Cytometry and plant sciences: a personal retrospective.

The plant kingdom is divided into the lower plants (blue-green algae, green algae, the bryophytes consisting of mosses and liverworts, and the euglenaphytes) and the higher plants (which mainly comprise the vascular plants). Higher plants are grouped into the pteridophytes (ferns) and their relatives, and the two classifications of seed plants, the angiosperms and the gymnosperms. The former comprises the monocotyledons and the dicotyledons, and the latter comprises the conifers and cycads (1). In this retrospective, I have restricted my coverage to the seed plants. Plants differ fundamentally from animals in a few general ways, one of which is the almost universal presence of a cellulosic cell wall. This is coupled to a method of cell division that involves partitioning of the daughter nuclei after mitosis by a phragmoplast, which expands and elaborates to join the parental cell walls, thereby effecting cytokinesis. Consequently, the cell walls of daughter cells remain topologically continuous with one another. Given that the entire mature form of a plant is achieved through regulated cell division and cell expansion, all cell walls can be viewed as a single continuum, termed the apoplast. The concept of flow cytometry and cell sorting arose in the late 1960s and 1970s from the study of natural single-cell suspensions, particularly those of the hematopoietic system. At first glance, these technologies did not seem applicable to higher plants, comprising complex three-dimensional tissue architectures of interlinked cells. In fact, only in the last few weeks did I become aware of what appears to be the first report, in German, of the use of flow cytometry for analysis of fluorescence signals from higher plant nuclei prepared from fixed tissues (1a), which involved use of ISAC member Wolfgang Gohde’s flow cytometer (2). My interest in flow cytometry and cell sorting started in 1976, when I took up a NATO postdoctoral fellowship at Stanford University. I was working in the laboratory of Dr. Peter Ray in the Department of Biological Sciences, about 300 yards away from the medical school. One of the topical interests of plant biologists at that time was the production from plant tissues of single-cell suspensions (termed protoplasts, prepared by enzymatic hydrolysis and solubilization of the cell wall) and their use for somatic hybridization. Kao and coworkers at Saskatoon had described the use of polyethylene glycol for the induction of high-frequency fusion of protoplasts (3), an observation that translated to animal cells and became a major technical basis for the emerging hybridoma technology. One persistent problem for plant biologists interested in somatic cell fusion was how to recognize the two parental sets of protoplasts that would be employed as fusion partners. Genetic mutants and transgenic lines resistant to various chemicals were not at that time available. In part of my graduate work at Cambridge, I had explored the use of generating antibodies directed against plant protoplasts with the hope of being able to find antibodies directed against plasma membrane proteins. At Stanford I became aware that Len Herzenberg had developed an interesting machine that was capable of recognizing and sorting cells based on surface fluorescence. I remember walking over to his laboratory to see a version of the fluorescenceactivated cell sorter (I think a FACS IV) installed there. I recognized the possibilities offered by the instrument, but also realized that it would be necessary to devise means to label specific plant cells by using fluorescent tags before the fluorescence-activated cell sorter could be used for sorting hybrid protoplasts. There also was a variety of technical questions that would require solving, including how to accommodate protoplasts having diameters close to, or in some cases greater than, that of the flow tips. To label protoplasts, it would be necessary to find two pairs of fluorochromes that had distinct absorption and emission spectra. Keller et al. (4) in 1977 had described the synthesis of lipid-linked derivatives of fluorescein and rhodamine. I and my colleagues synthesized these molecules and found that they could be used to prelabel cell cultures from which fluorescent protoplasts could subsequently be produced (5). Alternatively, we found it was possible to label protoplasts after preparation by using fluorescein isothiocyanate and rhodamine isothiocyanate (6), which are required for protoplasts prepared from tissues other than suspension cultures. It therefore seemed feasible that flow cytometry and cell sorting might be used for heterokaryon identification and cell sorting for their purification (7,8). For the work to be successfully accomplished, of course, we would need a flow sorter. In 1979, I put together a multi-user equipment proposal to the National Science Foundation requesting funds to purchase a cell