Amir Hossein Keshtkar; Noshin Fallahi; Mohammad Reza Abdollahi; Hassan Sarikhani; Hooshmand Safari; Zhaleh Mohseni Araghi
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 ...
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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.
