Document Type : Original Article
Authors
1 Master of student, Agriculture Biotechnology,
2 Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Iran
Abstract
Introduction: Despite the economic importance of saffron, significant research has not been done in the field of molecular biology and genetics of this plant. Sterility and triploid genome, along with lack of genetic diversity are main reasons for this lack of research. Optimization of saffron cell suspension is a great step forward. It will provide the possibility of studying the production of saffron secondary metabolites, gene transformation, and mutagenesis cellular level. There have been few reports of cell suspension culture in saffron, mainly for the production of secondary metabolites (Yoon et al., 2015; Moradi et al., 2020; Taherkhani et al., 2019). In order to optimize the production of crocin and phenolic compounds, Moradi and co-workers produced calli from saffron stigma and then created a suspension culture from these calli (Moradi et al., 2020). However, the production of embryogenic cell suspension in saffron has not been reported. The aim of the current research was to achieve a cell suspension system with acceptable growth in saffron. For this purpose, different explants were subjected to different hormonal treatments to determine the optimal callus formation and suspension culture media.
Materials and Methods: Saffron corms were used to prepare explants. The lateral and terminal buds and the base of the corms were removed and the central part of the corms was used to prepare explants. Then, the explants were sterilized with 2.5% Sodium hypochlorite for 15 minutes. Explants were grown on CIM1 to CIM5 media (Table 1). After 5 weeks, the percentage of callus formation and the fresh and dry weight of calli were measured. The calli obtained from CIM2c treatment were used to prepare suspension culture with three different types of culture media (Table 1). To compare the speed of cell growth in different cell suspension treatments, the optical density (OD) measurement method was used. For all the assays, data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey Honestly Significant Differences (HSD) Post-Hoc test using SPSS® statistical software, version 22 (IBM Corp., USA). The graphs were created using GraphPad Prism version 9 for Windows (GraphPad Software, California USA).
Results and Discussion: The results showed that the concentration of 2 mgL-1 of 2,4-D and 1 mgL-1 of BAP had the best performance in callus formation and callus fresh weight. The calli of this treatment were used for cell suspension culture. SM3 medium had the best growth rate in cell suspension culture, but it did not have cells with appropriate shape and form. SM2 medium had a suitable growth rate and cells with small size and dry color, which had the best performance in terms of quality. In order to increase the quality, phenol production control materials were used. Adding PVP increased the growth rate of cell suspension in the SM2 medium. As we have shown, among the auxins 2, 4-D has a greater effect than NAA, contrary to other studies reported (Ahmad et al., 2013; Verma et al., 2016; Safarnejad et al., 2016; Georgiev et al., 2009; Sharma et al., 2008).
Conclusion: The current research showed the effect of treatment of 1 mgL-1 of BAP and 2 mgL-1 of 2,4-D on increasing the percentage of callus formation and the weight of calli taken from the saffron corms. SM2 and SM3 treatments had the best cell suspension growth rate. However, the SM3 treatment had cells with high starch vacuoles. Nevertheless, the SM2 treatment had smaller cells containing pigments.
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