曼地亚红豆杉‘Hicksii’花粉活力检测条件优化和适宜储藏温度分析

Detection condition optimization of pollen viability of Taxus media 'Hicksii' and analysis on its suitable storage temperature

采用单因素实验设计对适宜于曼地亚红豆杉‘Hicksii’(Taxus media‘Hicksii’)花粉活力检测的TTC染色法和离体培养法的实验条件进行了选择,并采用优化条件研究了在25℃～-196℃条件下储藏13周花粉活力的变化。结果表明:采用TTC染色法测定的花粉活力均高于离体培养法。在TTC染色法的3个影响因素(染液pH、TTC浓度和染色温度)中,染液pH对检测结果有极显著影响,而染色温度和TTC浓度则无显著影响,但温度对染色速率有影响。在离体培养法的3个影响因素(培养基中蔗糖添加量、H3BO3和Ca(NO3)2浓度及培养温度)中,蔗糖添加量对检测结果有极显著影响,在含质量体积分数15%蔗糖的培养基上花粉活力最高,而在含质量体积分数20%和25%蔗糖的培养基上花粉均不能萌发;在含100和200 mg.L-1H3BO3的培养基中添加200 mg.L-1Ca(NO3)2均能显著提高花粉活力;培养温度对花粉萌发速率有影响但对花粉活力没有明显影响。TTC染色法的最优检测条件为:用5.0 g.L-1TTC染液(pH 7.0)在35℃下染色2.0 h;离体培养法的最优检测条件为:用含质量体积分数15%蔗糖、100 mg.L-1H3BO3和200 mg.L-1Ca(NO3)2的培养基暗培养4 d。在25℃、4℃、0℃、-20℃、-80℃和-196℃条件下储藏13周,‘Hicksii’花粉活力和保持时间有明显差异,其中,于-80℃和-196℃储藏3 d花粉就丧失活力;于25℃和-20℃储藏7周、4℃储藏10周,花粉仍有一定的活力;而在0℃条件下花粉活力最高,且储藏11周花粉仍有活力。推测曼地亚红豆杉‘Hicksii’花粉对低温的抗性较差,0℃为其适宜的储藏温度。

By single-factor experiment design, experimental conditions culture methods suitable for pollen viability detection of Taxus media of TTC ＇ Hicksii ＇ staining and in vitro were selected, and changes of ＇ Hicksii＇ pollen viability stored at conditions from 25 ℃ to -196 ℃ for thirteen weeks were detected by optimal conditions. The results show that the detection result of pollen viability by TYC staining method is higher than that by in vitro culture method. Among three influence factors （staining solution pH, TFC concentration and staining temperature） of TYC staining method, staining solution pH has an extremely significant influence on detection result, while staining temperature and TFC concentration have no significant influence but the former has an effect on staining speed. Among three influence factors （ sucrose addition, concentrations of H3 BO3 and Ca （ NO3 ） 2 in medium and culture temperature） of in vitro culture method, sucrose addition has an extremely significant influence on detection result. The pollen viability is the highest in medium containing 15% （mass-volume ratio） sucrose, while all of pollens can not germinate in media containing 20% or 25% （mass-volume ratio）sucrose. Addition of 200 mg . L-1 Ca （ NO3 ） 2 in media containing 100 or 200 mg . L-1 H3 BO3 can obviously improve pollen viability, and culture temperature has an effect on pollen germination speed but no obvious effect on pollen viability. The optimal detection condition of TYC staining method is taking 5.0 g . L-l TFC solution （ pH 7.0）, dyeing at 35 ℃ for 2.0 h. The optimal detection condition of in vitro culture method is using cuhure medium H3BO3 and 200 mg . L-1 Ca（ NO3 ）2, taking containing 15% （mass-volume ratio） sucrose, 100 mg. L-1 dark culture for 4 d. Stored at 25 ℃, 4 ℃, 0 ℃, -20 ℃, -80 ℃and -196 ℃ for thirteen weeks, pollen viability of ＇ Hicksii＇ and its retain time have significant differences. In which, pollens lost viability stored at -80 ℃ or -196 ℃for 3 d, pollens still have a certain viability stored at 25 ℃ or -20 ℃ for seven weeks and at 4 ℃ for ten weeks, while pollen viability is the highest stored at 0 ℃ and pollen still has a certain viability after stored at 0 ℃ for eleven weeks. It is conjectured that the resistance of pollen of T. media ＇ Hicksii＇ to low temperature is weak, and its suitable storage temperature is 0 ℃.