台灣穗花杉小孢子形成與花粉發育之研究

Abstract

台灣穗花杉 (Amentotaxus formosana Li) 為台灣單屬單種的裸子植物，分類上屬穗花杉科、穗花杉屬。本研究結合光學、穿透式電子顯微鏡、掃瞄式電子顯微鏡和螢光顯微鏡研究，並配合化學組織染色，完整敘述台灣穗花杉小孢子和花粉發育之過程。 台灣穗花杉在同一個花粉囊腔內的小孢子母細胞，進行減數分裂後，接著同時進行連續型和同時型之細胞質分裂，而產生多種不同形式的四分體，主要有長菱形、四面體形與四角形，少數為十字形、線形、T形之四分體。四分體小孢子的細胞核內有相當多雙層膜包覆之構造出現，細胞質內的胞器，如粒線體、色素體、高爾基氏體、粗內質網快速累積。四分體中期即開始有花粉外壁的堆積，首先由層狀的外壁內層先形成，再由顆粒狀的外壁外層堆積其上。四分體中期原本位於近心極之萌芽口，至四分體晚期時因四分小孢子的轉向，而使萌芽口朝向遠心極。小孢子由胼胝質釋放出來後，早期在其細胞質內先有大量澱粉粒累積，晚期則逐漸消失，可能被用為碳源和能量之來源。自由小孢子早期，於營養層之徑向與內切細胞壁開始出現烏氏體，初期為圓形或者為嘴喙形。烏氏體由低電子密度的中心脂質和外圍高電子密度的孢粉質所構成，其自營養層細胞以胞外方式釋出至囊腔。再接著，進入液胞小孢子時期，此時整個花粉外壁厚度雖持續增厚，但外壁內層的層數卻不再增加，並且開始有花粉內壁的形成。當液胞化的小孢子進行有絲分裂後，形成一個營養細胞和一個生殖細胞，並且外壁內層開始壓縮，但內壁則快速膨脹且明顯可見二層，內壁外層較平滑，內壁內層則為波浪狀。 成熟花粉粒，花粉表面附著大量烏氏體，其新鮮剛釋出的花粉粒含水量僅20.8%。以FCR進行成熟花粉粒活性測試，具有69.3 %的活性，經由水合一小時後的花粉，外壁開裂脫離，同時內壁快速吸水膨脹，此時花粉活性超過90%，顯示台灣穗花杉成熟花粉具良好的活性。成熟花粉經由一至二個月，分別儲存於低溫4℃和-20℃，一個月後，經由水合4小時後活性皆可達90 %；此外，儲存-20℃二個月的花粉，其水合4小時後活性值為61.3 %，儲存於4℃的花粉之活性值為51.4 %，顯示花粉儲存於-20℃較儲存於4℃有較佳之活性保存能力。 本研究首次發現裸子植物之四分體時期，其小孢子隨著胼胝質之瓦解而產生旋轉現象，此現象推測可能由於胼胝質瓦解速度較慢，因而產生拉力導致四分體小孢子旋轉。

Amentotaxus formosana Li (Amentotaxaceae) is an endemic and rare species of gymnosperms in Taiwan. In this study, the ultrastructural changes of its microspores by means of LM, SEM, TEM, fluorescent microscopy, and histochemical analysis during microsporogenesis were investigated. During meiosis, both simultaneous and successive cytokinesis occurred in the same microsporangium. In addition, most tetrads were rhomboidal, tetrahedral or tetragonal in shape; only a few were, decussate, linear and T-shaped. At the middle tetrad stage, many structures with double-membrane appeared in the nucleus, while many organelles, such as mitochondria, plastids, dictyosomes, and endoplasmic reticula appeared in the cytoplasm. Meanwhile, the lamellated endexine accumulated on the plasma membrane, and the granular ectexine subsequently appeared on the surface of lamellated endexine. Shortly before the microspores were released from the callose wall, the location of tetrad germinal pore moved from the proximal pole to the distal one, which suggested a rotation of the tetrad microspores. At early free microspore stage, starch grains accumulated but then disappeared toward the late stage, suggesting that the starch grains were possibly consumed as carbon and energy sources. The Ubisch bodies which appeared on the surface of the inner tangential wall of the tapetum were either spheroidal or elongated beak-shaped. All of the Ubisch bodies had a core with low electron density material and were enclosed by round, high electron density sporopollenin. This was a result of the exocytosis of Ubisch bodies from the degenerating tapetal cells. At the vacuolated stage, the ectexine continued to increase in its volume, while the endexine ceased to increase in its number of layers. Subsequently, the microspore divided into a vegetative cell and a generative cell. At the bicellular stage, the lamellate endexine was highly compressed, while the intine swelled rapidly and differentiated into a smooth outer layer and a wavy inner layer, respectively. At the mature pollen grain stage, the Ubisch bodies occurred on the surface of pollen grains. The water content of mature pollen grain was 38.5%. When those pollens grains were tested with FCR reaction, they had 69.3% viability. After 1hr rehydration in distal water, the exine and intine of the pollen grains split and expanded respectively and presented more than 90% viability. The results demonstrated the high viability of A. formosana pollen. Within the conservation test, fresh pollen grains were stored respectively at 4℃ and -20℃ for 1 and 2 months. The viability tests showed 1 month-stored pollen grains under two kinds of temperature treatments both had 90% viability after 4 hrs rehydration. However, viability of 2 months-stored had a higher value under -20℃ preservation than under 4 ℃ preservation. In addition, the pollen grains under -20℃ preservation increased gradually to 60.3%. The results suggested that the -20℃-stored pollen had better preserving ability. We firstly observed the inversion of microspore during microsporogenesis in A. formosana and presumed that this phenomenon resulted from a differential dissociation time of callose.