Flow Cytometric Analysis of Ploidy in Rhododendron (subgenus Tsutsusi)

Ploidy level was determined for six species and 88 cultivars of the Rhododendron subgenus Tsutsusi. High-resolution flow cytometry of nuclear DNA was performed on macerated plant tissue. All plants analyzed were diploid (2n = 26) with the exception of ‘Euratom’, ‘Euratom Orange’, and ‘Red Wing’, which were triploid (3n = 39), and ‘Casablanca Tetra’, which was found to be a cytochimera: mixoploid (2n + 4n) in the LI and LII, but tetraploid in the LIII. The described method has proven to be useful in screening a large population of rhododendrons. Analysis of different organs and plant tissues was easily accomplished through flow cytometry, and has proven useful in determining the ploidy of different histogenic layers. The genus Rhododendron, consisting of ≈800 species, belongs to the heather family (Ericaceae), and contains eight subgenera (Chamberlain and Rae, 1990; Cullen, 1991). The subgenus Tsutsusi comprises 66 species, with spring leaves semi-evergreen and summer leaves persistent. The many hybrids are generally known as evergreen azaleas (Cox and Cox, 1997). Huge numbers are grown as pot plants for home decoration, making it one of Belgium’s most popular ornamentals. Within the evergreen azaleas, four more or less well-defined groups of cultivated azaleas are distinguished: Belgian pot azaleas, Hirado, Kurume, and Satsuki azaleas. Belgian pot azaleas have been created from a relatively narrow genetic base of collector’s material, from botanical gardens and private collections brought from the Far East. Rhododendron simsii Planch. subgenus Tsutsusi, the species accepted as the primary ancestor, originates from hilly areas in China (Chang Jiang Valley), Thailand, Laos, and Myanmar. Apart Table 1. Cultigens of Rhododendron subjected to flow cytometry. R. simsii Doctor Heimann Julia Pink Dream R. indicum Donatine Kingfisher Professor Wolters R. scabrum Dorothy Gish Kirin Red Wing R. mucronatum Eclaireur Knut Erwen Reinhilde R. kiusianum Elsa Kaerger Kosmos Reinhold Ambrosius R. noriakianum Eric Danneberg Lara Rex Ademurasaki Directeur Van Slycken Laura Ashley Roi Leopold Ambrosiana Etoile de Belgique Leopold Astrid Rosali Adventsglocke Euratom Marianne Haerig Sachsenstern Anja Euratom Orange Mevr. A. Heungens Sayonara Aline Flamenco Mevr. E. Troch Schuman Apollo Friedhelm Scherrer Mme Bourlard Sima Avenir Gerda Keessen Wit Mme P.B. Van Acker Solitaire Aquarell Gilbert Mullaie Mme Petrick Spreeperle Bertina Gilles Mistral Stewartstownean Blue Danube Glaser Nummer 10 Niobe Tamira Carmen Gloire de St. Georges Nordlicht Tempérance Casablanca Tetra Heide Hanisch Omurasaki Terra Nova Christine Heiwa-no-Hikari Ostalett Vervaeneana Coelestine Hellmut Vogel Otto Weinachtsblume Comtesse de Kerkhove Herrmann’s Superba Palestrina Werner Muckel Concinna Hexe Panfilia Phoenix Cyriel Buysse Inga Paul Schaeme Doberlug James Belton Perle de Swijnaerde Triploid cultivars (3n = 39) of the Rhododendron collection of the Dept. of Plant Genetics and Breeding. Cytochimera: mixoploid (2n/4n) in the LI and LII histogenic layer; tetraploid (4n = 52) in the LIII histogenic layer. from R. simsii, at least three other species from the Tsutsusi subgenus [R. indicum (L.) Sweet, R. mucronatum (Blume) G. Don and R. scabrum G. Don], from Southeast Asia and Japan, may have contributed (Heursel, 1999). The efficiency of a breeding program is strongly enhanced by the knowledge of the ploidy level of the population. Taxonomists use nuclear DNA content to help determine the (phylogenetic) evolution of plant species. Estimations of nuclear genome sizes are also important for the mapping of plant genomes and the development of strategies for isolation of plant genes (Arumuganathan and Earle, 1991a). Conventionally, microscopic chromosome counting and Feulgen scanning microspectrophotometry, using meristematic mitotic