夏蓳與毛葉蝴蝶草種間雜交胚之拯救與多倍體化

Abstract

夏蓳(Torenia fournieri Linden)為倒地蜈蚣屬植物，是臺灣夏季重要花壇植物，現今市面上流通之夏蓳皆為國外品種，臺灣具有原生種源，應有潛力開發本土自有的品種。已知本屬植物存在種間雜交受精後障礙。本試驗以夏蓳與臺灣原生之毛葉蝴蝶草(T. benthamiana Hance)進行種間雜交，透過胚拯救成功獲得雜交後代、並調查後代之性狀，切片觀察雜交胚及胚乳之發育情形，探討溫度對雜交後代生長與開花之影響，並以秋水仙素誘導雜交後代多倍體化，以增加觀賞價值。 在日/夜溫25/20 oC環境下，以夏蓳‘Clown Rose’為種子親與毛葉蝴蝶草為花粉親，以雜交授粉後12、14、16或18天之胚珠培養於含3g•L-1之 Hyponex培養基上，以授粉後16和18天培養者有較高的發芽率，而授粉後12或14天進行培養者則發芽率不佳，顯示授粉後天數是影響胚拯救成功與否的關鍵。 在日/夜溫25/20 oC環境下，以夏蓳 ‘Clown Rose’為種子親與毛葉蝴蝶草為花粉親，於授粉後8和12天進行胚珠培養，在添加0%、3%、6%或12%蔗糖濃度之1/2MS培養基中，胚發芽率極低。若於授粉後16天進行培養，以0%、3%或6%蔗糖之1/2MS培養基較適合，胚可順利發芽並發育為小植株；12%蔗糖會抑制發芽。另以授粉後8、12和16天之子房進行切片觀察。夏蓳自交授粉胚與胚乳發育完整，授粉後8天胚乳已充滿胚囊。夏蓳與毛葉蝴蝶草雜交者之胚和胚乳細胞數少。夏蓳自交授粉後12天胚乳充實，胚發育已達魚雷期，而與毛葉蝴蝶草雜交者胚僅發育至球胚期。授粉後16天，夏蓳自交者已有成熟胚且珠被絨氈層明顯加厚，雜交者胚乳發育較慢，缺乏完整內皮層結構。 夏蓳‘Clown Rose’與毛葉蝴蝶草雜交之後代，株型性狀皆為半匍匐性，花瓣顏色介於紫色與紫羅蘭色之間，花冠筒中具條帶性狀且唇瓣近喉部有黃色斑點。 將夏蓳、毛葉蝴蝶草與雜交後代置於日/夜溫30/25 oC和15/13 oC之自然光照室，栽培兩個月。夏蓳栽培於15/13 oC者皆萎凋死亡，而雜交後代雖生長緩慢，但仍可維持開花，顯示雜交後代可耐低溫至13 oC。 為誘導夏蓳與毛葉蝴蝶草雜交後代多倍體化，以1.25、2.5和5 mM秋水仙素進行瓶內莖段處理24、48、72和96小時。結果顯示已1.25 mM秋水仙素處理有較高之培植體存活率及出瓶時株高。經選拔出之多倍體後代之花朵長寬、保衛細胞長度和花粉粒徑皆顯著高於二倍體，而氣孔密度顯著較二倍體為低。多倍體株型和花色與二倍體無顯著差異。有一個多倍體單株可自交結實，經播種後可順利發芽。將14個疑似多倍體化植株以流式細胞分析儀確認倍體數，僅有編號234單株為四倍體，其餘13株參試植株皆為具有二倍體與四倍體細胞的混倍體。

Wishbone flower (Torenia fournieri Linden) is one of the important summer bedding plants in Taiwan. There are Taiwan-native Torenia species, which could be used for breeding to develop domestic cultivars. However, post-fertilization barriers were known to exist when interspecific crossing. The objectives of the study were to 1) develop suitable conditions of ovule culture for producing progenies from crossing T. fournieri ‘Clown Rose’ and T. benthamiana, 2)investigate the floral traits of the progenies, 3) to observe endosperm and embryo development of selfed and crossed progenies, 4) determine the cold tolerance in progenies from crossing T. fournieri and T. benthamiana, and 5) induce polyploidization of the hybrid progenies with colchicines treatments to improve ornamental characteristics. The ovules were taken 12, 14, 16, and 18 days after pollination (DAP) from crossing T. fournieri ‘Clown Rose’ with T. benthamiana and were cultured on Hyponex medium. Germination percentage was higher (12%-31%) when ovules were taken at DAP 16 and 18, as compared with 0.4% at 12 and 14 DAP. Time after pollination is vital for the success of ovule culture of the crossed progenies. The ovules were taken 8, 12, and 16 DAP from crossing T. fournieri ‘Clown Rose’ with T. benthamiana and were higher cultured on 1/2MS medium with 0%, 3%, 6% or 12% sucrose. Germination percentage was higher when ovules were taken at 16 DAP than there at 8 or 12 DAP. Culture medium with 0%, 3%, or 6% sucrose increased germination percentage, while 12% sucrose significantly reduced germination. Capsules of self-pollinated T. fournieri ‘Clown Rose’, and those cross-pollinated with T. benthamiana were collected and sectioned at 8, 12, and 16 DAP. More embryo and endosperm development were observed in self-pollination than cross-pollination. At 8 DAP, embryo sac was fully occupied by endosperm in self-pollinated T. fournieri ‘Clown Rose’, whereas cross-pollinated ones had fewer embryo and endosperm cells. At 12 DAP, endosperm cells were enriched, and the embryo was at torpedo stage in self-pollinated T. fournieri ‘Clown Rose’. The cross-pollinated embryo remained at globular stage at 12 DAP. At 16 DAP, mature-seedlike embryo sac with thick-walled endosperm and enlarged endothelial cells were observed in selfed ‘Clown Rose’, whereas the development of cross-pollinated endosperm was significantly slower, without complete endothelial cells. The hybrid progenies from crossing T. fournieri ‘Clown Rose’ with T. benthamiana were semi-prostrate. Corolla color ranged between violet and purple-violet. All flowers had papillae petals with yellow dot and corolla tube with strip pattern. Torenia fournieri ‘Clown Rose’, T. benthamiana, and their hybrids were placed in natural light rooms with day/night temperature 30/25 oC and 15/13 oC for two months. T. fournieri ‘Clown Rose’ plants died when grown of 15/13 oC for two months, while the hybrid progenies flowered although grew slowly. To induce polyploidization of progenies from crossing T. fournieri ‘Clown Rose’ with T. benthamiana, the stem section of the hybrids were treated in vitro with 1.25, 2.5, or 5 mM colchicine for 24, 48, 72, or 96 hours. A low concentration (1.25 mM) of colchicine concentration increase survival rate and plant height of the explants. The colchicine-induced polyploidy progenies had bigger pollens and flowers, longer guard cells, and fewer stomata density than the diploid ones. A fertile polyploid hybrid was obtained, as the progeny produced seeds that could germination. The ploidy level of the colchicine-treated individuals were analysed by flow cytometry. These techniques confirmed that, of the 14 putative polyploids, only code no.234 was tetraploid, and thirteen were mixoploid.