Effect of Colchicine Tablets on Morphology of Torenia fournieri

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1 2013 International Transaction Journal of Engineering, Management, & Applied Sciences & Technologies. International Transaction Journal of Engineering, Management, & Applied Sciences & Technologies Effect of Colchicine Tablets on Morphology of Torenia fournieri Sasiree Boonbongkarn a, Thunya Taychasinpitak a*, Shermarl Wongchaochant a, and Shinji Kikuchi b a Department of Horticulture, Faculty of Agriculture, Kasetsart University, Jatujak, Bangkok THAILAND b Graduate School of Horticulture, Chiba University, Chiba, JAPAN A R T I C L E I N F O A B S T R A C T Article history: Received 02 August 2013 Received in revised form 06 September 2013 Accepted 10 September 2013 Available online 12 September 2013 Keywords: Polyploid induction; Cromosome; Flow cytometry; Morphological characteristics; Cytological characteristics. The effects of colchicine tablets on Torenia fournieri were studied. Leaves were cut and soaked in different concentrations of colchicine solution: 0, 5, 10, 15 and 20 ppm for 0, 1, 2 and 3 days. The survival rate decreased when colchicine concentration and treatment duration were increased. The stomata length was found to be greater in the putative polyploids. Flow cytometric analysis demonstrated that the nuclear DNA of putative polyploid Torenia plants was doubled relative to that of control diploid plants, and microscopy results confirmed that the chromosome number of the tetraploid plants was 2n = 4x = 36. The highest frequency of tetraploid induction was 6.67% at 15 ppm of colchicine solution soaked for 3 days. Morphological characteristics of tetraploid and diploid plants were compared. The results showed that growth of tetraploid plants were less than diploid plants. Tetraploid plants also had larger leaves and flower sizes when compared with diploid plants INT TRANS J ENG MANAG SCI TECH.. 1. Introduction Torenia spp. are dicotyledons, belonging to the class Magnoliopsida, order Scrophulariales and family Scrophulariaceae, known as the figwort family. The Scrophulariaceae comprise 306 genera and approximately 5850 species native to South East Asia, Africa and Madagascar. Yamazaki (1985) reported a total of 50 Torenia species, 20 species from Cambodia, Laos and 299

2 Vietnam, and 19 species from Thailand. Other reports indicate 40 species of Torenia (Fischer 2004; Spencer 2006). Torenia spp. have an extended history of cultivation and safe use as an ornamental plant and are usually found in nurseries, home gardens and landscaping situations. Torenia have been grown as a front border plant especially in lightly shaded areas and are popularly grown in hanging baskets or as trailing specimens in patio planters (Starman, 2005). Flowers of Torenia fournieri are reportedly edible and can be used as salad material (Shindu et al., 2008). Torenia fournieri (2n = 18) has also been used to study the location and movement of chromosomes and their centromeres in the early stages of embryogenesis in interspecific hybrid plants (Kikuchi et al., 2005). Torenia is generally a diploid plant. The basic chromosome number varies with species. Tetraploids can be induced by colchicine treatment of young seedlings and have relatively large flowers, offering potential for developing better horticultural varieties. However, these tetraploids exhibit significant reduction in pollen viability, seed setting and unequal distribution of chromosomes at anaphase when compared to their diploid progenitors (Tandon & Bhutani, 1965). Inducing polyploidy is an effective means for the generation of innovative germplasm resources suitable for selective breeding. In general, polyploid plants have larger leaves and flowers, and autopolyploids tend to be less fertile. Previously unsuitable habitats can sometimes be better tolerated by polyploids, compared to the original diploids (Hancock, 1997). Polyploids are superior to diploids in terms of a higher genetic adaptability and improved levels of resistance or tolerance to environmental stresses (Shao et al., 2003). Colchicine treatment is the classical method to induce doubling of chromosome number. Jensen (1974) reported the techniques used for chromosome doubling of barley haploid with colchicine. Preliminary morphological screening for putative polyploids stated that stomata length was the accurate indicator of polyploidy level in many plants. Wright (1976) also showed that stomatal measurement was a quick way to determine whether or most of the leaves on a branch were polyploidy. The research objectives were to study the optimum concentration and duration of colchicine solution exposure for polyploid induction in Torenia fournieri and to study the effect of colchicine solution from colchicine tablets on morphological and cytological characteristics and chromosome number variation of Torenia fournieri. 2. Materials and Methods Purple-flowered native Thai Torenia (Torenia fournieri) with a semi-erect, 300 S. Boonbongkarn, T. Taychasinpitak, S. Wongchaochant, and S. Kikuchi

