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標題: 紫錐菊花藥及小孢子培養與根尖染色體鏡檢之研究
Studies on anther and microspore cultures and root tip karyotype examination of purple coneflower (Echinacea purpurea L.)
作者: 林依霈
Lin, I-Pei
關鍵字: purple;紫錐菊;coneflower;microspore;culture;karyotype;examination;小孢子培養;染色體鏡檢
出版社: 農藝學系所
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紫錐菊 (Echinacea spp.) 為世界著名的保健作物,其為異交作物且大多具有自花不合和性。傳統自交方式純化後代不易,故擬利用花藥培養獲得雙單倍體 (doubled-haploid, DH) 植株。鑑於花藥培養亟需注意分化起源位置,故進行小孢子培養,期能提高效率,此外,亦針對紫錐菊染色體鏡檢流程進行改良,以提高其效率。試驗結果如下:


根尖染色體數鏡檢試驗中,適當的取樣時間會隨取樣月份而有所不同。以 0.08% 秋水仙素作為前處理搭配Farmer’s solution 的固定效果較佳。已固定的根尖加入醋酸洋紅後直接加熱至沸騰軟化 10 分鐘,再添加 1 ml 10% pectinase,加熱至 37℃、10分鐘,壓潰之可得良好鏡檢效果。

本試驗參考麥類的子房共培養方式進行小孢子培養,此為菊科作物之首例,以添加 77 g/l sucrose,pH 5.8 之修正後 N6 培養液表現較佳,30 天後即有膨大的小孢子出現,60 天後小孢子有近似分裂跡象。加入 10 枚雌蕊進行共培養,對出現小孢子膨大反應的效果為 38.9% (21/54),無共培養處理者為 22.2% (8/36),而 0.9 µM 與 1.4 µM 2, 4-D 的反應則分別為 17.8% (8/45) 與 46.7% (21/45)。本試驗雖未能獲得微癒合組織,但在菊科作物小孢子培養研究上已有所進展。

Purple coneflower (Echinacea purpurea L.) is one of the most popular health protection herbs recently and it is a cross-pollinated crop with dominant self-incompatibility. Considering the heavy burden of purification of inbred line via self-pollination in a long schedule, we tried to employ anther culture to get doubled-haploid plants. To promote efficiency the microspore culture was studied for avoiding paying attention to dissect where the regenerated plants were derived from. Furthermore, karyotype examination protocol was developed to aim at coneflower plantlets in vitro. The results are summarized as follows:
1. Observation of regenerated plantlets
The regenerated plantlets were categorized into six levels according to size. Characters including plant heights, stomata sizes and densities, chlorophyll contents of leaves, diameters of root's elongation region and karyotype examination for chromosome numbers of root tips were collected to study the relationships between those and size levels. The results indicated no obvious relationship could be followed and the karyotype examination for chromosome number was the most reliable criterion. One of the reasons for the results is the majority of regenerated plantlets are diploids and this phenomenon indicated that it were very important to make sure the regenerated plantlets derived from right places.

2. Protocol of karyotype examination
The optimum time of sampling root tips for karyotype examination was changing monthly. Root tips treated with 0.08% colchicine and then fixed in Farmer's solution brought a better effect on examination. The fixed root tips were stained and softened at the same time by adding aceto-carmine and heated directly up to boiling for 10 minutes, and then 1 ml 10% pectinase was added and kept at 37℃ for 10 minutes. After that, a satisfactory karyotype examination of squashed sample would be obtained.

3. Microspore culture
The microspores cocultured with ovaries, refered to wheat microspore culture, were tested and this is reported first for composite crop. The modified N6 medium with 77 g/l sucrose and pH 5.8 was better than other media in terms of emergence frequency of enlarged microspores. The enlarged microspores and dividing microspores appeared after culturing for 30 days and 60 days, respectively. The effect of adding 10 ovaries in coculture on emergence of enlarged microspores was 38.9% (21/54) in contrast to check with 22.2% (8/36). While the effect of 0.9 µM and 1.4 µM 2, 4-D were 17.8% (8/45) and 46.7% (21/45), respectively. Though it is unable to form microcallus in microspore cultures, the results did show some chances to promote it.
其他識別: U0005-2201200916232300
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