cells, have been conducted to determine chromosome number in plants (Bennet and Smith, 1976; Price, 1988; Price et al., 1980). However, these methods are time-consuming and laborious. Nuclear DNA content of rhododendrons is difficult to estimate with these techniques because of the small chromosome size in Rhododendron (McAllister, 1993). Furthermore, chromosome counts can be conducted only during a short, difficult-to-predict period of time during the growing season (Heursel, personal communication). Flow cytometric analysis offers a valuable and rapid alternative to traditional chromosome counts. Flow cytometry involves the analysis of fluorescence and light-scattering properties of single particles during their passage within a narrow, precisely defined, liquid stream (Dolezel, 1991). Heller (1973) was the first to use this technique for DNA analysis in plant cells. The technique is now widely used for many plant species (Arumuganathan and Earle, 1991b; Baert et al., 1992; Bennet and Leitch, 1995; Dolezel, 1997; Galbraith, 1990). Compared with conventional chromosome counting, the main advantages of flow cytometry are its accuracy, convenience, simplicity, low cost, and rapidity (De Laat et al., 1987; Dolezel, 1997; Galbraith, 1990; Galbraith et al., 1983). All previous ploidy analyses of the genus Rhododendron have employed conventional chromosome counting (Heursel and De Roo, 1981; Janaki Ammal et al., 1950; McAllister, 1993; Pryor and Frazier, 1970). The basic diploid chromosome number in Rhododendron is 26. Some lepidote species of the genus are highly polyploid, whereas the elepidote species are mostly diploid (Janaki Ammal, 1950). The objective of this research was to assess the ploidy level of the Tsutsusi collection maintained by the Dept. of Plant Genetics and Breeding at Melle, Belgium. Therefore, 88 Tsutsusi cultivars and six Tsutsusi species (R. mucronatum, R. scabrum, R. indicum, R. simsii, R. kiusianum Makino, and R. noriakianum Suzuki), hereafter collectively referred to as cultigens, were subjected to flow cytometric ploidy analysis. Materials and Methods Plant material. Of the 300 cultigens of Rhododendron present in the collection of the Dept. of Plant Genetics and Breeding, six Tsutsusi species and 88 cultivars were analyzed (Table 1). Preparation of suspensions of nuclei. Generally, analyses were performed on young leaves obtained from plants grown in the greenhouse. For some cultigens, flower petals and root tips were also analyzed. Measurements were performed on nuclei isolated from 5-mm discs of leaf tissue and each cultigen was analyzed in triplicate. Because of the clonal nature of asexually propagated azaleas, plants were randomly harvested within each cultivar. The leaf discs were chopped with a sharp razor blade at room temperature, essentially according to Galbraith et al. (1983). Because of the woody nature of Rhododendron, two different procedures were tested to optimize the preparation of nuclear suspensions. First, leaf discs were chopped in 400 μL of solution containing 0.1 M citric acid and 0.5% Tween-20 (pH ≈2.5) (Otto, 1990). The samples were then passed through a 100μm nylon filter. The residue on the filter was washed with 700 μL of a solution containing 0.4 M Na2HPO4 and 2 mg·L of 4 ́, 6-diamidino2-phenylindole (DAPI) (pH ≈8.5) (Otto, 1990). The nuclear suspension was then analyzed. Alternatively, samples were macerated with the LB01 buffer (Dolezel et al. 1989), containing 15 mM Tris-Cl (pH 7.5), 2 mM disodium ethylenedinitrilo tetraacetic acid (EDTA), 0.5 mM spermine tetrahydrochloride, 80 mM KCl, 20 mM NaCl, and 0.1% Triton X-100. The subsequent filtration and wash steps of the extraction method were as described by Dolezel et al. (1989). Flow cytometric analysis. For flow cytometry, the PARTEC Cell Analyser II (Partec, Münster, Germany) was used, equipped with the DPAC software (Data Pool Application