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International Transaction Journal of Engineering, Management, & Applied Sciences & Technologies
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a*
Department of Horticulture, Faculty of Agriculture, Kasetsart University, Jatujak, Bangkok 10900. THAILAND b Graduate School of Horticulture, Chiba University, Chiba, JAPAN
ARTICLEINFO
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.
ABSTRACT
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.
2013 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
*Corresponding author (T. Taychasinpitak) Tel: +66-2-579-0308 Fax: +66-2-579-1951. E-mail address: agrtyt@ku.ac.th. 2013 International Transaction Journal of Engineering, Management, & Applied Sciences & Technologies. Volume 4 No.4 ISSN 2228-9860 eISSN 1906-9642. Online Available at http://TuEngr.com/V04/299-309.pdf
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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.
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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.
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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 14-14-14 was added at the rate of 5 g per pot, and liquid fertilizer of formula 21-21-21 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.
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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).
10
15
20
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 mol 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.
Tetraploid
13.30 2.16b
20.80 2.84b
13.00 4.8 ns
1.80 0.14 ns
* * t-test 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
*Corresponding author (T. Taychasinpitak) Tel: +66-2-579-0308 Fax: +66-2-579-1951. E-mail address: agrtyt@ku.ac.th. 2013 International Transaction Journal of Engineering, Management, & Applied Sciences & Technologies. Volume 4 No.4 ISSN 2228-9860 eISSN 1906-9642. Online Available at http://TuEngr.com/V04/299-309.pdf
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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 width (cm) 3.77 0.15b Mean leaf length (cm) 0.31 0.05b Mean leaf thickness (mm) 0.31 0.04
Diploid
Tetraploid
4.10 0.13a
0.44 0.04a
0.48 0.11 ns
t-test * * 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 m) 306
S. Boonbongkarn, T. Taychasinpitak, S. Wongchaochant, and S. Kikuchi
Table 6: Stomata size of Torenia fournieri plants that were selected as putative polyploids from colchicine treatment, 120 days after treatment.
Plant Control Selected plant (colchicine concentration days number) 5-1-1 5-1-2 5-2-1 10-2-1 15-1-1 15-3-1 15-3-2 Mean stomata guard cell length SD (m) 22.77 0.48
28.34 3.82 27.22 0.95 28.61 2.68 29.17 3.01 30.81 0.84 31.43 1.59 31.11 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 =10m)
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
*Corresponding author (T. Taychasinpitak) Tel: +66-2-579-0308 Fax: +66-2-579-1951. E-mail address: agrtyt@ku.ac.th. 2013 International Transaction Journal of Engineering, Management, & Applied Sciences & Technologies. Volume 4 No.4 ISSN 2228-9860 eISSN 1906-9642. Online Available at http://TuEngr.com/V04/299-309.pdf
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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. 1952. Colchicine. The Alkaloid Chemistry and Physiology. 2: 261-329. Dermen, H. 1940. Colchicine polyploidy and technique. The Bot. Rev. 6: 599-635. Dolezel, J., 1997. Applications of flow cytometry for the study of plant genomes. Journal of Applied Genetics. 38:285302. Elliott, F.C. 1958. Plant Breeding and Cytogenetics. McGraw-Hill, Inc, New York. Espino F. J. and A. M. Vazquez. 1981. Chromosome numbers of Saintpaulia zonantha plantlets regenerated from leaves cultured in vitro with caffeine and colchicine. Euphytica. 30: 847-853. Fischer, E. (2004). The Families and Genera of Vascular Plants. Kadereit, ed. Springer-Verlag, New York. pp 333-391. Hancock, J.F., 1997. The colchicine story. Hortscience 32: 10111012. Jensen, C.J. 1974. Chromosome doubling techniques in haploids, pp. 153-190. 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: 29-32. 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: 228-235. 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: 37-43. Loureiro, J., Pinto, G., Lopes, T., Dolezel, J., Santos, C., 2005. Assessment of ploidy stability of somatic embryogenesis process in Quercus suber L. using flow cytometry. Planta. 221: 815822. Shao, J.Z., Chen, C.L., Deng, X.X., 2003. In vitro induction of tetraploid in pomegranate (Punica granatum). Plant Cell Tissue and Organ Culture 75: 241246. 308
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Shindu, K., Saito, E., Sekiya, M., Matsui, T., Koike, Y. (2008). Antioxidant activity of the flower of Torenia fournieri. Journal of Natural Medicine 62: 247-248. 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 92-94. Tandon, S.L., Bhutani, K. (1965). Morphological and cytological studies colchicine-induced tetraploids in Torenia fournieri Lind. Genetica. 36: 439-445. of
Yamazaki, T. (1985). A Revision of the Genera Limnophila and Torenia from Indochina. J Fac Sci Univ Tokyo III 13: 575-624. 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: 95101. Zhang, W., H. Hui, M. Leyuan, Z. Cong and Y. Xiyan. 2010. Tetraploid muskmelon alters morphological characteristics and improves fruit quality. Scientia Horticulturae. 125: 396400.
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 journals website.
*Corresponding author (T. Taychasinpitak) Tel: +66-2-579-0308 Fax: +66-2-579-1951. E-mail address: agrtyt@ku.ac.th. 2013 International Transaction Journal of Engineering, Management, & Applied Sciences & Technologies. Volume 4 No.4 ISSN 2228-9860 eISSN 1906-9642. Online Available at http://TuEngr.com/V04/299-309.pdf
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