عنوان مقاله [English]
Background and Objectives
Induced mutations have been used in a wide range of horticultural crops for more than half a century. This approach has opened up great opportunities for biotechnologists and plant breeders to produce new varieties with several new traits including resistance to disease, and tolerance to adverse environmental conditions or even produce new horticultural characters in some crops. The optimization of dosage of the physical and chemical mutagenic agents for inducing mutations is the first step in using this technique in plant breeding. This study was thus conducted to determine the optimum dosage of two mutagenic agents including gamma radiation as a physical agent and ethyl methanesulphonate (EMS) as a chemical agent in kiwifruit callus in in-vitro conditions. To that end, simple sequence repeat (SSR) markers were used to detect the mutations in the regenerated plantlets.
Materials and Methods
Having induced the callus in tissue culture media containing naphthaleneacetic acid, 6-benzylamino purine and meta-Topolin, the produced calli were exposed to different dosage of gamma radiation (0, 10, 20, 30, 40, 50 Gy) or different EMS concentrations (0, 0.4, 0.6, 0.8, 1, 1.2 % for 60 min). Then, the callis were transferred to the shoot regeneration media. The number of dead calli, and the size of live calli as well as the number of shoots generated in live calli were then recorded. Afterwards, the regenerated shoots were transferred to elongation media, and the DNA was extracted. The SSR markers analysis was followed on the randomly selected plantlets to detect the SSR markers using a Genetic Analyzer.
As expected, with increasing the dosage of gamma radiation, the number of dead calli increased and the callus growth was thus retarded. The lowest number of dead calli was, indeed, detected in the control and the 50 Gy dose resulted in the highest number of dead calli. The morphology of shoots generated in the treated calli and the SSR marker analysis showed that 50 Gy dose could be used as the optimum radiation dose to produce an acceptable mutation rate in kiwifruit callus. In turn, EMS as a chemical mutagenic agent showed a different pattern in increasing the number of shoots regenerated with increasing dosage, however the same trend happened with gamma radiation in the number of dead calli and live callus size. Considering the effect of EMS on callus and the number of shoots regenerated per callus, a treatment with 1% EMS for 60 min can be used to produce a high density mutant population in kiwifruit. The highest mutation rate (12.5 %) was observed in the 50 Gy gamma ray treatment with changes in 12 pairs of SSR markers among 25 loci. For EMS, this value was 2.6 % in 1% concentration treatment.
To detect the induced mutations using Gamma ray and EMS, the SSR markers can be applied. However, for the EMS mutation detection, more studies are needed to evaluate the efficiency of other techniques such as single nucleotide polymorphism (SNPs).
Bourgis, F., Guyot, R., Gherbi, H., Tailliez, E., Amabile, I., Salse, J. and Ghesquiere, A. (2008). Characterization of the major fragance gene from an aromatic japonica rice and analysis of its diversity in Asian cultivated rice. Theoretical and Applied Genetics, 117(3), 353-368.
Debenham, M. C., Seelye, J. F. and Mullan, A. C. (2016). An in vitro repository for clonal kiwifruit. Acta Horticulturae, 1113, 93-98.
Doyle, J. J. (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bulletin Botanical Society of Amrica, 19, 11-15.
Fisher, R. A. (1999). The genetical theory of natural selection: A complete variorum edition. Oxford, UK: Oxford University Press.
Fraser, L. G., Tsang, G. K., Datson, P. M., De Silva, H.N., Harvey, C. F., Gill, G. P. and McNeilage, M. A. (2009). A gene-rich linkage map in the dioecious species Actinidia chinensis (kiwifruit) reveals putative X/Y sex-determining chromosomes. BMC Genomics, 10(102), 1-15.
Gamborg, O.L., Miller, R. and Ojima, K. (1968). Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research, 50(1), 151-158.
Gaswanto, R., Syukur, M., Purwoko, B. S. and Hidayat, S. H. (2016). Induced mutation by gamma rays irradiation to increase chilli resistance to Begomovirus. Agrivita, 38(1), 24-32.
Hussain, M., Iqba, M. A., Till, B. J. and Rahman, M. (2018). Identification of induced mutations in hexaploid wheat genome using exome capture assay. Plos One 13(8), e0201918.
Karami, S. and Imanikhah, F. (2012). Crop species transposability of sorghum microsatellite markers in sugarcane and maize. Plant Productions, 34(2), 27-38. [In Farsi]
Khalil, F., Naiyan, X., Tayyab, M. and Pinghua, C. (2018). Screening of EMS-induced drought-tolerant sugarcane mutants employing physiological, molecular and enzymatic approaches. Agronomy, 8(10), 1-13.
Kiani, G. (2017). Identification of restoring fertility and maintainer rice varieties using SSR marker. Plant Productions, 40(1), 81-86. [In Farsi]
Kodym, A. and Afza, R. (2003). Physical and chemical mutagenesis. Plant Functional Genomics, 236, 189-203.
Krupa-Malkiewicz, M., Kosatka, A. Smolik, B. and Sedzik, M. (2017). Induced mutations through EMS treatment and in vitro screening for salt tolerance plant of Petunia × atkinsiana D. Don. Not Bot Horti Agrobo, 45(1), 190-196.
Mathew, L., McLachlan A., Jibran R., Burrit D. J. and Pathirana R. (2018). Cold, antioxidant and osmotic pre-treatments maintain the structural integrity of meristematic cells and improve plant regeneration in cryopreserved kiwifruit shoot tips. Protoplasma, 255(4), 1065-1077.
Mohan Jain, S. (2006). Mutation-assisted breeding for improving ornamental plants. Acta horticulturae, 714(714), 85-98.
Murashige, T. and Skoog, F. (1962). A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiologia Plantarum, 15(3), 473-497.
Novak, F. J., Afza, R., Duren, M. V. and Omar, M. S. (1990). Mutation induction by gamma irradiation of in vitro cultured shoot-tips of banana and plantain (Musa cvs). Tropical Agriculture, 67(1), 21-28.
Pathirana, R., Deroles, S., Hoeata, K., Montefiori, M., Tyson, J., Wang, T. and Hellens, R. P. (2014). Fast-tracking kiwifruit breeding through mutagenesis. Acta Horticulturae, 1127, 217-222.
Pestanana, R. K. N., Amorim, E. P., Ferreira, C. F., de Oliveira Amorim, V. B., Oliveira, L. S., da Silva Ledo, C. A. and Silva, S. D. O. (2011). Agronomic and molecular characterization of gamma ray induced banana (Musa sp.) mutants using a multivariate statistical algorithm. Euphytica, 178(2), 151-158.
Purnamaningsih, R. and Hutami, S. (2016). Increasing Al-Tolerance of sugarcane using ethyl methane sulphonate and in vitro selection in the low pH media. Hayati Journal of Biosciences, 23(1), 1-6.
Schuelke, M. (2000). An economic method for the fluorescent labeling of PCR fragments. Nature Biotechnology, 18(2), 233-234.
Sega, G. A. (1984). A review of the genetic effects of ethyl methanesulfonate. Mutation Research/Reviews in Genetic Toxicology, 134(2-3), 113-142.
Vavilov, N. I. and Dorofeev, V. F. (1992). Origin and geography of cultivated plants. Cambridge, UK: Cambridge University Press.
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