DEVELOPMENTAL STUDIES ON CABOMBA CAROLINIANA GRAY II. Floral Anatomy and Microsporogenesis BY D. PADMANABHANAND M. V. RAMn (University Botany Laboratory, Chepauk, Madras-5, India) Received March 2, 1966 (Communicated by Dr. K. Subramanyam, ~.A.sc.) ABSTRACT The flower in tTabomba caroliniana is trimerous. There are three free carpels and each encloses three ovules; of these one is attached to the midrib and the other two are laterally disposed. The thalamus exhibits a vascular plexus from which traces to perianth, stamens and carpels are given off. The stamen is traversed by a single median bundle. Each carpel is traversed by a single bundle which trifureates at its base forming one median which extends up to the tip without branching, and two laterals extending to the base of the style where they fuse and supply the median ovule. Among the two lower ovules one receives supply from both the median and one of the lateral bundles of the carpel and the other from only the other lateral. The tapetum is of the secretory type. Cytokinesis in the microsporocytes is of the successive type and the pollen grains are two-celled at the shedding stage. INTRODUCTION RECENTLY, there is ample evidence in favour of the concept that Ranales possess characters of the primitive angiosperms (see Moseley, 1961). Hence students of phylogeny pay much importance to this group of plants. Certain evolutionary trends in the specialization of stamens and carpels have already been postdated (Bailey and Swamy, 1951; Canright, 1960; Moseley, 1958, 1961) and there is need for additional information on the anatomy of the ranalean flower. While Moseley (1958, 1961) studied the floral vasculature of certain nymphaeaceous species, information on the floral morphology of Cabomba (Raciborski, 1894; Moseley, 1958) is meagre. Recently Ramji and Padmanabhan (1965) described the developmental features of the carpel 216 Developmental Studies on Cabomba caroliniana Gray--H 217 and ovule in Cabomba caroliniana Gray. The present study is on the floral anatomy and microsporogenesis of this plant. MATERIALS AND METHODS Floral materials of Cabomba caroliniana were collected from the garden pond of the University Botany Laboratory, Madras. Formalin acetic alcohol was used as a fixative and the materials were processed in tertiary butyl alco- hol series and embedded in paraffin wax (Sass, 1951). Serial sections were cut at 8-10 microns in thickness and stained with safranin and fast green. Mature stamens and carpels were cleared in 20% sodium hydroxide and stained with safranin for studying their vasculature. OBSERVATIONS F/ower.--In the trimerous flower (Figs. 1 and 18) the sepals and petals are arranged alternately and do not differ from each other. The petals have m lli m 7B •,~'a'J 1 t ,;I, ".;, s ?c 6 ii7 FIGS. 1-7. Cabomba caroliniana. Fig. 1. A flower with perianth lobe removed to show stamens and carpels, × 12. Figs. 2, 3. Adaxial and abaxial views of a stamen, ×30. Figs. 4, 5. Adaxial and abaxial views of a carpel, x30. Fig. 6. A carpel split exposing ovules, ×30. Fig. 7. Three-dimensional diagram of carpel illustrating vasculature, dorsal bundle in solid black and laterals in striped lines. Figs. 7 A-7 C. Diagrammatic representation of carpel vasculature. (/, lateral bundle; m, median bundle.) B4 218 D. PADMANABHAN AND M.V. RAMn B' 8 # \ # .._f j X, ,,,. lF l .Z.I ( Fzos. 8-26. Serial transections of flower illustrating floral anatomy. All figures, x 72. Developmental Studies on Cabomba caroliniana Gray--H 219 auriculate appendages at the base (Figs. 14 and 15). The stamens (Figs. 2 and 3) are arranged in two whorls of three each and their filaments are flattened. The anther consists of four elongated sporangia (Figs. 2 and 3). The gynoe- cium consists of three free carpels (Fig. 1) and each elongated, flask-shaped carpel is continued into a stylodium (style) which finally terminates at the stigma (Fig. 7). The stigmatic heads and the surface of the overies are beset with small dark hairs (Figs. 4-6). There are 5-7 main veins in the petals which bifurcate at their tips (Fig. 1). The carpel contains three ovules of which one is placed below the style, the other two are lower in position and laterally disposed (Fig. 6). Floral Anatomy.---The vasculature of the pedicel consists of three bundles each of which bifurcates at the base of the flower (Figs. 8 and 9). A vascular plexus is formed above the level of bifurcation (Figs. 10-13). Sepals and petals receive their supply from the plexus. The vasculature for each sepal and petal consists of a single trace which soon bifurcates and supplies the respective organs (Figs. 10-16). The staminal trace arises from the plexus at a higher level. Each stamen is traversed by a single bundle (Figs. 14-19).