Effect of colchicine concentration at two temperatures (۴ and ۲۸ °C) on the seed of fenugreek (Trigonella foenum-graecum) as a medicinal plant

Introduction: Polyploidy induction by mutagenic chemicals is considered to enhance the potential production of secondary metabolites in medicinal plant breeding programs. Colchicine is the most effective chemical used in polyploidy induction studies. Production of polyploid plants in fenugreek (Trigonella foenum-graecum L.) normally involves artificial chromosome doubling by colchicine treatment of seedlings or seeds (Alavi et al., ۲۰۲۲). Materials and methods: Seeds of Trigonella foenum-graecum were collected from Kermanshah, Iran. The seeds were surface sterilized by immersing in ۲۰% hypochlorite sodium solutions for ۱۰ minutes. In this study, five concentrations of colchicine (۰, ۰.۰۵, ۰.۱۰, ۰.۲۰ and ۰.۵۰%) and four duration treatments (۱۲, ۲۴, ۴۸ and ۷۲ hours) with two levels of temperature (۴ and ۲۸°C) were studied on fenugreek seeds. At the end of each period, samples were removed from colchicine solutions and were washed out in water for ۳۰ minutes. Seeds were then positioned in a germinator at ۲۸°C after being placed on wet filter papers in petri dishes that had been previously sterilized by autoclaving. A very small portion of the root of germinated seeds was cut for cytogenetic study of chromosomes. The seedlings were transferred to the greenhouse and cultured in plastic pots. Young leaves according to Huh et al., method (۲۰۰۵) were used for analysis of ploidy level by flow cytometry model (Partec, D-۴۸۱۶۱, Germany), equipped with an HBO lamp and UV laser. Results and Discussion: The analysis of variance showed that all characteristics for germination percentage and mixoploidy percentage were statistically significant at ۱% probability level. In this experiment, there no tetraploid plants were observed. There was significant difference between using zero and ۰.۰۵, ۰.۱, ۰.۲, ۰.۵ volume of colchicine concentrations to weight ratio for explant germination. After the control, the highest germination rate was found for ۰.۰۵% colchicine concentration at ۴°C and the germination rate decreased with an increase of colchicine concentration and increased exposure time of colchicine-treated seed. In general, mutagens cause a decrease in germination and survival rates of plants (Mensha, ۲۰۰۵). Due to the significant difference of all interaction effects in the ANOVA table and the impact on germination percentage of fenugreek seeds, it can be concluded that cold treatment had a very positive effect on increasing the seed germination percentage. No research has been found to compare the effect of colchicine treatments on plant seeds at different temperatures, but in general, researchers have found that low temperatures lead to an increased seed germination rate and survival percentage. Cold stratification or chilling under moist conditions has long been recognized as a useful method of treating seeds to improve the rate and the percentage of germinability (Alrumaihi et al., ۲۰۲۱; Outcall, ۱۹۹۱). A concentration of ۰.۲% colchicine for a ۱۲-hour duration at ۴°C was the best treatment for Mixoploidy percentage. Cytogenetic study on vetch root meristem indicated that colchicine treatment at a concentration of ۰.۲% for ۲۴ hours induces a mixoploidy plant (Tulay et al., ۲۰۱۰). In the control group, the diploid number of chromosomes in Trigonella foenum-graecum ۲n=۱۶ (Fig.۲) while the mixploid(۲n = ۲x = ۱۶, ۴n = ۳۲) chromosomes (Fig ۱,۲). The flow cytometry method is widely used for detection of plant ploidy levels (Dolezel et al., ۲۰۰۷). Results obtained from ploidy analysis of treated seeds by the flow cytometry device are showed at two ploidy levels, diploid and mixoploid (Figure ۳). The Flow cytometric analysis showed that several treated plants were mixoploids. In fact, there were certain plants with two levels of ploidy: a tetraploid level (۴n = ۳۲) and a diploid level (۲n = ۱۶). Flow cytometry evaluation by Omezzine and Bouaziz (۲۰۱۲) indicated that fenugreek seed treatment at ۰.۰۵% colchicine concentration for ۴ hours caused mixoploidy plants with four ploidy levels (۲x, ۴x, ۶x and ۸x).

کلیدواژه ها:

Polyploidy ، colchicine ، Cytogenetic ، Flow cytometry ، Fenugreek