Effect of Colchicine on Polyploidy Induction and Its Effects on Morphophysiological and Biochemical Properties of Fenugreek (Trigonella foenum-graecum)

Document Type : Research Article

Authors

• Amir Hossein Keshtkar ¹
• Noshin Fallahi ²
• Mohammad Reza Abdollahi ¹
• Hassan Sarikhani ³
• Hooshmand Safari ⁴
• Zhaleh Mohseni Araghi ⁵

¹ Bu- Ali Sina University

² Ilam University

³ Department of Horticulture Science, Faculty of Agriculture, Bu-Ali Sina University, Hamedan, Iran.

⁴ Department of Forests and Rangelands, Kermanshah Agricultural and Natural Resources Research and Education Center. I.R. Iran

⁵ College of Agricultire Islamic Azad University, Science and Research Branch, Tehran.

https://doi.org/10.22067/jhorts4.v33i1.71781

Abstract

Introduction: Polyploidy plays an important role in creation of genetic variability. Polyploidy induction by mutagenic chemicals such as colchicine is considered to enhance the potential of secondary metabolites production in herbs breeding. Colchicine is the most effective chemicals used in the polyploidy induction studies. The effect of colchicine is to form cells with two or multiply number of chromosomes resulting in a lack of germination and death of a large number of plant samples.  Flow cytometry analysis and cytogenetic studies were effectively used to assess the ploidy levels for fenugreek (Trigonella foenum-graecum) plants. In the beginning of 90 decade, a new type of adsorption technique called solid-phase micro extraction (SPME) has been developed by Pawliszyn and co-workers. This method compared to the traditional techniques, offers many advantages such as the high sensitivity and reproducibility, does not require solvent, and combines extraction and pre-concentration in a single step without pre-treatment of samples. Moreover, it is a fast and inexpensive method, requires low sample volume and it can be easily automated. Solid-phase micro extraction (SPME) uses a fine rod (fused silica or metal) with a polymeric coating to extract organic compounds from their matrix and it directly transfer them into the gas chromatograph injector for thermal desorption and analysis (Kaykhaii, 2008). The aim of this study was to investigate the effect of colchicine treatment on ploidy levels and compare some of the morphological, physiological, cytogenetical, flow cytometric analysis and biochemical characteristics in diploid and tetraploid fenugreek plants.
Materials and Methods: In order to investigate the effect of polyploidy induction by colchicine on Trigonella foenum-graecum medicinal species, an experiment was planned as a factorial completely randomized design with five concentrations of colchicine (0, 0.05, 0.1, 0.2, and 0.5%) for 12, 24, 48 and 72 hours. In this experiment the effect of colchicine was examined on the percentage of survival and tetraploidy of seed, root and terminal bud samples. Level of ploidy was identified in survival explants through root tip chromosome counting and flow cytometry of leaf samples. In addition to distinguish tetraploid from the diploids plants, morphological, physiological and biochemical characteristics were considered in treated plants. SAS and SPSS software programs were used to analysis of variance and comparison of means by Duncan's multiple test. Graphs were also drawn by EXCEL software.
Results and Discussion: The analysis of variance showed that all characteristic factors for survival percent and mixoploidy percentage were statistically significant. Survival percentage was decreased with increasing of colchicine concentration and increased exposure time of colchicine-treated seed. After the observation of morphological changes, the samples were considered to assess the ploidy levels by flow cytometry system. Results showed that 0.5% colchicine concentration had the highest survival rate after control treatment for the terminal bud. The highest percentage of mixoploidy was also observed in treated terminal buds with 0.1 and 0.2% of colchicine concentrations. Morphological, physiological, cytogenetic, flow cytometric analysis and biochemical studies confirmed that terminal bud treatment with 0.2% colchicine for 72 hours is the most effective treatment to induce tetraploidy in fenugreek plant. The results of GC/MS also indicated an increase in secondary metabolites content, but traits including growth rate and plant height of tetraploid reduced compared to the diploid plants. Result of this study showed a significant increase in chlorophyll a, b and total chlorophyll contents of tetraploid plants, which were higher than the levels of diploid plants.
Conclusion: Polyploidy induction using mutagenic chemicals is one of the methods to enhance the production of plant secondary metabolites. Colchicine is the most effective mutagenic chemical in inducing plant polyploidy. Although, flow cytometry is an expensive method, it is increasingly used for ploidy screening by analyzing of nuclear DNA content. In this study, both flow cytometry and chromosome microscopic examinations were used to test ploidy. The two methods were compared, and it was found that flow cytometry testing was fast and labor saving, especially in case of a large number of samples. Tetraploidy induction significantly affected different morphological, physiological, and biochemical characteristics of Trigonella foenum-graecum. These changes suggested that ploidy manipulation as a rapid and effective method for enhancing genetic diversity and metabolite production for this plant. SMPE method offers a number of practical advantages: smaller sample volume, simplicity of extraction and low cost, when compared to the other methods that are currently being used.