~ Above the level of stamens, the vascular plexus breaks up into three main bundles (Fig. 15) each of which supplies a carpel. At the base of the carpel the trace trifurcates giving rise to a median and a pair of laterals (Figs. 15-17). The median branch extends up to the style without branching (Figs. 16-26). The laterals traverse upwards and reach the base of the style where they fuse to supply the upper ovule (Figs. 20-25). One of the lower ovules receives a composite supply from a branch of the lateral and another of the median (Figs. 7, 7 A-7 C). The remaining ovule receives its supply from the lateral of the opposite margin. Occasionally, one of the lower ovules receives supply from the median alone. Thus, in Cabomba the ovules receive vascular supply from both the median and lateral traces. Microsporogenesis.--During early development, the young anther becomes four-lobed and in each lobe a microsporangium develops (Figs. 30:-32). The hypodermal archesporial cells differentiate in a row and the outer products develop into wall layers. The inner derivatives function as microsporocytes (Fig. 33). The sporangial wall is made up of four layers (Figs. 27-29); epidermis, endothecium, middle layer and tapetum. The endothecium fails to develop wall thickenings. The uninucleate single- layered tapetum (Fig. 27) during later stages becomes two-nucleate (Fig. 27). It is of the secretory type and shrivels at the time of microspore formation (Figs. 28 and 29). 220 D. PADMANABHAN AND M. V. RAMJ1 29 Q 34 31 .... '%.'! !!(~C~ ~.~ ~.~,~ 36 ~ 38 FIGS. 27-39. Figs. 27-29. T.s. anthers at successive stages of development. Figs. 30-32. Outline drawings of anther sections depicted in Figs. 27-29. Fig. 33. L.s. young anther showing sporogenous cells and wall layers. Fig. 34. Microsporocyte. Figs. 35-38. Formation of microspore tetrads. Fig. 39. Pollen grain at shedding stage. Figs. 27-29. ×180; Figs. 30, 31, ×120; Fig. 32, ×200; Fig. 33, ×540; Figs. 34-39, ×1,500. Developmental Studies on Cabomba caroliniana Gray--H 221 The microsporocyte (Fig. 34) enlarges prior to meiosis. After the first meiosis (Fig. 35) a cell plate is deposited between the daughter nuclei (Fig. 36). The spindles during the second division are oriented at fight angles (Fig. 36) or parallel to one another (Fig. 37). At the end of this division the two ceils are partitioned by walls so that four microspores are formed. The micro- spore tetrads show tetrahedral (Fig. 38) or isobilateral arrangement. The microspore nucleus divides before shedding and the generative cell is surrounded by a distinct membrane (Fig. 39). The vegetative cell is vacuo- late. The pollen grains are shed at the two-celled stage. DISCUSSION Bailey and Swamy (1951) point out that the characters of primitive angio- sperms are to be looked for in the Ranales. In particular, the vasculature of the carpel and stamen deserve special attention. However, much work is still necessary for a proper concept of the primitive ranalean flower. The floral morphology of the Nymphaeaceae, especially the carpels, provides valuable information from this point of view. Moseley (1958, I961) studied some members of the family and concluded that the carpels of Nymphaea and Brasenia which are grouped under the Cabomboideae of Nymphaeaceae possess free carpels in contrast to their partially fused condition in Nymphaea. The peduncle of Cabomba is traversed by three main bundles. The vasculature of the receptacle forms a plexus, and the anastomoses of bundles resemble a nodal plate. The receptacular vascular ring gives off traces to the sepals and petals. Each of these divides further to form the venation of the corresponding organ. Since there are no anastomoses in the petal traces, the venation pattern is an open system as in leaves. The organization of a receptacular plexus and vascular supply to sepals and petals resembles Nymphaea (Moseley, 1961). The present work confirms the observations of Moseley (1958) regarding vasculature of the stamen. The stamens of Brasenia also show a similar condition (Moseley, 1958) but for the dichotomy of the strand at the tip of the anther. Two vascular strands supply the gynoecium in Nymphaea. The carpels of Cabomba on the contrary receive one bundle each. The single carpellary trace trifurcates to form two laterals and a single median which traverses up to the stigma. The vascular supply to the ovules in Cabomba is derived from the laterals as well as the median bundles. Of the three ovules in a carpel two are situated at a lower level and the remaining one below the style. The vasculature for B~ 222 D. PADMANABHAN AND M. V. RAM~ the upper ovule is formed by the terminal fusion of the laterals whereas among the lower two ovules one receives supply from a branch of the lateral and the other from the fusion of a branch of the lateral with a branch of the dorsal.