水稻花粉活力檢測方法研究

Abstract

水稻是台灣最重要的糧食作物，其產量與米質會受高溫的影響而下降，在水稻的生長過程中以開花當日對高溫最為敏感，因此瞭解花粉的耐熱性是評估耐熱水稻品種的重要指標。本研究除最適化水稻花粉培養所需配方外，並比較花粉染色法、花粉培養與電阻抗式流式細胞儀 (impedance flow cytometry; IFC) 3 種花粉活力檢測方法，同時以IFC 檢測經不同溫度處理後花粉的活力變化。試驗以稉稻 「台南11 號」 (‘Tainan 11’;‘TN11’) 與耐熱秈型陸稻 (aus type) ‘Nagina 22’ (‘N22’) 為材料，針對花粉培養液中之蔗糖、硼酸及硝酸鈣逐項進行最適濃度測試，結果顯示2 品種之花粉皆以含有22.5% 蔗糖、60 mg L-1 硼酸與200 mg L-1 硝酸鈣之培養液可獲得最高之花粉萌發率。比較3 種染劑分別為二乙酸螢光素酯 (fluorescein diacetate; FDA)、2,3,5-氯化三苯基四氮唑 (2,3,5-triphenyltetrazolium chloride; TTC) 以及碘化鉀 (I2/KI) 對水稻花粉之染色率，結果顯示2品種皆以1% I2/KI 之染色率最高，TTC 之染色率最低，FDA 染色率居中。利用花粉培養、FDA 染色及IFC分析三種方法進行花粉活力檢測，在 ‘TN11’ 和 ‘N22’ 均顯示IFC 檢測與花粉培養或FDA 染色之結果無顯著差異，顯示IFC 可作為花粉活力快速檢測方法。進一步利用IFC 檢測花粉經25–45℃溫度處理15 min 後之活力變化，結果顯示 ‘TN11’ 之花粉活力隨著溫度上升明顯下降；而 ‘N22’ 在溫度上升至35℃時才開始下降，顯示 ‘N22’ 花粉對於高溫的耐受性較 ‘TN11’ 為高。 Rice is the major staple food in Taiwan, however, detrimental effects on rice grain yield and quality by the heat stress have been observed. Since anthesis is the most sensitive stage to high temperature in the rice life cycle, the viability of pollens under the heat stress will be an appropriate index for thermo-tolerance of the variety. In addition to establishing an in vitro pollen culture medium, a number of pollen staining methods as well as using an impedance flow cytometry (IFC) for pollen viability analysis were conducted in this study. Fresh pollens of a japonica rice cultivar ‘Tainan 11’ (‘TN11’) and a heat-tolerant aus type rice cultivar ‘Nagina 22’ (‘N22’) were used in all experiments. In order to establish a proper medium for pollen tube germination, the ultimate concentration of various important components including sucrose, boric acid and calcium nitrate was investigated in successive terms. The results showed that a medium containing 22.5% sucrose, 60 mg L-1 boric acid, and 200 mg L-1 calcium nitrate had the highest pollen tube germination rate of 76.0% in ‘N22’ and 78.7% in ‘TN11’. Three pollen staining methods including I2/KI, fluorescein diacetate (FDA), and 2,3,5-triphenyltetrazolium chloride (TTC) were compared in this study. The highest staining rate was obtained from I2/KI followed by FDA and the lowest rate was found from TTC. Three methods of in vitro pollen culture, FDA staining, and IFC analysis were investigated for pollen viability. Although no difference was found among these three methods on pollen viability in ‘TN11’, FDA resulted in a higher staining rate than the in vitro pollen culture in ‘N22’. However, there was no difference found on IFC with the other two methods in ‘N22’. It is concluded that IFC was not different from pollen staining or in vitro pollen culture in detecting pollen viability. Fresh pollens of the two rice cultivars were subjected to temperature from 25℃ to 45℃ for 15 min before analyzing by IFC. The results showed that pollen viability was significantly decreasing along with raising the temperature in ‘TN11’; however, no significant decrease was found until 35℃ , and no significant changes in pollen viability between 35℃ to 40℃ were found in ‘N22’. The results indicated that pollens of ‘N22’ had higher thermo-tolerance than that of ‘TN11’.