Morphological and anatomical characterisation of chemically induced polyploids in Spathiphyllum wallisii

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1 Morphological and anatomical characterisation of chemically induced polyploids in Spathiphyllum wallisii Ives Vanstechelman 1,2, Hein Vansteenkiste 3, Tom Eeckhaut 1,*, Johan Van Huylenbroeck 1 and Marie-Christine Van Labeke 2,4 1 Institute for Agricultural and Fisheries Research (ILVO), Plant Unit, Applied Genetics and Breeding, Caritasstraat 21, 9090 Melle, Belgium 2 Ghent University, Department of Plant Production, Coupure links 653, 9000 Gent, Belgium 3 Research Center for Ornamental Plants (PCS), Schaessestraat 18, 9070 Destelbergen, Belgium 4 Faculty Biosciences and Landscape Architecture, University College Ghent - Ghent University Association, Voskenslaan 270, 9000 Gent, Belgium tom.eeckhaut@ilvo.vlaanderen.be Keywords: cell, flow cytometry, nucleus, microscopy, stomata Abstract Tetraploids were induced in Spathiphyllum wallisii Regel (2n = 2x = 30) through in vitro application of mitosis inhibitors. Tetraploids were compared to the original diploid control plants. Polyploidization had a significant effect on plant anatomy and morphology. The stomatal area of diploids was smaller compared to the tetraploid plants. The leaf angle was smaller in diploids. The stomatal length and width, leaf thickness and angle and thickness of the spathum were positively correlated to the higher ploidy level. On the other hand, stomatal density, length/width ratio of leaf, spathum and spadix, number of shoots and leafs and length of the flower stalk decreased in tetraploids compared to the corresponding diploid controls. The leaf number of diploid plants was higher compared to tetraploids. Altogether, this study quantified the extended morphological changes of chromosome doubling in our model crop Spathiphyllum wallisii. INTRODUCTION In plant breeding, polyploidy is often a yearned trait because it is associated with enhanced vigor, altered morphology, increased sterility, higher pest or disease tolerance, and hybrid fertility restoring (Stebbins, 1971). Possible morphological consequences include larger and heavily textured flowers, delayed and/or prolonged flowering, increased width/length ratio of leaves, thicker leaves and stems and a more compact growth habit; however, their appearance is influenced by heterozygosity, gene interactions, gene dose effects, and epigenetic phenomena (Leitch and Bennett, 1997). Given these possible beneficial consequences, chemical polyploidization has become an almost routinely applied breeding technique in many ornamentals, also in Araceae (Cohen and Yao, 1996; Eeckhaut et al., 2004). These publications aim to optimize induction parameters to obtain the highest possible number of solid tetraploids. Ploidy chimeras are also frequently found, however, they are in most cases not interesting for applications in plant breeding. Plant chimeras were discarded in this particular research. 1

2 The monocotyledonous ornamental crop Spathiphyllum wallisii Regel (2n = 2x = 30 according to Marchant, 1973) belongs to the family of the Araceae. It can be easily propagated in vitro (Fonnesbech and Fonnesbech, 1979). In recent years, protocols for the induction of anther filament embryogenesis (Werbrouck et al., 2000), gynogenesis (Eeckhaut et al., 2001), and chemical polyploidization (Eeckhaut et al., 2004) have been established. Especially tetraploids were efficiently produced by several mitosis inhibitors on various tissues. Polyploidy has a lot of consequences for plant morphology. Polyploidy has an important consequence for cell and nuclear size. Polyploid plants have larger nuclear and cell sizes compared to diploid plants (Jovtchev et al., 2006; Kondorosi et al., 2000; Sugiyama, 2005). As a result of the bigger cells in polyploid plants, plant morphology shows a bigger and more compact habitus. Polyploidy increases leaf size mainly by increasing the cell elongation rate (Sugiyama, 2005). Chromosome doubling is also accompanied by increased thickness and a darker green coloration of leaves. The length/width ratio of leaves also decreases as a