DEVELOPMENT OF MALE REPRODUCTIVE STRUCTURES IN HELIANTHUS CILIARIS AND H. TUBEROSUS (ASTERACEAE)
Helianthus ciliaris DC. and H. tuberosus L. are wild perennial sunflower species from Ciliares and Divaricati sections respectively. They belong to the genus Helianthus L. of Asteraceae family (Schilling, Heiser, 1981). These species differ in appearance, flowering time and chromosome number. H. ciliaris used in our investigation was a tetraploid with chromosome number 2n = 68, H. tuberosus - a hexaploid with 2n = 102. The embryology of H. ciliaris is unknown, that of H. tuberosus is studied incompletely. We investigated the processes of their anther and pollen formation and their structure. The material of H. ciliaris was collected in the Kuban Experiment Station of the N. I. Vavilov All-Russian Institute of Plant Genetic Resources, where the samples of annual and perennial species of wild sunflower are cultivated. H. tuberosus was obtained from the park of the Komarov Botanical Institute. The flowers were not isolated, free (cross) pollination was permitted. The whole flower heads on the different developmental stages were collected and fixed in FAA (formalin, glacial acetic acid and 70° ethanol in 7 : 7 : 100 proportion). Then material was rinsed with 70° ethanol, dehydrated with the series of alcohols, processed with the sequence of alcohol-chloroform mixtures up to the pure chloroform and embedded in Histomix® (Biovitrum). Sections for light microscopy (4-10 pm) were stained with various dyes in some combinations: toluidine blue; Ehrlich’s haematoxylin with eosin or alcyan blue; Heidenhain’s iron haematoxyline with alcyan blue, «triple staining» and Schiff’s reagent according to Feulgen with alcyan blue (that is «triple staining» without Ehrlich’s haematoxylin processing). For mature pollen in whole mounts aceto-carmine was used. We have seen that initiation, development and structure of male reproductive structures meet the pattern characteristic of the genus Helianthus. The timing of the processes and their details, however, are specific. There are 5 stamens, similar in length, arranged in one whorl in the tubular flower of Helianthus. The initiation of flower elements occurs centripetally. Stamen initiation takes place after laying down of petals and before pistil formation. Anthers are tetrasporangiate, the pairs of locules in a mature anther are fused to form two thecae. Microsporangia on the adaxial (inner) side of the anther are a little smaller than on the abaxial (outer) side. The microsporangium wall develops according to dicotyledonous type. At first, archesporial cells begin to differentiate under the epidermal layer of anther primordium. After that, they divide periclinally to form parietal and sporogenous cells. The parietal cell is situated above the sporogenous one, outwards the microsporangium. Anticlinal divisions of the parietal cell initiate the parietal layer forming in the outer side of the microsporangium. The parietal layer in the inner side of the microsporangium (near the connective) forms from the cells situated under the sporogenous ones. Further, the parietal layer appears as a rather uniform layer of cells around the sporogenous ones. The dualism of its inner and outer parts origin has no significant display in future. The cells of the parietal layer divide to form two layers. Tapetum differentiates from the inner parietal layer at first, and later the divisions in the outer (subepidermal) layer lead to formation of the endothecium and the middle layer. The formed microsporangium wall consists of four layers: epidermis, endothecium, middle layer and tapetum. The middle layer is ephemerous. The tapetum is cellular, with reorganization to false periplasmodium (syncytium). The first signs of reorganization begin to appear at the final stages of the second meiotic division. The wall of mature anther consists of epidermis and endothecium with fibrous thickenings, the latter develop about the stage of two-celled pollen grain. During the flower formation and growth, the microsprangia of adjacent stamens aggregate with their contacting parts due to very close location. Usually, it concerns abaxial ones. The cells of epidermal layer take part in this aggregation. The first time we could see it was the beginning of the sporogenous cells division and formation of 3 layers of microsporangium wall. Anthers dehisce by longitudinal slits; the walls between microsprangia in thecae break down simultaneously. The edges of the adjacent anther slits come in touch at the middle level of anthers, which results in joining of their thecae. The anthers have flattened sterile appendages in their apical parts. These appendages appear to be open after anther dehiscence and may take part in pollen distribution. Microspore tetrad formation follows the simultaneous type. The cell wall does not initiate after the first meiotic division, the cytokinesis takes place after formation of four nuclei of future microspores. The tetrad of microspores arises within the mutual cell wall. This process is accompanied with callose walls formation around every microspore in the tetrad. The divisions inside the microsporangium happen asynchronously. Usually the tetrads of microspores are tetrahedral, sometimes isobilateral. Mature pollen grains are 3-celled, with a characteristic sculpture of exine. Beginning from the stage of tetrad, microspores, and afterwards pollen grains, show variability in their size and structure. Some of them are abnormal: too large or too small, distorted, with jammed or black-stained cytoplasm, with too thick wall.