Keywords

• GC/MS
• flow cytometry
• Mutation
• Secondary metabolites

20.1001.1.20084730.1398.33.1.11.7

References

1- Aasim M., Hussain N., Umer E.M., Zubair M., Hussain S.B., Saeed S.H., Rafique T.S. and Sancak C. 2010. In-vitro shoot regeneration of fenugreek (Trigonellafoenum-graecum L.) using different cytokinins, African Journal of Biotechnology, 42: 7174-7179.

2- Adaniya S., and Shira D. 2001. In vitro induction of tetraploid ginger (Zingiber officinalis Roscoe) and its pollen fertility and germinability, Scientia Horticulturae, 88: 277-287.

3- Al-Habori M. and Raman A. 2002. Pharmacological Properties in Fenugreek- The genus Trigonella (1st edition) by G.A. Petropoulos (ed.). Taylor and Francis, London and New York, 10: 163-182.

4- Andersson S.C. 2009. Carotenoids tocochromanols and chlorophylls in sea buckthorn berries (Hippophae rhamnoides) and rose hips (Rosa sp.). Swedish University of Agricultural Sciences, Alnarp, Sweden, 52:75-60.

5- Arnon D.I. 1949. Copper enzymes in isolated chloroplasts: polyphenol oxidase in Beta vulgaris, Plant Physiology, 24: 1-15.

6- Beck S.L., Dunlop W.R. and Fossey A. 2003. Stomatal length and frequency as a measure of ploidy level in black wattle, Acacia mearnsii (de Wild), Botanical Journal of the Linnean Society, 141 177–181.

7- Borgheei S.F., Sarikhani H., Chaichi M. and Kashi A. 2010. In vitro induction of polyploidy in lemon balm (Melissa officinalis L.), Journal of Medicinal and Aromatic Plants, 26:283-295. (in Persian with English abstract).

8- Dhawan O.P. and Lavania U.C. 1996. Enhancing the productivity of secondary metabolites via induced polyploidy. A review, Euphytica, 87: 81-89.

9- Ghiasvand A. R., Nasseri M, Farsizaeh S., Meshkatalsadat M.H., Sadeghi-Sarabi R., Shadabi S.H. and Borzoei M .2011. Chemical characterization of cultivated Tagetes minuta L. by use of ultrasound-assisted head space SPME and GC–MS, Chromatographia, 73:1031–1035.

10- Gu X. and Huang W. 2002. Testing the parsimony test of genome duplications: A counterexample, Genome Research, 12: 1-2.

11- Han D.S., Niimi Y. and Nakamo M. 1999. Production of doubled haploid plants through colchicine treatment of anther-drived haploid calli in Asiatic hybrid lilly, Journal of Japanese Society of Horticultural Sciencs, 68: 979-983.

