the develop- mental stages in the life-history are far from being satisfactory. An inves- tigation of several species of the genus has been taken up by the author and a few salient features in the life-history of Casuarina equiset~folia Forst have been embodied in this preliminary note. The archesporiurn of the microsporangium is subepidermal in origin and can be differentiated by rich cell contents and conspicuous nuclei. After the formation of the endothecium, wall layers and tapetum, the microspore mother cells undergo the usual stages of the reduction divisions and form quartets of microspores arranged tetrahedrally. The quartets round off and their nuclei undergo division into tube and generative cells The pollen grains at the shedding stage are binucleate. Each ovary contains two erect ovules which arise laterally from a basal placenta (Fig. 1). The ovules are bitegnmentary, the inner integument differentiating slightly earlier than the outer; these grow upwards and organise a micropyle. Simultaneously with the origin of the integuments a group of cells differentiate and become conspicuous in the subepidermal layer (Fig. 2); these divide transversely and the resulting cells towards the micropyle give rise to 5 or 6 parietal layers which make the sporogenous tissue deep seated; hand in hand with this the primary sporogenous layer also divides and increases in number. A conducting strand is organised (Figs. 3 and 4) at the chalaza and this extends upto the sporogenous mass. Some of the megaspore mother cells in the centre of the sporogenous mass also 187 188 B.G.L. Swamy contribute to the formation of the conducting strand instead of developing into embryo-sacs. The megaspore mother cells become elongated and undergo the usual meiotic divisions to give rise to a linear row of four megaspores (Fig. 5). Simultaneous development of more than one megaspore of the tetrad is a very common phenomenon. The stages in development of the embryo-sac are quite normal and typical. Usually at the two-nucleate stage, the em- bryo-sac elongates antipodally, grows into the chalaza in the form of a nar- row long tube and terminally develops a small pouch. The antipodal nuclei become surrounded by individual cytoplasmic sheaths and are very ephemeral; their position within the embryo-sac varies. The egg appara- tus which is always organised at the micropylar end of the embryo-sac exhibits a variety of forms. The polar nuclei remain juxtaposed, occupying a position beneath the egg apparatus or somewhere in the antipodal elongation of the embryo-sac. The pollen grain germinates on the filiform stigma and the pollen tube grows down the style and after travelling through the tissues of the ovary, enters the chalaza and there branches profusely. Ultimately the branch that contains the male nuclei makes its way between the antipodal tubular prolongations of the embryo-sacs on to the top of the particular embryo- sac that is destined to be fertilized (Fig. 7). This course of the pollen tube differs from those reported by the previous workers on Casuarina investigated by them. In those species, the general contention was that the pollen tube enters one of the antipodal swellings and travels within the embryo-sac to the bottom of the egg apparatus of that sac ol" that the pollen tube implants itself on any part of the embryo-sac membrane and discharges its contents into the sac at that point. Such a feature is not noticed by the present author in the species investigated by him. On the other hand, it was seen that the pollen tube follows a definite course in the nueellus and on reaching the micropylar tip of an embryo-sac, pierces the sac between the egg apparatus and the membrane of the sac and discharges its contents. One of the male nuclei fuses with that of the egg and the other one with the two unfused polars. Thus a clear " double fertilization" is accomplished (Fig. 6). In spite of the fact that as many as 20 embryo-sacs develop to maturity within each nucellus, only one of them is fertilized, resulting in the formation of one embryo in each fruit. It was observed that the triple fusion of the polars with the second male nucleus is in advance over that of the zygote. In some instances while the egg nucleus and the associated male nucleus Preliminary Note on Embryology of Casuarina equisetifolla, Forst 189 Figs, 1-6.--Fig. 1. Ovules of an ovary at the time of differentiation of the integuments and archesporium. >', 80. Fig. 2. Archesporium in the nucellus, x 1800. Fig. 3. A stage dur- ing the formation of the parietal and sporogenous tissue; note the conducting strand inside the sporogenous tissue. ~ 800. Fig. 4. Longitudinal section of the ovule depicting the conduct- ing strand. • Fig. 5. Tetrad of megaspores in which three have germinated, two- nucleate and four-nucleate embryo-sacs, x 1800. Fig. 6. Ovule at the time of fertilization, showing the course taken by the pollen tube (pollen tube is dotted), x 240. 190 B.G.L. Swamy Figs. 7-13.--Fig. 7. Double fertilization. • 1800. Fig. 8. A stage in the formation of endosperm with two-celled embryo, x240. Fig. 9. Proembryo. x1800. Fig. 10 and 10a. Stages in the development of the embryo. Fig. 10 x1800: Fig. 10a x1260. Fig. 11. Longi- tudinal section of the mature embryo, x40. Fig. 12. A stage in the formation of nucellar embryos. • 1260. Fig. 13. t~ermination of a polyembryonate seed. x 5. Preliminary Wote oJz L'mbryo&gy ofCasuarina equisetifolia, Forst 191 were still at the stage of fasion, the primary endosperm nucleus had already divided four or five tim es. The wall formation of the endosperm commences from the micropylar end and proceeds centrapetally towards the chalazal end. When the cellular endosperm fills the micropylar half of the embryo-sac, the zygote undergoes its first division. It is not unusual to find cellular endosperm above and free nuclear divisions taking place at the chalazal region of the same sac (Fig. 8). Still later the endosperm tissue increases in bulk by inter- calary divisions especially at the micropylar region around the embryo. The first wall laid down in the zygote is transverse. A proembryo con- sisting of a linear row of three cells is organised (Fig. 9). The basal cell enlarges and its nucleus gets hypertrophied. The middle cell divides to form a suspensor consisting of four to five cells (Fig. 10). The terminal cell under- goes the fisrt division by a vertical wall followed by quadrant and octant stages. The mature embryo has well-differentiated radical, stern-tip and two cotyledons (Fig. l l). Casuarina equisetiJolia often exhibits a range of variations which have not been recorded previously. Two ovules sectioned from a particular tree invariably showed double nucelli with common integuments. From the very begining the development of the female gametophytes in the two nucelli was similar and independent. The two nucelli were in the majority of cases unequal in size. After fertilization of an enbryo-sac in a particular nucellus was over, the second nucellus degenerated. Sometimes two pollen tubes enter the same ovule and double fertiliza- tion is accomplished in two embryo-sacs, resulting in the formation of two embryos in a single ovule. Occurrence of two proembryos side by side was noticed in some cases. Failing fertilization, some of the nucellar cells in certain ovules showed developmental stages in the formation of additional embryos (Fig. 12). Fig. 13 represents a case of the germination of a polyembryonate seed. Further details and their relation to the systematic position of the family will be discussed in the full paper, to be published elsewhere. LITERATURE CITED Frye, T. C. .. "The Embryo-sac of Casuarina stricta," Bot. Gaz., 1903, 36, 101-13. Juel, H. O. .. " Ein Beitrag zur Entwicklungsgeschte der Samananlagen yon Casuarina," Flora, 1903, 92, 284-93 9 Treub, M. 9 " Sur les Casuarinees et leur place dans le Systeme Naturel," Ann..lard. Buitenzorg, 1891, 10, 145-231. B3a .