consequence of the higher ploidy level and the internode length differs in plants with different ploidy levels (Kermani et al., 2003). Our objectives were (1) to induce tetraploid derivatives of diploid Spathiphyllum wallisii genotypes through the action of mitosis-arresting chemicals and subsequent flow cytometric selection and (2) to study morphological and anatomical effects of polyploidization in the model crop Spathiphyllum wallisii. MATERIAL AND METHODS Plant Material, General in vitro Practices and Greenhouse Conditions Stock cultures of 5 Spathiphyllum wallisii Regel genotypes were provided by 2 different companies. The basal medium for Spathiphyllum micropropagation was based on MS (Murashige and Skoog, 1962) macrosalts and enriched with Nitsch (Nitsch and Nitsch, 1969) microsalts, 555 µm myo-inositol, 230 µm NaFeEDTA, 0.89 µm thiamine HCl, 167 µm sucrose and 7 g/l agarose (ph 5.8). For multiplication, µm BA (N6- benzyladenin) was added. All media were autoclaved (121 C, 30 min, 500 hpa). Melijars (De Proft et al., 1985) contained 100 ml medium/jar. Cultures were maintained at 23 ± 2 C under a 16 h photoperiod at 40 µmol m -2 s -1 photosynthetic active radiation, supplied by cool white fluorescent lamps (OSRAM L36W/31). For morphological characterization, plants were acclimatized and grown in the greenhouse for 6 months. Chemical Polyploidization and Flow Cytometric Ploidy Measurements Chemical polyploidization was done according to Eeckhaut et al. (2004). Afterwards, each individual shoot was screened by flow cytometry using a Partec PAS III (De Schepper et al., 2001). Detected tetraploid or mixoploid shoots were multiplied on multiplication medium and tetraploids were preserved for this research. Twelve weeks later, flow cytometric evaluation of the newly formed shoots was performed again to confirm the stability of the induced tetraploids. Anatomical and Morphological Characterization 1. Microscopical Analysis. All samples were observed under a Leica DM IRB microscope equipped with a fluorescence module and a DAPI filter (excitation 355 nm). Digital recordings and computer aided measurements were performed with a Leica Image Manager 500 camera and corresponding software. 2

3 2. Stomatal Analysis. Epidermal strips were taken from diploid and derived tetraploid Spathiphyllum wallisii genotypes after 7 weeks of growth in the greenhouse. The youngest fully formed leaf was sampled 10-fold. Nail polish was applied on the lower epidermis, pulled off with cellotape after drying and applied on a microscopical glass slide. The stomatal dimensions (length, width and area) were measured in 5 replications per stomatal strip. The relative area of the stomata was calculated as the area of an ellipse (Relative area = *a*b). Stomatal density was measured by counting the number of stomata on 25 randomly chosen microscopic fields under a 20x objective. Statistical power analysis was done for determining the minimum number (N min ) of microscopic fields to consider. The formula for N min was calculated according to (N min = *s 2 ; with = the value for (25-1) measures of freedom with p = 0.05; X = the mean number of stomata counted on the 25 microscopic fields; s = standard deviation). N min is the number of stomatal fields required to analyze to obtain a confidence interval of 95%. An N min value of 32 was calculated. The number of stomata was counted on 32 microscopic fields per genotype. 3. Leaf Cell Size Analysis. Leaves of in vitro plants were dissected from the diploid and tetraploid Spathiphyllum genotypes. Leaves were fixed overnight in a fixation fluid containing 18:1:1 70% ethanol:glacial acetic acid:formaldehyde. In successive dehydration steps in a mixture of 70% ethanol and butanol, ethanol was gradually replaced by butanol. Five dehydration baths were used during at least 8 h, namely 16:3:1 ethanol: butanol: water, 13:7 ethanol: butanol, 9:11 ethanol:butanol, 1:3 ethanol:butanol and butanol. Afterwards, the samples were embedded in liquid paraffin. The samples were stained with safranin and FastGreen according to Johansen (1940). The samples were fixed in Canada balsam and covered with a cover slide. Cell