12- Hanzelka P. and Kobza F. 2001. Genome induced mutation in Challistephus chinensis Nees. Effect of colchicines application on the early plant development, Zahradnictvi Horticultural Science, 28: 15-20.

13- Hasani M.A., Mirzaei M. and Omid Baigi R. 2010. An investigation of the effect of autotetraploidy on essential oil content and some of quntitative and qualitative characteristics of basil medicinal plant (Ocimum basilicum L.), Iranian Journal of Horticultural Science, 41 (2): 111-118. (in Persian with English abstract).

14- Hidvegi M., El-Kady A., Lasztity R., Bekes F. and Simon-Sarkadi L 1984. Contribution to the nutritional characterization of fenugreek (Trigonella foenum-graecum L. 1753), Acta Alimentaria., 13(4): 315-324.

15- Hoseini H., Chehrazi M., Nabatiahmadi D. and Mahmodisarvestani M. 2012. Induction polyploidy in Vinca plants and changes in phenotypic properties. First National Conference Playbooks Achieving Sustainable Development, 5pp (In Persian).

16- Jesus L.D. 2003. Effect of artificial polyploidy in transformed roots of Artemisia annua L. Plant Cell Reports, 21: 809-813.

17- Kaykhaii M. and Saffari F. 2008. Application of polypyrrole coated stainless-steel wire to the determination of aliphatic amines using headspace solid-phase microextraction, Journal of Sciences, 2: 111-117.

18- Kitson F.G., Larsen B.S. and McEwen C.N. 1996. The fundamentals of GC/MS. p 3-35 In: Gas Chromatography and Mass Spectrometry: A Practical Guide. F.G. Kitson et al. (ed.) Academic Press. San Diego, CA.

19- Lavania U.C. and Srivastava S. 1991. Enhanced productivity of tropane alkaloids and fertility in artificial autotetraploids of Hyoscyamus niger L., Euphytica, 52: 73-77.

20- Lavania U.C. 1998. Enhanced productivity of the essential oil in the artificial autotertraploid of Vetiver (Vetiveria zizanioides L. Nash). Euphytica, 38: 271- 276.

21- Lavania U.C. 2005. Genomic and ploidy manipulation for enhanced production of phyto-pharmaceuticals, Plant Genetic Resources, 3(2): 170–177.

22- Madon M., Clyde M.M., Hashim H., Mohdyusuf Y., Mat H. and Saratha S. 2005. Polyploidy induction of oil palm through Colchicine and oryzalin treatments, Journal of Oil Palm Research, 17:110-123.

23- Malekzade Shafarodi S., Ghani A. and Habibi M. 2010. The study of induction of polyploidy in basil (Ocimum basilicum) plant, Journal of Horticulture, 25: 469-461. (in Persian).

24- Marzougui N., Boubaya A., Elfalleh W., Ferchichi A. and Beji M. 2009. Induction of polyploidy in Trigonella foenum-graecum L.: Morphological and chemical comparison between diploids and induced autotetraploids, Acta Botanica Gallica, 156: 379-389.

25- Marzougui N., Guasmi F., Elfalleh W., Boubaya A., Touil L., Ferchichi A., Lachieheb B. and Beji M. 2010. Physiological performa nce of induced autotetraploid genotype of Trigonella foenum-graecum L. compared with diploid genotypes, Acta Botanica Gallica, 157: 117-126.

26- Mathura S., Fossey A. and Beck S. 2006. Comperative study of chlorophyll content in diploid and tetraploid black wattle (Acacia mearnsii). Journal of Forestry, 79: 381- 388.

27- Massumi M., Fazeli M., Alavi S. and Ajani Y. 2007. Chemical constituents and antibacterial activity of essential oil of Prangos ferulacea (L.) Lindl. fruits, Iranian Journal of Pharmaceutical Sciences, 3(3): 171-6.

