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 (1), 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 fluorescence-activated 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 sorter, with the major purpose being for the sorting heterokaryons for production of somatic hybrid plants. The grant was approved later that year, and I started the necessary homework to find out what instruments were available for the $126,000 amount of the award. Three manufacturers were producing suitable instruments: Coulter, Becton-Dickinson, and Ortho. Ortho took themselves out of the running early on, based on cost, and we chose Coulter based on the availability of a their new model, the EPICS V. It turned out we were to get the seventh instrument off the production line. The EPICS V was delivered around September 1980. Gary Durack was the Coulter field service representative at the time, and remembers calling Coulter to find out how to program the MDADS. Unfortunately, the software had not been completed at that time, so we were loaned a one-parameter pulse height analyzer, which at least enabled us to learn the basics of flow cytometry. The fact that I was forced into single-parameter analyses over this initial period had the unforeseen and, in retrospect, highly productive effect of forcing our attention onto one-dimensional analysis of the plant cell cycle. I had already been interested in the idea of analyzing the plant cell cycle, based on work that we had started to investigate the behavior of tobacco leaf protoplasts placed in culture. Over a period of 2 days or so, the protoplasts initiated cell wall formation and entered into the cell division cycle, producing clusters of undifferentiated cells. By using Hoechst 33258 staining of fixed protoplasts and quantitative measurement of nuclear fluorescence with a jury-rigged photomultiplier attached to a fluorescence microscope, I was able to follow the onset of DNA synthesis and found that leaf protoplasts initiated the cell cycle within about 30 h and, after resynthesizing a cell wall, entered into cell division (9). Measurements made in this way were inaccurate and time consuming, so, with the availability of the flow cytometer, we started to examine fluorescence emission from fixed protoplasts. The EPICS was equipped with a 5-W multiline argon laser and separate optics for ultraviolet light, which were inconvenient to switch out. Because mithramycin could be excited at 457 nm, mithramycin had been successfully employed in combination with other dyes (ethidium bromide or propidium iodide) for animal cell cycle analyses, and plant cells generally lacked pigments absorbing and emitting in this part of the spectrum, we chose this DNA-specific fluorochrome for our work. We were able to establish rather quickly that fixed tobacco leaf protoplasts produce a readily distinguishable signal representing the nuclear DNA fluorescence, with a reasonable coefficient of variation of approximately 7%. Immediately before our work, ISAC member Awtar Krishan (10) had described the use of hypotonic citrate for mammalian cell lysis before flow cytometric cell cycle analysis, and Christensen et al. (11) had introduced the use of detergents, so we also examined the suitability of similar approaches using plant protoplasts. The eventual composition of the eventual lysis buffer included 45 mM MgCl2 (required for mithramycin binding), 30 mM sodium citrate, 0.1% Triton X-100, and 20 mM MOPS for buffering at pH 7. Freshly prepared protoplasts treated with this lysis buffer and stained with mithramycin produced DNA histograms of very high quality, with coefficients of variation for the G1 peak at approximately 2–3%. Using this methodology, we then went back to analysis of tobacco protoplasts during the 2-day period of culture, with the additional use of 2,6-dichlorobenzonitrile as an inhibitor of cell wall formation. 2,6-Dichlorobenzonitrile acts at the level of cellulose synthase by preventing formation of a coherent cellulose cell wall. It has little or no effect on re-initiation of the cell cycle by leaf protoplasts in culture (12). To release free nuclei from the cultured protoplasts, it was important to prevent cell wall formation around the protoplasts. Having charted this process to our satisfaction, we sent a manuscript to Plant Physiology describing the method of plant cell cycle analysis using flow cytometry, a first of its type, and the use of this method for the analysis of the initial stages of leaf protoplast development in culture. The paper came back with the major criticism that our method was not general to plant tissues, i.e, we could not ensure that protoplasts would be released from all cells that were present in the tissue of interest. To address this criticism, I realized that it was not necessary to make protoplasts at all—nuclei could be released from plant tissues simply by homogenization, assuming the process of homogenization was sufficiently gentle. We devised such a method by using single-edged razor blades and manual chopping. Each slice of the razor blade has the effect of cutting open the cells, thereby releasing the nuclei into suspension, along with the remaining cellular organelles and a variety of other forms of debris. Large material is then removed with nylon filters, and the clarified homogenate is then stained with mithramycin and run through the flow cytometer. Armed with a couple of razor blades, over a couple of hours my technician at the time (Kristi Harkins) and I reduced a large number of plant species within the teaching greenhouse to homogenates, and we were able to measure genome sizes for most of them by flow cytometry, including chicken red blood cells as an internal standard. The resultant publication describing the method, which appeared in Science in 