Cytogenetic Characterization of Zinnia Species and Cultivars
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Floriculture and Ornamental Biotechnology ©2007 Global Science Books Cytogenetic Characterization of Zinnia Species and Cultivars Jackrit Anantasaran1 • Max B. Schröder2** • Klaus Eimert2 • Kamnoon Kanchanapoom1* 1 Department of Biology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112, Thailand 2 Department of Botany, The Geisenheim Research Center, Geisenheim, Germany Corresponding author : * [email protected], ** [email protected] ABSTRACT Zinnia is a tropical ornamental plant, and its cultivar classification is complicated. The typical characters such as height, flower size and leaf shape are of a quantitative nature, and under strong environmental control. In some cases, unequivocal determination of cultivars may be difficult using morphological features only. So in this study, cytological, cytogenetical and molecular biological characteristics of Zinnia have been analyzed. The DNA content of Zinnia angustifolia (cv. ‘Starbright’), Zinnia elegans (cv. ‘Short Stuff’, ‘Dreamland’, ‘Jupiter’, ‘Border Beauty’, ‘Jungle’, ‘Piccolo’, ‘Sinnita’), Zinnia haageana (cv. ‘Chippendale Daisy’, ‘Persian Carpet’) and the F1 hybrid cv. ‘Profusion’ (Z. elegans x Z. angustifolia) has been analyzed by flow cytometry, and compared to morphological features (guard cell size, number of chloroplasts per guard cell, number of chromosomes). The three analyzed species Z. angustifolia, Z. haageana and Z. elegans could be clearly distinguished by their DNA content. The nuclear DNA content ranged from 1 pg (2C) in Z. angustifolia cv. ‘Starbright’ up to 5 pg in the tetraploid Z. elegans cv. ‘Jungle’. Genetic diversity among and between Zinnia varieties was accessed by RAPD, confirming a higher genetic identity in Z. elegans cv. ‘Dreamland’ when compared to Z. haageana cv. ‘Persian Carpet’. _____________________________________________________________________________________________________________ Keywords: cytogenetics, flow cytometry, RAPD, Zinnia spp. INTRODUCTION were grown in the greenhouse under natural conditions. Zinnia is a beautiful ornamental plant. Novel flower colors Flow cytometry of this plant are of great commercial and aesthetic value. Many new cultivars are created either by hybridization (cv. Nuclei were isolated from fresh, young leaves (of Zinnia species ‘Profusion’, Z. elegans x Z. angustifolia) or by X-ray muta- or cultivars, and of Raphanus sativus at comparable developmen- genesis (Venkatachalam 1991). Zinnias are used as ethno- tal stages) by chopping with a sharp razor blade, and stained for botanical plant in some regions of North America, e.g. Z. flow cytometry with either propidium iodide (PI, CyStain® PI Ab- angustifolia as a poison, and Z. elegans as an anodynic and solute P, PARTEC GmbH, Münster, Germany) or 4, 6-diamidino- menstrual medicinal herb. Interestingly, zinnia is a good 2-phenylindole (DAPI, CyStain® UV Precise P, PARTEC GmbH, model system to study the in vitro interactions of different Münster, Germany). In every sample a minimum of 10,000 nuclei cell types and the consequences for commitment to a par- was analyzed with a Partec PAS flow cytometer (Partec GmbH, ticular cell fate such as tracheary elements (TE) (Pesquet et Münster, Germany). The coefficient of variation (CV) was esti- al. 2006). mated with the WinMDI software (Version 2.0.4); only samples The identification of cultivars or breeding lines is very with a CV <5% were accepted. important in all horticultural and agricultural species in PI staining: Young leaves (about 1 cm2) were chopped in 400 μl order to protect the rights of plant breeders (Wolff et al. nuclei extraction buffer at room temperature. The nuclei suspension 1995). In Zinnia cultivars are usually identified by flower- was filtered through a 42-μm nylon filter, and 1.6 ml of staining ing trials as well as by morphological characteristics (e.g. buffer (containing 100 μg PI) and 3 μl RNase were added. flower shape and size, leaf and growth morphology). How- DAPI staining: Nuclei were extracted and stained using the ever, those characteristics may be affected by environ- high resolution DNA Precise P kit (PARTEC). Young leaves mental factors. To overcome these problems, in the present (about 1 cm2) were chopped in solution A of the kit. 0.5 ml of 1% study we analyzed Zinnia species and cultivars on the geno- PVP was added. The nuclei suspension was filtered through a 42- typic level (DNA content, DNA sequence). μm nylon filter, and 1.5 ml of solution B was added. Two samples of 5 plants per cultivar were analyzed. The fol- MATERIALS AND METHODS lowing species/cultivars were used as internal standard: Raphanus sativus cv. ‘Saxa’ for DNA content estimation (PI staining), and Plant materials comparative analyses of Z. angustifolia, Z. elegans cv. ‘Dream- land’ and the hybrid ‘Profusion’ (DAPI staining); the Z. angusti- Seeds of all plants analyzed in the present study were provided by folia cv. ‘Starbright’ and for comparative analyses of the DNA the AFM Flower Seed Co., Ltd. (Chiang Mai). The following 11 content in cultivars of Z. elegans (DAPI staining). Further on, cultivars were studied: Z. angustifolia cv. ‘Starbright’; Z. haage- intraspecific variation in cultivars of Z. elegans was studied by ana cvs. ‘Persian Carpet’, ‘Chippendale Daisy’; Z. elegans cvs. mixing samples in the following order ‘Border’/‘Dreamland’, ‘Short Stuff’, ‘Dreamland', ‘Jupiter’, ‘Border Beauty’, ‘Jungle’, ‘Dreamland’/‘Giant’, ‘Giant’/‘Sinnita’, ‘Sinnita’/‘Jupiter’, ‘Jupi- ‘Giant’, ‘Sinnita’; and a hybrid (Z. elegans x Z. angustifolia) cv. ter’/‘Short stuff’. ‘Profusion’. Raphanus sativus cv. Saxa seeds used as internal standard for flow cytometry were kindly provided by Dr. Jaroslav Dolezel, Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, Olomouc, Czech Republic and Received: 23 April, 2007. Accepted: 15 August, 2007. Original Research Paper Floriculture and Ornamental Biotechnology 1(2), 125-130 ©2007 Global Science Books Size of guard cells and number of chloroplasts RESULTS Small parts of the lower epidermis of young leaves were manually The DNA content (2C) of the species and cultivars of the removed, placed in a drop of water on a microscopic slide genus Zinnia varies between 1.00 to 4.94 pg (Table 1). The following the method as described by Dorè (1986), and measured flow cytometric analyses could clearly show that there is a under a light microscope (DM 5000 B, LEICA, Wetzlar, Germany). significant difference between the DNA content of Z. an- The number of chloroplasts per guard cell was counted under a gustifolia, Z. haageana and Z. elegans. No significant dif- fluorescence microscope (DM 5000 B, LEICA, Wetzlar, Germany). ference could be found between the cultivars of Z. elegans Four leaves per two plants per cultivar were analyzed. (Fig. 1) with only one exception; cv. ‘Jungle’ has about two times the amount of DNA of the other analyzed Z. elegans Chromosome counting cultivars. By analyzing mixed samples of two different cul- tivars in Z. elegans we could show that the DNA contents in For chromosome counting root tips from greenhouse plants were this group are identical; the histograms always consist of used. The root tips were pre-treated for 4 hours in saturated PDB one peak only (Fig. 2A shows the mixed sample of the cul- (para-dichlorobenzene), and then fixed 24 hours in 1:3 glacial ace- tivars ‘Dreamland’/‘Giant’ only; the other combinations are tic acids: 95% ethanol. After a passage through 75% ethanol the identical, and therefore, they are not shown). The DNA root tips were hydrolyzed in 1 N HCl for 15 min at 60°C, and content of the interspecific hybrid ‘Profusion’ represents ap- stained with Feulgen solution for 4 hours, squashed in aceto-car- proximately the sum of the DNA contents of both its par- mine, and analyzed under a light microscope. Five cells per culti- ents (Z. angustifolia and Z. elegans, Fig. 2B). var were analyzed. The species and cultivars analyzed by flow cytometry have also been studied with regards to the size of their DNA extraction guard cells of the lower leaf epidermis, and the number of chloroplasts per guard cell as well (Fig. 3; Table 2). Chro- Total DNA from leaves of young Zinnia seedlings was isolated mosomes were counted in the three Zinnias as well as in the following a modified method used for Asparagus officinalis (Eim- putative tetraploid Z. elegans cv. ‘Jungle’ (Table 3). ert et al. 2003). Two volumes of DNA extraction buffer containing Five primers, comprising UBC 65, 87, 88, 89 and 95, 50 mM Tris-HCl (pH 8.0), 312.5 mM NaCl, 20 mM EDTA (pH 8), were selected for RAPD analysis. The rational behind the 1% sarkosine and 7 M urea were added to 100-500 mg leave tis- choice is explained in the discussion. A typical RAPD pro- sues, and ground gently at room temperature in a mortar with a file is shown (Fig. 4). The percentage of polymorphic loci pestle. The homogenate was centrifuged for 10 min at 12,500 rpm of Z. elegans cv. ‘Dreamland’ is 41.84 and of Z. haageana at room temperature in order to remove tissue fragments. The cv. ‘Persian Carpet’ is 65.31. Thus, diversity within the pop- supernatant was extracted twice with phenol: chloroform: isoamyl ulation of Z. haageana cv. ‘Persian Carpet’ was higher than alcohol (v/v 25:24:1). Half volume of 5 M NaCl was added to the within the population of Z. elegans cv. ‘Dreamland’ (Fig. 5). supernatant and mixed. Two volumes of 95% ethanol were added and mixed gently. The homogenate was centrifuged for 10 min at DISCUSSION 12,500 rpm at 4°C. DNA was washed with 70% ethanol and air- dried. Finally, the DNA was dissolved in 50 μl of TE. The DNA Zinnia has been used as a model system for analyzing isolated from each cultivar was run in a 1.3% agarose gel to check tracheary element differentiation as well as for analyzing the quality and was also scanned in a spectrophotometer and ab- programmed cell death as a part of xylem differentiation sorbances (A) were noted at 260 and 280.