3 semi-recumbent habit was the subject for this research. Polyploidy was induced using colchicine from medicinal tablets for treating gout manufactured in Switzerland and purchased at a pharmacy in Thailand. Each tablet contains 0.6 mg colchicine along with lactose, magnesium stearate and starch. 2.1 Polyploidy Induction Leaves were cut from native Torenia fournieri and for the different treatments, the petioles were soaked in 0, 5, 10, 15 and 20 ppm colchicine solution for 0, 1, 2 and 3 days (30 leaves per treatment), after which they were placed upright in peat moss for rooting. Once roots were established, the leaf cuttings were transferred to 4-inch pots containing a mixture of sand, rice husk charcoal, coconut coir, coconut husk chips, and manure 1:1:1:1:1/2. Slow-release fertilizer of formula was added at the rate of 5 g per pot, and liquid fertilizer of formula was also provided once a week at the concentration of 30 g per 20 L of water. The survival rate was recorded weekly. 2.2 Selection of Putative Polyploids When the plants had grown for 90 days, putative polyploids were selected based on morphological characteristics, such as slower growth, darker green leaves, thicker, larger and rougher leaves, or larger flowers. The stomata size was examined by examining impressions of the abaxial sides of leaves under a light microscope. 2.3 Flow Cytometry One young leaf (mature but newly emerged) of Torenia fournieri for each specimen to be tested was chopped in a Petri dish with 500 µl of Partec CyStain (a one-step extraction and DAPI stain solution) and filtered through a 30 µm filter before being analyzed in a Partec PAII flow cytometer. 2.4 Karyotype by DAPI staining Chromosome preparation with flower bud and the use of FISH for meiotic chromosomes were performed according to the procedures described in Kikuchi et al., The slides were mounted with Vectashield (Vector) containing 5 mg/ml 4, 6-diamidino-2-phenylindole (DAPI) for staining of chromosomes. All images were captured with an Olympus BX61 fluorescence microscope equipped with a cooled-ccd camera (Photometrics CoolSNAP fx:roper Scientific) and processed using the Meta imaging series 5.0 software (Universal Imaging Corporation). 301

4 2.5 Comparison of Morphological and Cytological Characteristics of Diploid and Tetraploid Torenia Stem cuttings (about 7 cm long) were taken from 5 plants each of the diploid and confirmed tetraploid native Torenia and placed in peat moss for rooting. After the stem cuttings were established, they were transferred to 4-inch pots containing a mixture of sand, rice husk charcoal, coconut coir, coconut husk chips, and manure 1:1:1:1:1/2. Slow-release fertilizer of formula was added at the rate of 5 g per pot, and liquid fertilizer of formula was also provided once a week at the concentration of 30 g per 20 L of water. The experiment was a Completely Randomized Design (CRD) with 2 treatments (diploid and tetraploid) and 5 replicates. Growth characteristics were recorded, comprising plant height, spread, number of branches, stem thickness, leaf width, length and thickness and flower characteristics such as flower width and length and petal thickness. 2.6 Statistical Analysis Statistical differences among data were tested to 95% level confidence using Duncan s New Multiple Range Test (DMRT). 3. Results and Discussion 3.1 Survival Rate Following Colchicine Treatment After leaves were cut from native Torenia fournieri and the petioles/leaf stalks were soaked in 0, 5, 10, 15 or 20 ppm colchicine solution for 0, 1, 2 or 3 days, and then placed in peat moss for rooting, the survival rate was recorded after 90 days. The control group had the highest mean survival rate, and the survival rate dropped with increasing concentration of colchicine and increasing exposure time. The survival rate dropped to zero for the 20 ppm concentration and 3-day exposure time (Table 1). Report the survival rates of colchicine-treated shoots were lower, with the extent of the reduction depending on the colchicine concentration and treatment duration (Jiranapapan et al. 2011). This is consistent with the findings of Espino and Vazquez (1979), who reported that high concentrations of colchicine could cause tissue necrosis when parts of leaves of African violet were soaked in different concentrations of colchicine solution. This is because colchicine does not only have an effect on cell division but spreads throughout the cell, interfering with cellular mechanisms and causing toxicity at high concentrations (Dermen, 1940). Colchicine apparently affects the viscosity of the cytoplasm so the cell cannot function normally (Cook and Loudon, 1952). 302 S. Boonbongkarn, T. Taychasinpitak, S. Wongchaochant, and S. Kikuchi

