五倍體藍莓NTU295少籽化機制之研究

Abstract

商業栽培藍莓(Vaccinium spp.)罕見五倍體種類，然此類別之藍莓具有延長產季、果實成熟一致性以及果實少籽的優良特性。本試驗材料NTU295，源於六倍體兔眼藍莓‘Powderblue’雜交四倍體南高叢藍莓‘Misty’所得五倍體子代，其果實成熟期介於兔眼藍莓與南高叢藍莓；果實呈深藍色且種子數量少。為瞭解NTU295少籽的機制，本研究透過除雄、自交與回交授粉處理，觀察授粉後胚胎發育情形，評估其花粉親和性與受精能力，並結合胚珠(ovule)、胚囊(embryo sac)、胚(embryo)與胚乳(endosperm)之發育觀察，以釐清其少籽現象發生之可能原因。此外，本研究為分析NTU295開放授粉子代之倍體數與染色體數目，以探討染色體不成對之潛在對胚珠敗育的影響。 結果顯示，自交與回交處理於授粉後72小時皆可觀察到花粉於柱頭萌發，然僅有親本之花粉管可進一步延伸入胚珠週圍，並完成雙重受精，持續接合子發育，但不同胚珠間之發育程度差異明顯，部分順利發育出胚乳以及吸器構造；授粉四週後，多數胚珠敗育，但仍可見少數胚珠之胚持續發育。相較之下，自交處理之花粉管於72小時內未能進入子房與胚珠組織，接合子發育亦較為遲緩，且四週後多數胚珠呈現嚴重萎凋，同時觀察到胚、胚乳未正常形成及吸器構造發育不完全之情形。 流式細胞儀分析的結果顯示，NTU295開放授粉子代多呈現非整數倍體之個體，部分子代經根尖染色體計數觀察亦證實染色體為奇數條，且介於60至72條之間，證實NTU295在減數分裂過程中染色體分離異常、無法正常配對，為NTU295少籽之原因之一。綜上所述，NTU295之少籽現象可能與其五倍體基因組背景所導致之減數分裂異常，以及部分自交不親和反應造成胚和胚乳發育不全所導致。

Pentaploid 5x blueberries (Vaccinium spp.) are rare in commercial cultivated blueberry categories. Regardless, 5x blueberries may carry desirable traits including, extended harvest season, uniform fruit ripening and low seed count. The plant material used in this study, NTU295, is a pentaploid breeding line derived from a cross between the hexaploid rabbiteye blueberry ‘Powderblue’ and the tetraploid southern highbush blueberry ‘Misty’. The fruit ripening period of NTU295 falls between its parents. The berries of NTU295 are dark blue with few seeds. The study aimed to elucidate seedlessness mechanism in NTU295. Pollen compatibility and fertilization capability of NTU295 were tested after emasculation, self-pollination, or backcross-pollination treatments, and embryonic development was observed after pollination. The development of ovule, embryo sac, embryo, and endosperm was examined to elucidate the potential causes of this low-seeded phenomenon. Furthermore, the ploidy level and chromosome number of NTU295 open-pollinated progenies were analyzed to investigate the potential effects of chromosomal imbalance. Results showed that pollen germination on the stigma occurred 72 hours after both self- and backcross pollination treatments. However, only the backcross treatment resulted in pollen tube penetration into the ovular region, followed by double fertilization, and subsequent zygotic development. Nevertheless, variations in developmental progression among ovules were observed, with some ovules successfully forming endosperm and haustoria structures. By four weeks after pollination, ovule abortion became more pronounced, although a limited number of embryos continued to develop. In contrast, in the self-pollination treatment, pollen tubes failed to reach the ovary and ovule tissues within 72 hours, and embryo development was arrested. Most ovules exhibited severe degeneration after four weeks, accompanied by failure of embryo and endosperm formation and incomplete formation of haustoria. Flow cytometric analysis of NTU295 open-pollinated progenies revealed the presence of aneuploid individuals. Root tip chromosome observations further confirmed irregular chromosome numbers in some individuals that possessed odd chromosome numbers ranging from 60 to 72. These results suggest abnormal chromosome segregation and irregular chromosome pairing during meiosis in NTU295 resulted in the production of unbalanced gametes, which is likely one of the major causes of its low-seeded phenotype. Collectively, the seedless or low-seeded phenotype of NTU295 is likely attributable to meiotic abnormalities associated with its pentaploid genome constitution, as well as partial self-incompatibility that leads to impaired embryo and endosperm development.