花椰菜小孢子培養技術之研究

Studies on Microspore Culture of Brassica oleracea var. botrytis L.

夏奇鈮(Chi-Ni Hsia) ； 陳威臣(Uei-Chern Chen) ； 曹進義(Chin-Yi Tsao) ； 周宜玫(Yi-Mei Chou) ； 林子凱(Tzu-Kai Lin)

《台灣農業研究》 66卷2期 (2017/06) Pp. 94-104

https://doi.org/10.6156/JTAR/2017.06602.02

摘要

花椰菜 (Brassica oleracea var. botrytis) 是台灣的重要蔬菜，但有關花椰菜小孢子培養的研究甚少。本研究以台灣花椰菜商業一代雜交品種 (F_1) 為材料，測試各品種小孢子培養之反應，並建立胚誘導及胚發芽所需條件，生產雙單倍體 (doubled haploid; DH) 植株作為培育純系之用。花椰菜8 個F_1 品種之小孢子於不同批次，以相同接種濃度 (4 × 10^4 microspores mL^(-1)) 培養於NLN-13 培養液中，並以32℃分別處理1 d 或3 d，8 品種中以「農友極早生」及「慶農S-65」有較高之胚形成率，而「慶農H-42」、「慶農H-46」及「農友嬌雪」則未有胚形成。「農友極早生」以含有13% 蔗糖之全量及半量NLN 鹽類濃度培養基與32℃處理時間 (1、2 及3 d)組合進行複因子試驗，結果顯示32℃處理時間、培養基鹽類濃度以及兩者間的交感效應影響皆極為顯著 (P ＜0.01)，其中以1/2 NLN-13 培養基配合32℃處理1 d 可獲得12.0 胚/皿最高。將「農友極早生」及「慶農S-65」小孢子培養而來之子葉期胚繼代於B5 培養基中培養4 wk，發現僅有少數胚正常發芽 (2.1% 及4%)，多數胚未能正常生長且伴隨有二次再生組織長出，將未正常發芽之培植體依其形態分類後，先繼代於含有BA 之培養基中預培養1 wk，再繼代於不含生長調節劑之培養基中培養4 wk，結果兩品種皆以胚軸上長有再生組織之培植體有最高之小苗分化率，分別為「農友極早生」的66.7%，及「慶農S-65」的86.9%。以流式細胞儀檢測「農友極早生」及「慶農S-65」出瓶小苗之染色體倍體數，兩品種之2 倍體比率分別為58.8% 及56.3%，DH 植株比例皆超過50%。

關鍵字

花椰菜 ； 小孢子 ； 雙單倍體

並列摘要

Brassica oleracea var. botrytis is one of the most important vegetables in the world. However, very few studies on cauliflower microspore culture were conducted in Taiwan. The objective of this study was to establish a microspore culture system- an efficient technique to produce inbred lines for breeding elite F_1 hybrids, starting from cultivar screening for embryogenesis and followed by modifying conditions for germination of microspore-derived embryos. Microspores were inoculated with 4 × 104 microspores mL^(-1) in a NLN-13 medium, then incubated at 32℃ for 1- or 3-day. A total of 8 commercial F_1 hybrids were surveyed. Among them, 'Chinglong S-65' and 'Known-you Farmers extra early' had the highest embryogenesis response. However, there were 3 cultivars were found without any embryo. A multiple factor experiment using two culture media in combination with three duration treatments at 32℃ was further conducted using 'Known-you Farmers extra early'. The effect of 32℃ treated duration, medium salt strength, and their interactions were all significant (P ＜ 0.01) on embryogenesis. Among six treatments, 1/2 NLN medium in combination with 1-day culturing at 32℃ had the highest embryogenesis with 12.0 embryos/petri dish. Low germination rates of 2.1% and 4.0% for 'Known-you Farmers extra early' and 'Chinglong S-65' were obtained, respectively, from microspore- derived embryos cultured directly on a hormone-free B5 medium for 4-week. Various types of tissues derived from cotyledonary embryos were subcultured consistently on a BA containing medium for 1-week preculture before on the hormone-free medium for another 5 weeks culturing. Higher shoot germination rates of 66.7% and 86.9% for 'Known-you Farmers extra early' and 'Chinglong S-65' were observed, respectively, from the particular type of explant-elongated hypocotyl tissues with secondary organogenesis. Ploidy level of regenerated plants from 'Known-you Farmers extra early' and 'Chinglong S-65' were analyzed using flow cytometer, and diploid rate of 58.8% and 56.3% were obtained, respectively.

並列關鍵字

Cauliflower ； Microspore ； Doubled haploid