1983 (13), has since been cited more than 415 times. We did not at all appreciate the impact of this method would have on basic and applied plant biology and agriculture. Before this time, analyses of ploidy, genome size, and the cell cycle involved some kind of light microscopy requiring counting of chromosomes or was based on quantitative microspectrophotometry using Feulgen staining. These techniques were very time consuming and were comparatively inaccurate and less sensitive. Now, flow cytometric methods are used routinely for all of these measurements and forms the major methodologic underpinning of the searchable database of plant nuclear DNA contents established at the Royal Botanical Gardens at Kew (http://www.rbgkew.org.uk/cval/homepage.html). The ease of sampling means that large populations can be routinely analyzed, and we extended the method to the analysis of haploids in tissue culture (14), of natural variation in cytotype distributions (15), and for addressing issues in angiosperm evolution (16). Flow cytometry also can be used for quality control monitoring of the ploidy of commercial seeds and of different germ plasm accessions, for analysis of novel crosses and identification of wide-hybrids, and for monitoring euploidy in plants emerging from protoplast fusion, tissue culture, and genetic engineering procedures; for a complete discussion, see Galbraith et al. (17). Our laboratory and other investigators since 1983 have extended the scope of ploidy-based measurements, based on their high accuracy, to include the use of different fluorochromes (18, 19) and analysis of systemic and tissue-specific endoreduplication (20-25). The contributions of ISAC members Dick Kowles and Friedrich Srienc in the identification of endoreduplication in maize endosperm are particularly noteworthy, as are those of Spencer and in the flow methods to large of plant species ISAC member was involved in early flow analyses of Flow cytometry also has been used for the identification of and for a see Galbraith et al. (17). of it can be used for of in plants of plant genome sizes using flow cytometry is more than that of ploidy, it use of internal having DNA analysis of this was developed by ISAC member Spencer using various plant analysis of plant genome sizes also that the method of staining be to composition the of ISAC member Spencer at did work in this In of analysis of the cell cycle, the DNA histograms produced by flow cytometry can be used for of cell cycle and early work from ISAC members Spencer and and their to development of cytometric methods in this (18, including of nuclear and DNA contents with the particularly interesting of of nuclei as a of cell cycle and analysis of the of the cell cycle In of the contributions of and ISAC member Wolfgang have been particularly their development of flow has the of methods of plant nuclear DNA analysis into the laboratory and It be that the method produce within which the of the comprise a This is rather different than the in flow analysis of animal cell suspensions, within which the of interest comprise the of the of by the flow cytometer. For flow this field for the first time, instrument for of the plant nuclei within can be It be that the method the plant also produce DNA histograms from and from mammalian tissues and The DNA within of course, is into In with plant chromosomes in general not staining when using fluorochromes with different For the chromosomes are also and of similar a of has been made in the development of techniques for flow analysis and sorting of plant plants with such as that had chromosomes of sizes such as the chromosomes in or that were to in cell culture ISAC member Spencer was involved in early to chromosomes and in the development of for the in of the flow for different species ISAC member was one of the first to sorting from the and maize and ISAC member flow in similar work by using chromosomes ISAC members and and their coworkers have made some of the in this through methods for of large of chromosomes from of including and and for flow sorting of and in some cases pairs of chromosomes For some this the use of specific that have the effect of producing within which all pairs are of different sizes and therefore can be Alternatively, in with can be used to chromosomes can be prepared from the chromosomes and coworkers have been methods for for specific to the chromosomes and for preparation of large Our grant proposal for purchase of the EPICS V in had as its purpose the sorting of protoplasts for of somatic hybrid plants. For this to be it was necessary that protoplasts through the flow cytometer. flow at that time were in a problem for protoplasts for the species and we were using in our work, in flow from Coulter, and then and to our the process of formation by the and a of for sorting large These include in the and in the jury-rigged to the to of the of of to accommodate the large of by the large flow that could be used for and as My to my that protoplasts could through the flow cytometer, could be and could be in culture back into plants ISAC member and coworkers described using as a means to a problem with large such as maize or mammalian cell The idea this was the fluorescent of the two parental protoplast populations with distinguishable fluorescent These protoplasts would then be by flow cytometry, and the heterokaryons to the presence of The method was not with methods of leaf protoplast but we found that simply the protoplasts during protoplast preparation with of fluorescein isothiocyanate and rhodamine isothiocyanate to the production of protoplasts that could be readily the fluorescence microscope, and that these could be by the flow cytometer. It was then a of protoplast fusion and the cell sorter to and the heterokaryons sets of These were