Application‐based guidelines for best practices in plant flow cytometry

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.

Biological invasions are a major ecological and socio-economic problem in many parts of the world. Despite an explosion of research in recent decades, much remains to be understood about why some species become invasive whereas others do not. Recently, polyploidy (whole genome duplication) has been proposed as an important determinant of invasiveness in plants. Genome duplication has played a major role in plant evolution and can drastically alter a plant's genetic make-up, morphology, physiology and ecology within only one or a few generations. This may allow some polyploids to succeed in strongly fluctuating environments and/or effectively colonize new habitats and, thus, increase their potential to be invasive. We synthesize current knowledge on the importance of polyploidy for the invasion (i.e. spread) of introduced plants. We first aim to elucidate general mechanisms that are involved in the success of polyploid plants and translate this to that of plant invaders. Secondly, we provide an overview of ploidal levels in selected invasive alien plants and explain how ploidy might have contributed to their success. Polyploidy can be an important factor in species invasion success through a combination of (1) 'pre-adaptation', whereby polyploid lineages are predisposed to conditions in the new range and, therefore, have higher survival rates and fitness in the earliest establishment phase; and (2) the possibility for subsequent adaptation due to a larger genetic diversity that may assist the 'evolution of invasiveness'. Alternatively, polyploidization may play an important role by (3) restoring sexual reproduction following hybridization or, conversely, (4) asexual reproduction in the absence of suitable mates. We, therefore, encourage invasion biologists to incorporate assessments of ploidy in their studies of invasive alien species.

Flow cytometry (FCM) using DNA-selective fluorochromes is now the prevailing method for the measurement of nuclear DNA content in plants. Ease of sample preparation and high sample throughput make it generally better suited than other methods such as Feulgen densitometry to estimate genome size, level of generative polyploidy, nuclear replication state and endopolyploidy (polysomaty). Here we present four protocols for sample preparation (suspensions of intact cell nuclei) and describe the analysis of nuclear DNA amounts using FCM. We consider the chemicals and equipment necessary, the measurement process, data analysis, and describe the most frequent problems encountered with plant material such as the interference of secondary metabolites. The purpose and requirement of internal and external standardization are discussed. The importance of using a correct terminology for DNA amounts and genome size is underlined, and its basic principles are explained.