Confocal microscopic observations on microtubular cytoskeleton changes during megasporogenesis and megagametogenesis in Phaius Tankervilliae (Aiton) B1

Abstract

In nun orchid (Phaius tankervilliae (Alton) B1. ) embryo sac development follows the monosporic pattern. Changes in the pattern of organization of the microtubular cytoskeleton during megasporogenesis and megagametogenesis in this orchid were studied using the immunofluorescence technique and eonfocal microscopy. At the initial stage of development the microtubules in the arehesporium were randomly oriented into a network. Later the archesporial cell elongated to form the megasporocyte. The cytoskeleton in the elongated megasporoeyte was radially organized in which microtubules extending from the nuclear envelope to the peripheral region of the cell. The megasporoeyte then underwent meiosis 1 to form a dyad. The dyad cell at the chalazal end was larger than the cell at the micropylar end. Microtubules in the dyad cell were radially oriented. The dyad underwent meiosis to give rise to a linear array of four megaspores (i. e. tetrad formation). The chalazal-far most megaspore survived and became the functional megaspore, which contained a set of randomly oriented microtubules. The microtubules in the other 3 megaspore disappeared as the cells degenerated. The functional megaspore then underwent mitotic division giveing rise to a 2 nucleate embryo sac. The nuclei of the 2-nucleate embryo sac were separated by a set of longitudinally oriented microtubules which ran parallel to the long axis of the embryo sac. Each nucleus in the embryo sac was surrounded by a set of perinuelear microtubules. The gnucleate embryo sac again underwent mitotic division to form a 4-nucleate embryo sac. The division of the two nuclei was synchronous. But the orientation of the division plan of the two spindles was different (i. e. the spindle microtubules at the chalazal end ran parallel with the long axis of the embryo sac and those at the mieropylar end ran at right angle to the axis of the embryo sac). The 4 nuclei of the 4-nucleate embryo sac were all tightly surrounded by randomly oriented microtubules. Later the paired nuclei at the micropylr end and at the chalazal end as well underwent mitotic division in seguence. At this time when the embryo sac had reached the 8-nucleate embryo sac stage. The pattern of organization of the microtubules was very complex. Initially the nuclei were surrounded by a set of randomly oriented microtubules, but after the two polar nuclei had moved to the central region of the embryo sac, three different organizational zones of microtubules appeared, viz: a randomly oriented set of microtubules surrounding each nucleus in the chalazal zone: a set (in the form of a basket) of cortical microtubules which surrounded the vacuoles and the two polar nuclei in the central zone and a loosely knitted network of microtubules surrounding the nucleus that later became the egg cell nucleus in the micropylar zone. The two nuclei that would become the nuclei of the synergids were surrounded by a set of more densely packed mierotubules. Towards far the most micropylar end some microtubules formed thick bundles. The site of appearance of these thick bundles coincided with the site of development of the filiform apparatus. The pattern of microtubule organization after cellularization (i. e. at the beginning of embryo sac maturation) did not change much. The author's results indicated that various patterns of microtubule organization observed in the developing embryo sac of nun orchid reflected the complexity and dynamism of the embryo sac.

光鏡的觀察確定了鶴頂蘭(Phaius tankervilliae (Aiton) B1. )胚囊發育屬單袍子寥型。應用免疫熒 光標記技術及共焦鏡觀察了胚囊發育過程中微管分佈的變化。當孢原細胞初形成時，細胞內的微管呈網狀分佈。之後，孢原細胞體積增大發育為大孢子母細胞。大孢子母細胞延長，進入減數分裂I 。微管由分裂前的網狀分佈變為輻射狀排列。二分體的兩個細胞內的微管分佈一樣，呈輻射狀。四分體的近珠孔端的3個大孢子解體，細胞內的微管消失。靠合點端的功能大孢子內有許多微管呈網狀分佈。當功能大孢子進入第一次有絲分裂時，細胞內的微管由網狀變為輻射狀，從核膜伸展至週質。再經兩次有絲分裂形成八核胚囊。在核分裂之前微管一般是呈網狀分佈並緊包圍著核。在分裂期間二核和四核胚囊都呈極性現象，微管系統也呈極性分佈。微管在八核胚囊內的分佈變化情形特別複雜。首先，八核分別作不同程度的移動，其中兩個核移向胚囊中央，珠孔端和合點端的3 個核分別互相靠攏，形成3個區，即中央區、​​反 足區和卵器區。胚囊未形成區時.8個核都被網狀分佈的微管包圍著。當胚囊明顯分成區時，反足區內的微管仍作網狀分佈。中央區的微管分佈則趨疏鬆，形成籃形結構，包圍著液泡和兩個極核。在卵器區內卵核被1層微管網絡包圍著。而1對助細胞核除被較密集的微管網絡包圍之外，近珠孔端部位還形成一堆粗而扭曲的微管束。這個微管束的出現部位與絲狀器的形成部位吻合。胚囊成熟時，胚囊內的微管系統不再有很大的變化。