5 3.2 Selection of Polyploids and Stomata Guard Cell Size When the leaf cuttings had grown for 90 days, putative polyploids were selected based on changes in morphological characteristics, such as slower growth, darker green leaves, thicker, larger and rougher leaves, or larger flowers. When the plants were 120 days old the stomata size was examined to select those plants with larger stomata than the control. The mean stomata guard cell length of the putative polyploid plants was 31.43±1.59 µm, which was longer than the mean of the control group (22.77±0.48 µm) (Table 6 and Figure 2, A and B). This was in accordance with the findings of Ye et al. (2010) who applied different concentrations of colchicine for different durations to the shoot tips of 3 varieties of crape myrtle sprouts during cotyledon stage ( Zi Wei, Hong Wei and Yin Wei varieties). They found that the morphological and cytological characteristics of large leaf size, greater leaf thickness, darker green leaves, larger stomata, lesser stomata density on the abaxial leaf surface, and more chloroplasts in the guard cells were all indicators that could predict the presence of tetraploids in the large colchicine-treated population. In another study on polyploidy induction in Torenia fournieri, colchicine solution at concentrations of 0.05, 0.10, 0.20% were applied to seed-grown seedlings with 4-6 leaves for a duration of 10 hours. Tetraploids were obtained from the 0.20% colchicine treatment, and they had characteristics that were deemed valuable for breed development, such as larger flowers, larger leaves, larger anthers and larger stomata (Tandon and Bhutani, 1965). Table 1: Survival rate of native Torenia fournieri leaf cuttings 90 days after planting (control and colchicine treatments) 3.3 DNA Volume Analysis by Flow Cytometry Flow cytometry was used to measure the DNA volume of putative polyploidy plants that were selected based on morphological characteristics, such as slower growth, darker green leaves, thicker and larger leaves and flowers, and larger stomata size. Flow cytometry was 303

6 carried out with a Partec PA II flow cytometer, and the histograms showed that the ploidy level of some of the colchicine-treated plants had increased from 2n, as in the control, to 4n or tetraploid (Figure 4). Flow cytometry is now routinely used for ploidy analysis and is regarded as the most accurate tool for ploidy determination (Loureiro et al., 2005). Advantages of using flow cytometry to estimate ploidy level include the ease of plant sample preparation, requiring just a few milligrams of tissue, and multiple samples can be analysed in one working day (Dolezel, 1997). 3.4 Chromosome Counting Cytological analysis revealed that the diploid native Torenia in the control group had the expected chromosome number of 2n = 2x = 18 (Figure 3 A) and the 7 tetraploid plants from the colchicine treatment had twice as many chromosomes 2n = 4x = 36 (Figure 3B). Previously reported screened the 14 regenerated plants with morphological characteristics that could indicate polyploidy In addition, we confirmed that the 14 regenerated plants carried 34 chromosomes Thus, our treatment produced 14 amphidiploid plants from the 600 detached leaves treated with colchicine (Jiranapapan et al. 2011). However colchicine is an antimitotic mutagen that has the property of disrupting normal cell division during mitosis by reacting with microtubule proteins and preventing the formation of spindle fibers, so chromosomes can not migrate to opposite poles of the cell during anaphase, resulting in tetraploids cells (Elliott, 1958). Table 2: Frequency of polyploids induced in native Torenia fournieri 90 days after colchicine treatment Colchicine concentration (ppm) Duration Total plants treated (plants) Tetraploids Tetraploid induction rate (%) (days) (plants) Frequency of Polyploidy Data on the number of tetraploid plants induced by colchicine treatments at the different concentrations and different exposure times tested is shown in Table 2. In this research, a total of 7 tetraploid plants were formed. The most effective treatments were 5 ppm colchicine for S. Boonbongkarn, T. Taychasinpitak, S. Wongchaochant, and S. Kikuchi

