Ascari Lorenzo, Cristofori Valerio, Macrì Federico, Botta Roberto, Silvestri Cristian, De Gregorio Tommaso, Huerta Eloy Suarez, Di Berardino Marco, Kaufmann Silvan, Siniscalco Consolata
Department of Life Sciences and Systems Biology, University of Turin, Turin, Italy.
Department of Agriculture and Forest Sciences, University of Tuscia, Viterbo, Italy.
Front Plant Sci. 2020 Dec 8;11:615922. doi: 10.3389/fpls.2020.615922. eCollection 2020.
Impedance flow cytometry (IFC) is a versatile lab-on-chip technology which enables fast and label-free analysis of pollen grains in various plant species, promising new research possibilities in agriculture and plant breeding. Hazelnut is a monoecious, anemophilous species, exhibiting sporophytic self-incompatibility. Its pollen is dispersed by wind in midwinter when temperatures are still low and relative humidity is usually high. Previous research found that hazelnut can be characterized by high degrees of pollen sterility following a reciprocal chromosome translocation occurring in some cultivated genotypes. In this study, IFC was used for the first time to characterize hazelnut pollen biology. IFC was validated via dye exclusion in microscopy and employed to (i) follow pollen hydration over time to define the best pre-hydration treatment for pollen viability evaluation; (ii) test hazelnut pollen viability and sterility on 33 cultivars grown in a collection field located in central Italy, and two wild hazelnuts. The accessions were also characterized by their amount and distribution of catkins in the tree canopy. Pollen sterility rate greatly varied among hazelnut accessions, with one main group of highly sterile cultivars and a second group, comprising wild genotypes and the remaining cultivars, producing good quality pollen. The results support the hypothesis of recurring reciprocal translocation events in cultivars, leading to the observed gametic semi-sterility. The measured hazelnut pollen viability was also strongly influenced by pollen hydration ( = 0.83, ≤ 0.0001) and reached its maximum at around 6 h of pre-hydration in humid chambers. Viable and dead pollen were best discriminated at around the same time of pollen pre-hydration, suggesting that high humidity levels are required for hazelnut pollen to maintain its functionality. Altogether, our results detail the value of impedance flow cytometry for high throughput phenotyping of hazelnut pollen. Further research is required to clarify the causes of pollen sterility in hazelnut, to confirm the role of reciprocal chromosome translocations and to investigate its effects on plant productivity.
阻抗流式细胞术（IFC）是一种多功能的芯片实验室技术，能够对多种植物物种的花粉粒进行快速且无标记分析，为农业和植物育种带来了新的研究可能性。榛树是雌雄同株、风媒传粉的物种，表现出孢子体自交不亲和性。其花粉在仲冬时节气温仍较低且相对湿度通常较高时通过风力传播。先前的研究发现，一些栽培基因型中发生的相互染色体易位会导致榛树花粉高度不育。在本研究中，首次使用阻抗流式细胞术来表征榛树花粉生物学特性。通过显微镜下的染料排除法对阻抗流式细胞术进行了验证，并用于：（i）随时间跟踪花粉水合作用，以确定用于花粉活力评估的最佳预水合处理方法；（ii）对种植在意大利中部一个收集田中的33个品种以及两种野生榛树的榛树花粉活力和不育性进行测试。这些种质还通过其在树冠层中柔荑花序的数量和分布来表征。榛树种质间的花粉不育率差异很大，有一组主要是高度不育的品种，另一组包括野生基因型和其余品种，能产生优质花粉。结果支持了品种中反复发生相互易位事件的假说，导致观察到的配子半不育现象。测得的榛树花粉活力也受到花粉水合作用的强烈影响（r = 0.83，P ≤ 0.0001），在湿度箱中预水合约6小时时达到最大值。在花粉预水合的大致相同时间，活花粉和死花粉能得到最佳区分，这表明榛树花粉需要高湿度水平来维持其功能。总之，我们的结果详细阐述了阻抗流式细胞术在榛树花粉高通量表型分析中的价值。需要进一步研究以阐明榛树花粉不育的原因，确认相互染色体易位的作用，并研究其对植物生产力的影响。