analysis was done by microscopically measuring the cell area. 4. Epidermal Peelings And Analysis Of Epidermal Nuclei. The protocol for epidermal peelings was adapted from Boudolf et al. (2004). Leaves of diploid and tetraploid Spathiphyllum plants were dissected and fixed in Carnoy fluid (ethanol 95 % and glacial acetic acid, 3:1) for 2 h at room temperature and stored in 70 % ethanol at 4 C. Fixed tissue was first soaked in water and then in 0.5 M EDTA (ph 8). Leaves were placed with the abaxial side on a microscopic glass slide and held in place with a forceps. All adherent tissues were scraped with a fine scalpel. A drop of DAPI at a concentration of mg/ml in McIlvaine s buffer, ph 4.1 was placed on the epidermal peeling. Peelings were mounted in vectashield mounting medium (Vector laboratories, Burlingame, CA) and the nuclear area was measured. 5. Analysis Of Vegetative Parameters. Vegetative parameters were measured after 7 weeks and after 6 months of growth in the greenhouse. After 7 weeks, the length/width ratio of the leaves, the angle of the leaf basis and the leaf thickness were measured in 10 replications. For the length/width ratio, image analysis using Image J was used ( Leaf thickness was measured with a digital vernier caliper. The same parameters were measured after 6 months of growth in the glasshouse. The actual number of leaves and shoots was also counted. 6. Analysis Of Generative Parameters. Different parameters determining flower morphology were measured. The angle of the flower basis was measured with a protractor. The length of the flower stalk, length/width ratio of the spathum and spadix, 3

4 thickness of the spathum and thickness of the flower stalk, at 10 cm behind the spathum, were measured. Statistical Analysis All data were statistically analyzed by a T-test (P < 0.05) using SPSS 16. For each parameter, a minimum of 20 measurements/cultivar were performed. RESULTS AND DISCUSSION All results are summarized in Table 1. Since there was no significant interaction between cultivar and ploidy level, the mean results of 5 diploid genotypes were compared to the mean results of their 5 tetraploid counterparts. Microscopic cell analysis showed that cell areas in tetraploid Spathiphyllum plants are significantly larger than in diploid Spathiphyllum plants. This is in accordance with literature data that show that polyploid plants have larger cells than diploid plants (Kondorosi et al., 2000; Jovtchev et al., 2006). Diploid and tetraploid cells could be clearly distinguished by significant differences between their respective cell nuclei areas, as tetraploid nuclei were significantly larger. This is also demonstrated by Jovtchev et al. (2006). The efficient use of epidermal peelings to determine nuclear DNA levels is consistent with earlier experiments on Arabidopsis (Boudolf et al., 2004). Stomatal analysis showed that the stomatal density in diploid plants is larger than those of tetraploid plants, which is owing to the larger cells in the tetraploid plants. The stomatal length, width and area were also significantly larger compared to the diploid ancestral Spathiphyllum plants. Morphological differences are visualized in Fig. 1. Adult plants after 6 months of growth in the greenhouse showed a more homogeneous growth than plants with 7 weeks of growth in vivo. Leaves of tetraploid plants had a smaller length/width ratio. This phenomenon is also described by Kermani et al. (2003). The angle of the leaf basis and the leaf thickness were also significantly larger in tetraploids compared to diploids. Two other vegetative morphological parameters were only analyzed in plants that grew 6 months in the greenhouse. The number of leaves and the number of shoots were significantly larger in the diploid ancestral plants compared to their tetraploid counterparts. Measurements on the generative part of the plant showed that the length of the flower stalk was not significantly different between diploid and tetraploid plants. However, the flower stalk thickness was significantly larger in diploid plants. The spathum and the spadix length/width ratio were significantly smaller in the tetraploids. The spathum is