subsequently into plants and were as somatic at the level The methods described were general in all that is required are pairs of protoplasts that can be into plants. ISAC member and coworkers flow analysis and sorting of heterokaryons work from including contributions from ISAC members and the general of flow sorting for of heterokaryons of the of flow sorting for somatic in the have for see and In of the commercial development of a highly means for formation by using large flow has the that the plant for sorting large cells and cell The produced by Becton-Dickinson, particularly The of that I have for heterokaryons identification and sorting are or with the plant cells to fluorescent tags for protoplasts. We found that fluorescent could also be used for protoplast analysis and within the cells of tissues themselves which is highly fluorescent in the red and can be excited with a variety of laser We found that flow analysis of could be used to measure the of within protoplasts and to protoplast diameters based on time of We then used these to flow protoplasts from different cell and and to cell of ISAC member has the development of flow cytometric methods for analysis of and cell in plants. This work protoplasts for analysis of the presence of of nuclear DNA through propidium of to the of the plasma membrane through V and the of within DNA through analysis has up with ISAC member for flow cytometric analyses of species and of membrane to the of the in protoplast by Spencer and were the first to use flow cytometry for analysis of the fluorescence of protoplasts of cell cultures of with the idea of of protoplast sorting for the of cultures producing high of these important and also devised flow cytometric methods to examine the of protoplast plasma to the my has put into the development of that could be with techniques to plant cell of interest. These included the production of antibodies directed against plasma membrane analysis of the behavior of mammalian plasma membrane as the within plants and, the and of the green fluorescent of the fluorescent particularly for flow cytometric analysis a means for cells organelles that can be cell or tissue based on that is regulated by and This means that one can use flow sorting to protoplasts in a cell which can then be used for analyses, such as using et and or of this of is that the process of protoplast production not the measurements that are subsequently to be In this appears to be the the used for protoplast production and the of cells and the plasma membrane and cell wall of cells are that cellular it is not possible to protoplasts from all cell For this we have also the cell of nuclei by using with the idea of using flow analysis and sorting of fluorescent nuclei within for of This which is in the on the flow methods for nuclear genome analysis described and the for the method of homogenization and nuclear sorting is and can be on flow analysis and sorting of nuclei are than those of protoplasts to the of the variety of organelles found in higher plant cells, only the are to the presence of ISAC member did some of the first work in the flow analysis of and was able to and prepared from and maize by using and light and fluorescence emission ISAC member took this work by using and light and measurements to the of and, in combination with with fluorescein to from and from various membrane ISAC member was involved in the of the fluorescence signals produced by Flow analysis and sorting methods have been developed since for purification of from and cells of maize based on in fluorescence spectra. which are generally grouped with of the of can be using flow cytometry, as described by ISAC member Friedrich Srienc and his coworkers Flow analysis of which are in their natural use of fluorochromes or through in of was the first to the use of for the of at the surface and rhodamine to membrane and by of and and in to with work by ISAC member on the of in was The fluorescence in its natural We have found that can be to nuclei of various higher and lower plant species by using a nuclear signal from an tobacco This of as a to the of the the nuclear signal that of the of nuclear The fluorescent nuclei can be readily and in of transgenic tobacco plants in nuclear of the transgenic are achieved by to and We have developed methods for analysis of within nuclei Alternatively, nuclear can be used for production of fluorescent that can be to Given the to produce transgenic plants within specific cell such as cells and cells it be possible to the of and all plant cell We are that these methods are applicable to animal cells. Given the in the number of that has over the it is to ensure that one is in all contributions of ISAC members to the plant flow over that period has been most in the of ISAC I have restricted to in by the of Science and for For more and for in the field of plant cytometry, the is to the For specific the The plant kingdom in general has interested a of ISAC since most ISAC have had and more or less on plants, including Plants have the to the development of of flow cytometry and cell sorting other than those that I have For of I have not to include lower plants in this but it is that the development of a flow cytometric monitoring by ISAC member monitoring of the different and found in the In of within the plant the of flow cytometry and cell sorting in and of cytometry in general only to These techniques means to and populations of cells and of our attention on the different cells of plants and of the within these different cells, the of these techniques to development of the field no to I my of the work of the members of the Galbraith and which the achieved over the would not have been of this work was by from the and this is

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