28- Mehrafarin A., Rezazadeh S.h., Naghdi Badi H., Zand E. and Noormohammadi G.h. 2011. A Review on biology, cultivation and biotechnology of fenugreek (Trigonella foenum-graecum L.) as a valuable medicinal plant and multipurpose, Journal of Medicinal Plants, 1(37): 6-24.

29- Mensha J.K., Obadoni B.O., Akomeah P.A., Ikhajiagbe B. and Ajibolu J. 2007. The effects of sodium azide and colchicine treatments on morphological and yield traits of sesame seed (Sesame indicum L.), African Journal of Biotechnology, 6: 534-538.

30- Moradi P., Kashi A., Hasandokht M.R., Khosroshahi M. and Khalighi A. 2008. Study of genetic diversity of indigenous Iranian fenugreek masses based on cytological traits, Plant and Ecosystem,, 21: 33-46.( in Persian).

31- Nezhadali A., Akbarpour M., Zarrabi Shirvanb B. and Mousavic M. 2010. Comparison of volatile organic compounds of Thymus vulgaris using hydrodistillation and headspace solid phase microextraction gas chromatography mass spectrometry, Journal of the Chinese Chemical Society, 57:40-43.

32- Razavi S.M., Zahri S., Zarrini G., Nazemiyeh H. and Mohammadi S. 2009. Biological activity of quercetin-3-O-glucoside, a known plant flavonoid. Russian Journal of Bioorganic Chemistry, 35(3): 376-8.

33- Riasat M., Karaptyan Zh. and Nasirzadeh A. 2003. Study of karyotype of some species of the genus Trigonella Fars Province. Iranian Journal of Rangelands and Forests Plant Breeding and Genetic Research, 11:127-145 (in Persian with English abstract).

34- Rubuluza T., Nikolova R.V., Smith M.T. and Hannweg K. 2007. In vitro induction of tetraploids in Colophospermum mopane by colchicines. South African Journal of Botany, 73: 259–261.

35- Quan K., Guolu L., Qigao G. and Xiaolin L. 2004. Polyploid induction of Arctium lappa by colchicine, Plant Physiol Communi, 40:157-158.

36- Saeedi K.A. and Omidbaigi R. 2009. Evaluation of content and composition of fatty acids, totalphenolic content of Kelussia odoratissima Mozaff. seed, Journal of Medicinal and Aromatic Plants, 25 (1): 113- 9.

37- Saharkhiz M.J. 2005. The effects of some environmental factors and ploidy level on morphological and physiological characteristics of feverfew (Tanacetum parthenium L.) medicinal ornamental plant. Ph.D. Thesis, Tarbiat Modares University, pp 173. (in Persian with English abstract).

38- Sari N., Abak, K. and Pitrat, M.1999. Comparison of ploidy level screening methods in watermelon, Science Swedish University of Agricultural Sciences, 41: 361–390.

39- Singleton VL. and Rossi I.A. 1995. Colorimetry of total phenolics with phosphor-molybdic phosphotungstic acid reagents, American Journal of Enology and Viticulture, 16:144-58.

40- Takamura T., and Miyajima I. 1996. Colchecine induced tetraploids in yellow-flowered cyclamens and their characteristics, Scientia Horticulturae, 65:305-312.

41- Thao N.T.P., Ureshino K., Miyajima I., Ozaki Y. and Okubo H. 2003. Induction of tetraploids inornamental Alocasia through colchicine and oryzalin treatments, Plant Cell, Tissue and Organ Culture, 72: 19-25.

42- Wu H.Z., Zheng S., He Y., Yan G., Bi Y. and Zhu Y. 2007. Diploid female gametes induced by colchicine in oriental lilies. Scientia Horticulturae, 114: 50-53.

43- Yavari S. 2007. The study of the effect of colchicine on morphological, physiological, and active ingredients of (Melissa officinalis L.) medicinal plant. MSc. Thesis, Department of Horticulture. University of Tarbiat Modarres, Tehran. 140 pages. (in Persian with English abstract).