花椰菜雙單倍體誘變平台建立

Establishment of a Doubled Haploid Mutagenesis Platform in Cauliflower

夏奇鈮(Chi-Ni Hsia) ； 陳威臣(Uei-Chern Chen) ； 許秀惠(Shiow-Huey Hseu) ； 林楨祐(Chen-Yu Lin) ； 曹進義(Chin-Yi Tsao) ； 林子凱(Tzu-Kai Lin)

《台灣農業研究》 70卷3期 (2021/09) Pp. 182-195

https://doi.org/10.6156/JTAR.202109_70(3).0003

摘要

本研究採用雙單倍體（doubled haploid; DH）作為小孢子供體，於小孢子培養過程中施以誘變處理，建立花椰菜雙單倍體誘變平台。誘變處理包括以紫外光（ultraviolet; UV）處理4個不同結球天數花椰菜DH品系之小孢子，DH品系源自「慶農S-65」小孢子培養；以及利用ethyl methanesulphonate（EMS）處理上述UV試驗所得之小孢子胚，並將上述兩誘變處理所得DH植株種植於田間，觀察誘變株發育階段的變化。此外，又以EMS處理「慶農S-65」花蕾並取其內小孢子進行培養，比較不同濃度處理對小孢子胚分化之影響。誘變結果顯示，以UV處理小孢子10 s及20 s，4個DH品系之胚分化率為其對照組的23.9－65.1%，但2種光照時間處理的差異不顯著。接續觀察經UV處理之小孢子胚發芽率，結果顯示有不同程度之下降，為其對照組之50.9－78.8%。DH32之小孢子經UV處理後長出之胚，接續以0.125－0.500% EMS震盪處理10 min，小孢子胚之發芽率降為對照組的36.4－56.8%，但3個EMS濃度處理間差異不顯著。取「慶農S-65」花蕾以0.025－0.100%EMS以靜置浸泡或添加Tween 20加震盪方式處理5 min或10 min，結果顯示2種處理時間之胚分化率皆以0.025% EMS為最高。但採用震盪處理時，胚分化率會因處理濃度或時間延長而明顯下降。將4個不同結球天數DH品系單誘變小孢子（UV處理）及利用雙誘變（UV處理小孢子+EMS處理小孢子胚）所得之DH誘變株與其對照植株定植田間，於栽培84 d後調查DH誘變株之發育階段並比較與對照組之差異。綜合而言，誘變株發育階段之改變以提前發育比例較延後者為高，可作為未來預測誘變方向之參考。本誘變平台以DH品系為供體於小孢子培養過程中進行誘變處理獲得DH誘變株，除可縮短誘變育種時間並提高選拔效率外，DH具有高度一致性之特性則有助於突變體之辨識。

關鍵字

花椰菜 ； 小孢子培養 ； 雙單倍體 ； 單倍體誘變

並列摘要

An integrated haploid mutagenesis platform through doubled haploid (DH) donor plants providing microspores in cauliflowers was established in this study. Ultraviolet (UV) irradiation was applied to microspores of four DH lines derived from 'Chinglong S-65' and each line with various heading mature days. Embryos derived from UV treated microspores of DH32 as stated above were treated with ethyl methanesulphonate (EMS) subsequently for double mutagenesis. EMS was also applied on flower buds of 'Chinglong S-65' before microspore culturing for mutagenesis. The results showed that embryo production (embryos/petri dish) from two UV exposure duration in all four DH lines was significantly lower, accounting for 23.9% to 65.1% decrease compared to the control. However, no significant difference was observed on embryo production between the two UV exposure duration. The results also showed that embryo germination rate of the UV treated microspores decreasing 50.9 to 78.8% compared to that of the control. Cotyledonary embryos of DH32 derived from the UV 10 s treatment were soaked in 0.125-0.5% ethyl methanesulphonate (EMS) for 10 min. Embryo germination rates decreased in all EMS treatments by 36.4% to 56.8% compared to that of the control. However, no significant difference was found among tree EMS treatments. Flower buds of 'Chinglong S-65' were soaked in 0.025-0.1% EMS for 5 or 10 min with or without shaking for mutagenesis before microspore culturing. The results showed that regardless exposure time, the highest embryo production was obtained in buds treated with 0.025% EMS. However, embryo production decreased as exposure time increased and shaking was applied. DH plants derived from the UV or double mutagenesis (UV plus EMS) of four of DH lines were cultivated in the field and data were recorded at 84 d after cultivation. Field observation concluded that percentage of early growth rate was higher than that of delaying based on developmental performance of DH mutants in the four DH lines. Our results not only provide useful information for prediction of mutant tendency in cauliflower but also demonstrate that haploid mutagenesis is capable of shortening time span and enhancing selection efficiency on breeding. Additionally, this study demonstrated that trait screening on mutants was more efficient than using the highly uniform DH as donor plants for haploid mutagenesis.

並列關鍵字

Brassica oleracea var. botrytis L. ； Microspore culture ； Doubled haploid ； Haploid mutagenesis