Flow cytometric analyses of somatic and pollen nuclei in midday flowers (Aizoaceae)

Endoreduplication (continuous DNA replication without chromosomal or cellular division) is known to be extraordinarily pronounced in a number of succulent plants, including midday flower species. Cytological investigations in different genotypes of the Aizoaceae genera <i>Lampranthus</i> and <i>Delosperma</i> were performed with three objectives: characterization of endoreduplication in several plant organs; determination of absolute DNA contents (pg/2C); and screening for unreduced male gametes. Flow cytometric determinations of absolute DNA contents using internal standards were carried out on nuclei extracted from pollen tubes. Further analyses of pollen nuclei were intended to verify the occurrence of unreduced gametes in the two genera. In contrast to the majority of angiosperms, in which endoreduplication is often restricted to certain tissues or cell types, the investigated succulents primarily consisted of cells with elevated DNA contents. Absolute DNA contents ranged from 1.6 to 2.36 pg/2C in <i>Lampranthus</i> and from 1.18 to 3.68 pg/2C in <i>Delosperma</i>. In flow cytometric analyses of pollen nuclei, signals corresponding to 1C, 2C and 3C DNA levels were detected. Microscopic observations of sample suspensions revealed that, instead of unreduced gametes, pairs of sperm nuclei and intact male germ units (MGUs) were the primary cause for the detection of signals higher than 1C. Thus, flow cytometric pollen nuclei analysis turned out to be a valuable tool to determine cell DNA contents in endocycling plants, but entails the risk of false-positive results in screenings for unreduced gametes due to persistent nuclei associations.核内复制（Endoreduplication，即不发生染色体或细胞分裂的持续性DNA复制过程）在许多多肉植物中表现尤为显著，其中包括日中花类群。本研究针对番杏科（Aizoaceae）Lampranthus属与Delosperma属的不同基因型开展细胞学分析，设定三大研究目标：一是对多种植物器官中的核内复制现象进行表征；二是测定绝对DNA含量（pg/2C）；三是筛选不减数雄配子（unreduced male gametes）。本研究利用内参标准品，通过流式细胞术（Flow cytometric）对花粉管提取的细胞核进行绝对DNA含量测定。后续针对花粉细胞核的分析，旨在验证这两个属中是否存在不减数雄配子。与多数被子植物的核内复制通常仅局限于特定组织或细胞类型不同，本次研究的多肉植物主要由DNA含量升高的细胞构成。Lampranthus属的绝对DNA含量范围为1.6~2.36 pg/2C，Delosperma属则为1.18~3.68 pg/2C。在花粉细胞核的流式细胞术分析中，检测到了对应1C、2C及3C DNA水平的信号。对样本悬浮液的显微观察结果显示，导致检测到高于1C信号的主要原因并非不减数雄配子，而是成对的精子细胞核与完整的雄性生殖单位（male germ units, MGUs）。由此可见，流式细胞术花粉细胞核分析虽可作为测定发生核内复制的植物细胞DNA含量的有效工具，但由于细胞核关联现象持续存在，该方法在筛选不减数雄配子时存在产生假阳性结果的风险。