青花菜小孢子培養之胚誘導及胚發芽條件建立

Development of Microspore Embryogenesis and Plant Regeneration of Brassica oleracea var. italica

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

《台灣農業研究》 66卷3期 (2017/09) Pp. 184-192

https://doi.org/10.6156/JTAR/2017.06603.02

摘要

十字花科之青花菜(Brassica oleracea var. italica)是營養價值很高的蔬菜，本研究以台灣青花菜商業一代雜交品種(F_1)為材料，測試不同F1 之小孢子培養反應，並建立小孢子之胚誘導及胚發芽所需條件，用以生產雙單倍體(doubled haploid; DH)植株作為培育純系之用。利用5種F_1青花菜以每mL含有4 × 10^4個小孢子濃度接種於NLN-13培養液中，並以32℃處理1 d，其中3品種有胚形成，2品種未有反應，胚誘導率以「慶農B-45」最高。比較取自「農友天綠」不同長度花蕾之小孢子，結果顯示取自4.0-5.0 mm長度花蕾之小孢子，平均可獲得17.1個胚/皿，顯著較取自3.0-3.9 mm長度花蕾之7.0胚/皿為高(P ＜ 0.01)。「慶農B-45」以含有13%蔗糖之NLN培養基(全量及半量鹽類濃度)與32℃處理時間(1、2及3 d)組合進行小孢子培養。結果顯示以全量NLN培養基在32℃處理1 d獲得6.9胚/皿最高。將「慶農B-75」子葉期胚施以1-5 d之脫水配合溫度處理，處理後之胚先接種於含有生長調節劑之固態培養基預培養1 wk，再繼代於不含生長調節劑之培養基中培養4 wk後，調查胚發芽之比率。結果顯示子葉期胚於25℃以濾紙脫水1 d或於4℃以濾紙脫水1-5 d皆有助提高發芽率(35.0-48.3%)。將「慶農B-75」由小孢子發育而來之23株小苗以流式細胞儀檢測其染色體倍體性，結果顯示DH及單倍體之比率分別為60.9%及34.8%。DH植株之比率超過60%，有助於育種實務上之利用。

關鍵字

青花菜 ； 胚分化 ； 雙單倍體

並列摘要

Brassica oleracea var. italica is an important vegetable in the world with rich nutrition for human health. The objective of this study was to establish a microspore culture system using elite broccoli F1 hybrids from Taiwan to produce doubled-haploid (DH) lines for breeding purpose. Microspores taken from 5 commercial F1 hybrids were surveyed for embryogenesis. Microspores were inoculated with 4 × 10^4 microspores/mL into NLN-13 medium in combination with a heat shock treatment at 32℃ for 1 d before moving to 25℃ for culturing. Embryo induction was found derived from 3 out of 5 cultivars, among them ‘Chinglong B-45’ had the highest embryogenesis rate. However, two cultivars were found without any embryogenesis. Microspores of ‘Known-you Tender green’ were taken from two different bud lengths for embryogenesis. A higher embryo induction rate (17.1 embryos/ petri dish) was obtained from 4.0-5.0 mm length of bud than that of from 3.0-3.9 mm length of bud (7.0 embryos/petri dish). An experiment using two culture media in combination with three durations at 32℃ was conducted using 'Chinglong B-45'. Among six treatments, NLN medium with 1-d culturing at 32℃ had the highest embryogenesis with 6.9 embryos/petri dish. Various stress treatments were conducted using cotyledonary embryos derived from 'Chinglong B-75' to improve germination. Treated embryos were precultured on the hormone containing medium for 1-week followed by 4 wk culturing on a hormone-free medium for shoot germination. A low shoot germination rate of 1.7% was obtained from the control which was without any stress treatment. In contrast, shoot germination rate of 46.7% and 48.3% were obtained from filter paper dry treatments at either 25℃ for 1 d or at 4℃ for 5 d, respectively. Ploidy levels were analyzed using flow cytometry for a total of 23 microspores derived plants of 'Chinglong B-75'. Rates of 34.8% and 60.9% were obtained for haploid and diploid, respectively. A high DH production rate of 60% derived from the microspore culture would facilitate breeding program in practice.

並列關鍵字

Broccoli ； Embryogenesis ； Doubled haploid