7 day and 15 ppm for 3 days, both of which yielded 2 tetraploids, or an induction rate of 6.67% (Table 2). The highest frequency (8%) of polyploidy induction was seen in the treatment with 15 µmol mol-1 colchicine for 2 days. (Jiranapapan et al. 2011). Figure 1 Comparison of diploid (left) and induced tetraploid Torenia fournieri (right): (A) leaves; (B) branches; and (C) flowers. 3.6 Morphological Comparison of Diploid and Tetraploid Native Torenia When stem cuttings were made from 5 plants each of the diploid and confirmed tetraploid native Torenia, the growth parameters of plant height, plant width, number of branches, stem thickness, leaf width, length and thickness were compared. After 90 days the mean height of the tetraploids plants was less than the diploids, but the mean plant width, leaf length and leaf thickness of the tetraploids were all greater than the diploids to a statistically significant degree. There was no statistically significant difference in mean number of branches, leaf width or stem thickness between the diploid and tetraploid plants (Table 3 and Figure 1A). Table 3: Comparison of growth parameters of 90-day-old stem cuttings of diploid and tetraploid Torenia Mean height Mean spread Mean number of branches Mean stem thickness Ploidy (cm) (cm) (branches) (cm) Diploid ± 2.08 a ± 2.17 a ± ± 0.11 Tetraploid ± 2.16 b ± 2.84 b ± ± 0.14 t-test * * ns ns Note ns means not statically significant * means statistically different to the confidence level of 95% by Duncan s New Multiple Range Test (DMRT) Visual comparison showed that the leaves of tetraploids plants from stem cuttings were darker green, larger and thicker than the diploid group (Table 4 and Figure 1B). As for the floral characteristics, at 90 days the flowers on tetraploids were wider than flowers on diploids to a statistically significant degree. There was no statistically significant difference in mean 305

8 flower length or petal thickness between the diploid and tetraploid plants (Table 5 and Figure 3C). This is the consistent with the results of Zhang et al (2010), who induced polyploidy in the diploid (2n = 24) Cucumis melo pure line M01-3 through the application of colchicine. They reported that the tetraploid melon plants were noticeably taller, with larger leaves and flowers as well as thicker stems. Tetraploids can be induced by colchicine treatment of young seedlings and have relatively large flowers offering potential for developing better horticultural varieties. However these tetraploids exhibit significant reduction in pollen viability, seed setting and unequal distribution of chromosomes at anaphase when compared to their diploid progenitors (Tandon & Bhutani 1965). Table 4 Comparison of mean leaf sizes of 90-day-old stem cuttings of diploid and tetraploid Torenia fournieri. Ploidy Mean leaf Mean leaf Mean leaf width length thickness (cm) (cm) (mm) Diploid 3.77 ± 0.15 b 0.31 ± 0.05 b 0.31 ± 0.04 Tetraploid 4.10 ± 0.13 a 0.44 ± 0.04 a 0.48 ± 0.11 t-test * * ns Note ns means not statically significant * means statistically different to the confidence level of 95% by Duncan s New Multiple Range Test (DMRT) Table 5 Comparison of mean flower sizes of 90-day-old stem cuttings of diploid and tetraploid Torenia fournieri Note ns means not statically significant * means statistically different to the confidence level of 95% by Duncan s New Multiple Range Test (DMRT) Figure 2: Comparison of stomata of diploid Torenia fournieri (A) and tetraploid Torenia fournieri (B) (bar= 20 µm) 306 S. Boonbongkarn, T. Taychasinpitak, S. Wongchaochant, and S. Kikuchi

9 Table 6: Stomata size of Torenia fournieri plants that were selected as putative polyploids from colchicine treatment, 120 days after treatment. Plant Mean stomata guard cell length ± SD (µm) Control ± 0.48 Selected plant (colchicine concentration days number) ± ± ± ± ± ± ± 1.27 Figure 3: Comparison of the chromosome number of diploid Torenia fournieri (2n=18) (A) and an induced tetraploid Torenia fournieri (2n=36) (B) (bar =10µm) Figure 4: Flow cytometry histograms showing the DNA content of diploid (A) and tetraploid (B) Torenia fournieri 4. Conclusion The concentration and duration of colchicine treatment affected the survival rate of native Torenia fournieri. Survival rate decreased with increasing colchicine concentration and exposure time. Flow cytometry analysis demonstrated that the amount of DNA in the tetraploid plants had doubled. The treatments of 5 ppm colchicine for 1 day and 15 ppm colchicine for 3 307

