Suda Jan, Trávnícek Pavel
Department of Botany, Charles University, Benátská 2, Prague, Czech Republic.
Cytometry A. 2006 Apr;69(4):273-80. doi: 10.1002/cyto.a.20253.
Only fresh plant material is generally used for rapid DNA ploidy estimation by flow cytometry (FCM). This requirement, however, substantially limits convenient FCM application in plant biosystematics, population biology, and ecology. As desiccation is a routine way for sample preservation in field botany, potential utilization of dehydrated tissues of vascular plants in FCM research was examined.
Standard DAPI protocol was employed to evaluate the performance of 60 air-dried species, spanning more than 100-fold range of nuclear DNA amounts. Multiploid Vaccinium subg. Oxycoccus was selected as model taxon for detailed investigation and cytotype comparison.
A majority of analyzed plants yielded distinct peaks with reasonable coefficients of variation after several months of storage at room temperature. Fluorescence intensity of nuclei isolated from desiccated tissues was highly comparable with that for fresh material, allowing reliable DNA ploidy estimation. Deep-freezer preservation substantially extended Vaccinium samples lifetime (at least to 3 years) and maintained high histogram resolution.
The introduced approach eliminates the need for fresh material in many vascular plants and thus opens new prospects for plant FCM. Convenient cytotype investigation in field research and retrospective ploidy determination in already herbarized samples are among the principal advantages.
流式细胞术（FCM）快速估计DNA倍性通常仅使用新鲜植物材料。然而，这一要求极大地限制了FCM在植物生物系统学、种群生物学和生态学中的便捷应用。由于干燥是野外植物学中样本保存的常规方法，因此研究了维管植物脱水组织在FCM研究中的潜在用途。
采用标准DAPI方案评估60种风干植物的性能，这些植物的核DNA含量范围超过100倍。选择多倍体酸果蔓亚属作为详细研究和细胞型比较的模式分类群。
大多数分析的植物在室温下储存几个月后产生了具有合理变异系数的明显峰值。从干燥组织中分离的细胞核的荧光强度与新鲜材料的荧光强度高度可比，从而可以可靠地估计DNA倍性。超低温保存大大延长了酸果蔓样本的寿命（至少3年），并保持了高直方图分辨率。
引入的方法消除了许多维管植物对新鲜材料的需求，从而为植物FCM开辟了新的前景。野外研究中方便的细胞型调查和已制成标本的样本中的回顾性倍性测定是主要优点之一。
