甘藍小孢子培養影響因子之優化研究

甘藍是台灣最重要的葉菜類蔬菜，本研究之目的在提高甘藍小孢子培養技術的效率，用以建構甘藍雙單倍體（doubled－haploid；DH）生產系統，加速甘藍育種流程。篩選市售甘藍共14個栽培種，其中13個栽培種之小孢子在培養後具有胚分化反應，以取自2.50－3.50 mm大小花蕾具有高於或等於3.51－4.50 mm大小花蕾之胚分化率，其中以「金春」和「新威」栽培種的胚分化率最高，分別為15.89及10.17個胚／皿。比較NLN [Nitsch & Nitsch medium modified by Lichter（1981）]培養液中鹽類濃度及活性炭與瓊脂醣懸浮液組成對於「金春」和「新威」甘藍小孢子胚分化率的影響，兩栽培種皆以1／2 NLN較全量NLN培養液為佳，但活性炭與瓊脂醣懸浮液處理間則不具顯著差異。比較「金春」和「新威」甘藍小孢子在含有13%蔗糖或麥芽糖之培養液中培養，或先以含有16%蔗糖之培養液培養2 d後，再將蔗糖濃度降至10%繼續培養，共3種處理，結果顯示兩栽培種皆以含13%蔗糖處理可獲得最高之胚分化率。比較32℃及35℃高溫處理1 d後，再分別於22℃或25℃中靜置培養對「新威」小孢子胚分化的影響，結果顯示僅第一階段高溫處理的影響效果顯著，其中以32℃處理1 d再續以25℃培養可獲得之胚數最高，為每皿14.00個胚。在胚發芽率方面，「新威」甘藍的子葉期胚經4℃脫水1 wk後，接種於含有16 g L^(-1)洋菜的培養基中，發芽率可達91.81%；但「金春」使用相同處理之發芽率僅27.77%。將4℃脫水1 wk後之「金春」小孢子胚接種於含有不同濃度洋菜的培養基中於室溫培養，結果顯示10－16 g L^(-1)洋菜未能提高「金春」之發芽率；但若將小孢子胚先接種於16 g L^(-1)洋菜培養基，同時置於4℃培養1 wk，之後移至室溫繼續培養，可將「金春」之發芽率提高至94.00%。以流式細胞儀分析「金春」和「新威」甘藍的DH植株比例，分別為53.4%和12.5%，顯示不同栽培種小孢子胚染色體之自然倍加比率差異大。

關鍵字

甘藍；小孢子胚分化；雙單倍體

並列摘要

Cabbage (Brassica oleracea var. capitata) is the most important leafy vegetable in Taiwan. In order to shorten breeding process of F1 cabbage, an optimized protocol on microspore culture to produce doubled-haploid (DH) lines has been established in this study. Microspores derived from bud with size of 2.50-3.50 mm or 3.51-4.50 mm were compared. Results showed that the smaller-sized buds had either better than or equal to that of the larger-sized buds on embryogenesis among 14 screened cultivars. The results also showed that 13 out of 14 cultivars responded to embryogenesis induction. Moreover, among them 'Jin Chuen' and 'Shin Wei' had the highest microspore embryogenesis with 15.89 embryos/petridish and 10.17 embryos/petridish, respectively. An experiment testing 1/2 or full strength NLN salt medium in combination with active charcoal-agarose suspension on microspore embryogenesis was conducted using cv. 'Jin Chuen' and 'Shin Wei'. The results showed that 1/2 NLN was superior to full strength NLN [Nitsch & Nitsch medium modified by Lichter (1981)] on microspore embryogenesis in both cultivars. However, no significant effect of charcoal-agarose suspension on microspore embryogenesis was shown. Types of carbohydrate and its concentration in the microspore medium were tested on microspore embryogenesis including either using 13% sucrose or 13% maltose for 4-wk culture interval, or using 16% sucrose for 2 d culturing before changing to 10% sucrose culturing for the rest of time on embryogenesis. The highest embryogenesis rate was found by using 13% sucrose for all culture interval on both cultivars. Two stages temperature treatments on microspore embryogenesis were also investigated by culturing microspores in the first stage at either 32℃ or 35℃ for 1 d followed by culturing either at 22℃ or 25℃ for 2 wk. The results showed that only the high temperature in the first stage had significant effect on embryogenesis and the highest embryogenesis was observed by 1 d culturing at 32℃ following at 25℃ for the rest culturing time. A high embryo germination rate of 91.81% was obtained from 'Shin Wei' by desiccating cotyledon stage embryos on a filter paper at 4℃ for 1 wk before subculturing onto the B5 medium containing 16% agar (B5A-16) for germination. However, a low germination rate of 27.77% was obtained from 'Jin Chuen' by using the same treatment and no improvement on the germination rate was found by increasing agar concentration in the germination medium from 10% to 16%. An improved high germination rate up to 94.00% was obtained on 'Jin Chuen' by directly culturing embryos on the B5A-16 medium without filter paper desiccation at 4℃ for 1 wk before moving to 25℃. Ploidy levels of microspore derived plants were analyzed using flow cytometry and DH rates of 53.4% and 12.5% were obtained for 'Jin Chuen' and 'Shin Wei', respectively.

並列關鍵字

Brassica oleracea. var. capitata ； Microspore embryogenesis ； Doubled haploid

國際替代計量

甘藍小孢子培養影響因子之優化研究

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