10 days were both found to be effective for inducing polyploidy in Torenia fournieri, because both treatments resulted in 2 tetraploids, which were confirmed to have the doubled chromosome number of 2n=4x=36. A comparison of the morphological and cytological characteristics of diploid and tetraploids Torenia fournieri showed that the tetraploids grew more slowly and were shorter than the diploids, but had greater spread, darker green leaves, larger and thicker leaves, and larger flowers. The stomata size of the tetraploids was also larger than the diploids. 5. References Cook, J.W. and L.D. Loudon Colchicine. The Alkaloid Chemistry and Physiology. 2: Dermen, H Colchicine polyploidy and technique. The Bot. Rev. 6: Dolezel, J., Applications of flow cytometry for the study of plant genomes. Journal of Applied Genetics. 38: Elliott, F.C Plant Breeding and Cytogenetics. McGraw-Hill, Inc, New York. Espino F. J. and A. M. Vazquez Chromosome numbers of Saintpaulia zonantha plantlets regenerated from leaves cultured in vitro with caffeine and colchicine. Euphytica. 30: Fischer, E. (2004). The Families and Genera of Vascular Plants. Kadereit, ed. Springer-Verlag, New York. pp Volume VII, JW Hancock, J.F., The colchicine story. Hortscience 32: Jensen, C.J Chromosome doubling techniques in haploids, pp In K.L. Kasha (ed.). Haploids in higher plants-advances and potential. University of Guelph, Canada. Jiranapapan, J., Kikuchi, S., Manochai, B., Taychasinpitak, T., Tanaka, H., Tsujimoto, H. (2011). A simple method chromosome doubling suing colchicine in Torenia (Linderniaceae), and the behavior of meiotic chromosome in amphidiploids. Chromosome Science 14: Kikuchi, S., Kishii, M., Shimizu, M., Tsujimoto, H. (2005). Centromere-specific repetitive sequences from Torenia, a model plant for interspecific fertilization and whole-mount FISH of its interspecific hybrid embryos. Cytogenetic and Genome Research 109: Kikuchi, S., Matsui, K., Tanaka, H., Ohuishi, O., Tsujimoto, H. (2008). Chromosome evolution among seven Fagopyrum species revealed by flouresence in situ hybridization (FISH) probed with adnas. Chromosome Science. 11: Loureiro, J., Pinto, G., Lopes, T., Dolezˇel, J., Santos, C., Assessment of ploidy stability of somatic embryogenesis process in Quercus suber L. using flow cytometry. Planta. 221: Shao, J.Z., Chen, C.L., Deng, X.X., In vitro induction of tetraploid in pomegranate (Punica granatum). Plant Cell Tissue and Organ Culture 75: S. Boonbongkarn, T. Taychasinpitak, S. Wongchaochant, and S. Kikuchi

11 Shindu, K., Saito, E., Sekiya, M., Matsui, T., Koike, Y. (2008). Antioxidant activity of the flower of Torenia fournieri. Journal of Natural Medicine 62: Spencer, R. (2006). Horticultural flora of South Eastern Australia. Spencer, R. (eds). UNSW Press, Melbourne. pp 271. Starman, T. W.(2005). Focus on vegetative annuals: Torenia. Greenhouse Grower Tandon, S.L., Bhutani, K. (1965). Morphological and cytological studies of colchicine-induced tetraploids in Torenia fournieri Lind. Genetica. 36: Yamazaki, T. (1985). A Revision of the Genera Limnophila and Torenia from Indochina. J Fac Sci Univ Tokyo III 13: Y.M. Ye, J. Tong, X.P. Shi, W. Yuan, G.R. Li. (2010). Morphological and cytological studies of diploid and colchicine-induced tetraploid lines of crape myrtle (Lagerstroemia indica L.). Scientia Horticulturae. 124: Zhang, W., H. Hui, M. Leyuan, Z. Cong and Y. Xiyan Tetraploid muskmelon alters morphological characteristics and improves fruit quality. Scientia Horticulturae. 125: Thunya TAYCHASINPITAK is an Associate Professor in Department of Horticulture, Faculty of Agriculture, Kasetsart University, Bangkhen, Bangkok, THAILAND. He is teaching and researching in floriculture and floriculture crop improvement. Dr. Shermarl WONGCHAOCHANT is a lecturer in the Department of Horticulture, Faculty of Agriculture, Kasetsart University, Bangkhen, Bangkok, THAILAND. She earned an B.S. (Agriculture)(HONS.) from Chiang Mai University, Thailand, an M.A. and a Ph.D. (Plant Biotechnology) from Osaka Prefecture University, Japan. She is teaching and researching in floriculture crop improvement and plant molecular genetics. Dr. Shinji KIKUCHI is an Assistant Professor in Graduate School of Horticulture, Chiba University, Chiba, JAPAN. He is teaching and researching in Chromosome Science and Plant Breeding Sasiree BOONBONGKARN is a graduate student in Department of Horticulture, Faculty of Agriculture, Kasetsart University, Bangkhen, Bangkok, THAILAND. Her research encompasses plant breeding technology. Peer Review: This article has been internationally peer-reviewed and accepted for publication according to the guidelines given at the journal s website. 309