The study used the tetrazolium reduction method to quantitatively determine phloem–cambium cell viability in Eucalyptus. Circular sections of bark with underlying phloem and cambium were cut from mature E. obliqua and samples ranging in mass from 1 to 30 mg were exposed for 1 min to temperature treatments ranging from 20 to 85 °C and kept for 20–22 h at room temperature in 0.8% 2,3,5-triphenyl tetrazolium chloride (TTC) to test for cell viability. The 1,3,5-triphenyl tetrazolium formazan (TPF) formed was cold extracted with ethanol and quantified as absorbance at 485 nm.
The TTC reduction method reliably quantified a decline in cell viability with rising temperature in tissue sections that included vascular cambium, and identified 60 °C as the critical temperature for cambium–phloem cells of Eucalyptus species. Cell viability, calculated as [TPF Treatment °C]/[TPF 20 °C], declined by 90% between 20 and 85 °C. The cell viability results confirmed that significant tissue necrosis occurred in Eucalyptus at temperatures between 50 and 70 °C, after 1 min of in vitro tissue heating. The decline in cell viability with increasing temperature shown by the TTC method was consistent with an independently derived count of live cells following temperature treatment and neutral red staining.
摘要：
桉树树干维管形成层和次生韧皮部热致细胞坏死的定量试验
桉树(Eucalyptus)树干暴露在森林火灾辐射热中会杀死形成层细胞及其内嵌的再生分生组织，阻止树木萌枝和恢复。目前尚无组织水平的方法来量化热处理对桉树形成层细胞活力的影响。本研究的目 标包括：(1)采用并验证四氮唑还原法检测桉树细胞活力；(2)应用该方法确定斜叶桉(Eucalyptus oblique)形成层细胞活力的阈值水平，进而确定临界温度。采用四氮唑还原法量化该桉树韧皮部-形成层细胞活力。 从斜叶桉成树上切下带有形成层和韧皮部的圆形树皮切片，将质量为1-30 mg不等的样品在20–85°C 的温 度处理中放置1分钟，并在室温条件下在0.8%的2,3,5-氯化三苯基四氮唑(TTC)中保存20–22小时以检测 细胞活力。用乙醇冷萃取得到1,3,5-三苯基四唑甲臜(TPF)，在485 nm处测定吸光度。结果表明，TTC还原 法准确地量化了组织切片(包括维管形成层)中细胞活力随温度升高而下降的情况，并确定60°C 为桉树物种形成层-韧皮部细胞的临界温度。细胞活力按[TPF 处理温度]/[TPF 20°C]计算，在20-85°C之间下降90%。细胞活力结果证实，在50-70°C的温度区间，经过1分钟体外组织加热，桉树的组织坏死显著 增加。TTC 方法显示细胞活力随温度升高而下降，这与温度处理和中性红染色处理后独立获得的活细胞计 数一致。
关键词:
细胞活力,
临界温度,
桉树,
中性红,
2,3,5-氯化三苯基四氮唑