for CA-II; Partec, Münster, Germany). After filtration, the nuclear suspensions were passed through the flow chamber, which was filled with a sheath fluid (deionized water). The nuclei traversed the focus of an intense beam of light, produced by the highpressure mercury vapor damp lamp. At a wavelength of 365 nm, the nuclei, stained with DAPI, fluoresce. The excitation light is collected by a lens and converted to pulses of electric current by a photomultiplier. The electronic signals are then digitized and the binary data are stored as one-dimensional histograms (256 channel positions). The fluorescence inFig. 1. Histograms of relative DNA content obtained after flow cytometric analysis of nuclei isolated from (A) leaf tissue of the diploid azalea ’Nordlicht’ (channel number 50), and (B) petals of the triploid azalea cultivar Euratom (channel number 75). tensity is correlated directly with the amount of DNA that is stained with DAPI. Flow cytometry is a relative method of analysis. To calibrate the output, the first sample introduced in the flow cytometer is the external standard, which, in this study, was the azalea cultivar Nordlicht, a confirmed diploid (Heursel and De Roo, 1981). The voltage of the photomultiplier was adjusted in order to fix the signal of the diploid standard at channel position 50. During the course of the flow cytometric measurements of cultigens of unknown ploidy, the external standard was analyzed every 12 samples. This enabled us to determine if peak positions, i.e., diploid azalea signals at channel position 50, triploid and tetraploid signals at positions 75 and 100, respectively, were stable. Results and Discussion The objective of introducing flow cytometry in Rhododendron is to be able to analyze quickly large numbers of samples. Because of the woody nature of Rhododendron, the preparation of nuclear suspensions is of critical importance in the flow cytometric procedure. The quality of the histograms (Fig. 1) produced following lysis in 0.1 M citric acid/0.5% Tween-20 or with the LB01 buffer (Dolezel et al. 1989) was comparable, and the CV was <5%. Because the first procedure is less laborintensive, all further experiments were conducted in this way. Standard flow cytometry of diploid tissue resulted in a histogram with two peak regions. The first region consists of cells in the G0/G1 phase of the cell cycle corresponding to the 2C DNA content. The second, smaller peak, at twice the channel position of the 2C peak, consists of cells in G2 or M phases. Cells between the two peaks are at some point in the S phase. The surface area of the peaks is correlated with the number of cells that excite at the same fluorescence intensity, thus containing the same amount of DNA. Fig.1 shows the histogram of a leaf sample of a diploid azalea genotype. All plants included in this study were determined to be diploid (2n = 26), with the exception of four genotypes: ‘Euratom’, ‘Euratom Orange’, ‘Red Wing’, and ‘Casablanca Tetra’. Flow cytometric analysis of roots, leaves, and petals revealed that ‘Euratom’, ‘Euratom Orange’, and ‘Red Wing’ were triploid (3n = 39), with a peak at position 75, which is in agreement with earlier reports (Heursel and De Roo, 1981; Pryor and Frazier, 1970) (Fig. 1). The cultivars Euratom and Red Wing arose from a cross of ‘Hexe’ x ‘Apollo’ and ‘Hexe’ x ‘Willem van Oranje’, respectively. ‘Hexe’ and ‘Apollo’ are diploid. ‘Willem van Oranje’ is no longer available and there are no reports on its ploidy level. However, the parents of ‘Willem Van Oranje’ are ‘Mme Petrick’ and ‘Apollo’, both of which were found to be diploid. This suggests that ‘Willem Van Oranje’ is probably a diploid as well. The origin of the triploid state of ‘Euratom’ and ‘Red Wing’ remains uncertain. As both cultivars are descendants of ‘Hexe’, ‘Hexe’ may produce unreduced gametes (Heursel and De Roo, 1981; Pryor and Frazier, 1970).