significantly wider in tetraploids compared to their diploid ancestral genotypes. In the present study, the morphological changes of chromosome doubling in Spathiphyllum wallisii. were investigated The typical anatomical and morphological differences of tetraploid plants are also demonstrated in our model crop, and quantified for the first time. In future work, the effect of ploidy level on stress resistance will be investigated. Acknowledgements The authors thank IWT-Flanders for their financial support, Deroose Plants and Floreac for kindly offering Spathiphyllum genotypes. The technical help of Pepijn De Raeymaecker, Ingrid Proven, Annemie Stocké and Johan Ongena is highly appreciated. Literature Cited Boudolf, V., Vlieghe, K., Beemster, G., Magyar, Z., Acosta, J., Maes, S., Van Der Schueren, E., Inzé, D. and De Veylder, L The Plant-Specific Cyclin-Dependent 4

5 Kinase CDKB1;1 and Transcription Factor E2Fa-DPa Control the Balance of Mitotically Dividing and Endoreduplicating Cells in Arabidopsis. Plant Cell 16: Cohen, D. and Yao, J In vitro chromosome doubling of nine Zantedeschia cultivars. Plant Cell Tissue Organ Cult. 47: De Proft, M., Maene, L. and Debergh, P Carbon dioxide and ethylene evolution in the culture atmosphere of Magnolia cultured in vitro. Physiol. Plant. 65: De Schepper, S., Leus, L., Mertens, M., Van Bockstaele, E., Debergh, P. and De Loose, M Flow cytometric analysis of ploidy in Rhododendron (subgenus Tsutsusi). HortScience 36: Eeckhaut, T., Werbrouck, S., Dendauw, J., Van Bockstaele, E. and Debergh, P Induction of homozygous Spathiphyllum wallisii genotypes through gynogenesis. Plant Cell Tissue Organ Cult. 67: Eeckhaut, T., Werbrouck, S., Leus, L., Van Bockstaele, E. and Debergh, P Chemically induced polyploidization of Spathiphyllum wallisii Regel through somatic embryogenesis. Plant Cell Tissue Organ Cult. 78: Fonnesbech, M. and A. Fonnesbech In vitro propagation of Spathiphyllum. Scientia Hort. 10: Johansen, D Plant Microtechnique. McGraw-Hill, New York and London. Jovtchev, G., Schubert, V., Meister, A., Barow, M. and Schubert, I Nuclear DNA content and nuclear and cell volume are positively correlated in angiosperms. Cytogenetics Genome Res. 114: Kermani, M. J., Sarasan, V., Roberts, A. V., Yokoya, K., Wentworth, J. and Sieber, V. K Oryzalin-induced chromosome doubling in Rosa and its effect on plant morphology and pollen viability. Theoretical Applied Genet. 107: Kondorosi, E., Roudier, F. and Gendreau, E Plant cell-size control: growing by ploidy? Current Opinions Plant Biol. 3: Leitch, I. and Bennett, M Polyploidy in angiosperms. Trends Plant Science 2: Marchant, C Chromosome variation in Araceae: V. Acoraceae to Lasieae. Kew Bulletins 28: Nitsch, J. and Nitsch, C Haploid plants from pollen grains. Science. 163: Stebbins, G Chromosomal evolution of higher plants. Edward Arnold Ltd, London, UK. Sugiyama, S-I Polyploidy and Cellular Mechanisms Changing Leaf Size: Comparison of Diploid and Autotetraploid Populations in Two Species of Lolium. Ann. Botany 96: Werbrouck, S., Eeckhaut, T. and Debergh, P Induction and conversion of somatic embryogenesis on the anther filament on Spathiphyllum Schott. Acta Hort.. 520: Figures 5

6 Fig 1. A diploid and a tetraploid Spathiphyllum wallisii plant after 6 months in vivo. Tables Table 1. Microscopical and morphological parameters in diploid and tetraploid Spathiphyllum wallisii measured after 7 weeks (7w) and 6 months (6m) of growth in the greenhouse. The data are means of 5 diploid and tetraploid genotypes, respectively. Diploid Tetraploid Microscopical parameters cell area (µm 2 ) * nuclear area (µm 2 ) * stomatal length (µm) * stomatal width (µm) * stomatal area (µm 2 ) * stomatal density (mm -2 ) * Morphological parameters (7w) length/width ratio leaf * angle of leaf basis ( ) * thickness leaf (mm) * Morphological parameters (6m) length/width ratio leaf * angle of leaf basis ( ) * thickness leaf (mm) * number of leaves * number of shoots * length of flower stalk (mm) flower stalk thickness (mm) * spathum length/width * spadix length/width * angle of spathum bases ( ) * * Diploids and tetraploids are significantly different (T-test; P < 0.05) 6