Document Type : Original Article

Authors

1 PhD in Plant Genetics and Breeding, Faculty of Agriculture, University of Zanjan, Zanjan, Iran.

2 Professor, Department of Agronomy and Plant Breeding, Sabzevar Branch, Islamic Azad University, Sabzevar, Iran.

Abstract

Introduction: Saffron, a member of the lily family, is highly valued for its low water requirements, potential for job creation, and medicinal properties. Despite the high costs associated with traditional saffron cultivation, the use of tissue culture is recommended to enhance cultivation practices and produce disease-free seedlings. In vitro cultivation can significantly boost the production rate of new seedlings, with success largely dependent on the hormonal composition used. Additionally, carbon nanotubes, which have various applications in biology, have demonstrated a positive impact on plant growth. This study employs the response surface methodology (RSM) to optimize the saffron culture medium with carbon nanotubes and to identify the appropriate hormonal composition.
 
Materials and Methods: This research was conducted using in vitro culture techniques and a RSM design, comprising four replications. The factors investigated included concentrations of 2,4-D (0.2-0.5 mg/L), BAP (1.5-6 mg/L), and multi-walled carbon nanotubes (30-70 μg/mL). The culture medium and equipment were sterilized in an autoclave at 120°C and 1.5 atmospheres. Pedicel shells were removed, washed, and then immersed in Vitex solution for 30 minutes. The explants were placed in culture dishes and maintained under controlled light and temperature conditions. After five weeks, callus induction characteristics, such as callus induction percentage, diameter, and fresh weight, were measured. The Box-Benken statistical design was utilized for data analysis, and necessary transformations were applied based on software recommendations. A quadratic function was generally selected for the fitted model, even in cases of non-significance, and response surface curves along with contour diagrams were used to interpret the interaction effects of the traits. Tissue culture optimization aimed at maximizing callus formation, fresh weight, and diameter, with the highest model fitness identified as the optimal environment
Results and Discussion: The study's findings indicated that the concentrations of nanocarbon and 2,4-D significantly affected the number of calluses produced. At lower concentrations of 2,4-D, increasing BAP resulted in a higher number of calluses, while at higher concentrations, this effect remained positive. The maximum number of calluses was achieved with specific combinations of 2,4-D and BAP concentrations. High levels of BAP and carbon nanotubes increased the callus production percentage to 174%, whereas at lower concentrations, it was below 85%. The ratios exhibited a saddle function, with the highest number of calluses observed at low concentrations of 2,4-D and nanocarbon. Additionally, the linear effects of 2,4-D and carbon nanotube concentrations were significant at the 1% level, while the effect of BAP was not significant. Elevated concentrations of 2,4-D negatively impacted callus diameter, whereas increasing nanocarbon at high concentrations contributed to an increase in diameter. Ultimately, raising the concentration of 2,4-D led to an increase in callus induction percentage from 79.68% to 92.185%, although high concentrations had a detrimental effect. The highest callus induction percentage was noted with a specific combination of 2,4-D and carbon nanotubes.
 
Conclusion: The experimental results demonstrated that carbon nanotubes and the hormones BAP and 2,4-D significantly influenced saffron callus induction. High concentrations of 2,4-D exhibited the most substantial effect on the number of calluses produced. Furthermore, the application of BAP hormone up to a certain concentration increased callus number, but higher concentrations resulted in a decrease. Finally, the percentage of callus induction was positively affected by carbon nanotubes, while other characteristics did not show significant effects

Keywords

Ahmadi, J., Mohammadi, R., & Groosi, Gh. (2014). In vitro Micropropagation of Catharanthus roseus (Linn.) G. Don via shoot multiplication. Cellular and Molecular Research (Iranian Journal of Biology), 27(1), 14-25.
Ahmadian, Z., & Niazmand, R. (2016). Extraction of active components from saffron petal with the help of ultrasound and optimization of extraction conditions. Innovative Food Technologies4(1), 121-135.
Arbab, M. (2014). Effect of multi walled carbon nanotubes on in vitro callus induction and plant regeneration in withania coagulants (Master’s thesis). Islamic Azad University of Sabzevar, Sabzevar, Iran.
Assareh, M. (1998). In vitro culture plant regeneration through organogenesis, Somatic embryo genesis and photoautotrophic micro propagation of some Eucalyptus (Ph.D. thesis). National University of Ireland, Dublin, Ireland.
Bhagyalakshmi, N. (1999). Factors influencing direct shoot regeneration from ovary explants of saffron. Plant Cell Tissue Organ Cult, 58, 205–211.
Choob, V.V., Vlassova, T.A. and Butenko R.G. (1994). Callusogenesis and morphogenesis in generative organ culture of the spring flowering species of Crocus L. Russ J. Plant Physiol, 41, 712–716.
Ding, B., Bai, S., Wu, Y. & Wang, B.K. (1979). Preliminary report on tissue culture of corms of Crocus sativus. Acta. Bot. Sin., 21, 387.
Etedali, E., Moghadam, M., Khosrovshahli., M., Motlabi, A., Valizadeh, M., & Javidfar, F. (2004). The effect of medium and explant genotype on the induction and growth of rapeseed callus in mediums containing and without hormones, Quarterly Journal of Agricultural Science, 14(2), 95.
Ghodake, G., Seo, Y. D., Park, D., & Lee, D. S. (2010). Phytotoxicity of carbon nanotubes assessed by Brassica juncea and Phaseolus mungo. Journal of Nanoelectronics and Optoelectronics5(2), 157-160.
Ghorbani, M., & Jami, M. (2017). Effect of carbon nanotubes on callus formation of Marfona and Sinora potato varieties, The 4th National Congress on Development and Promotion of Agricultural Engineering and Soil Sciences of Iran papers, Environment and natural resources. [in Persian].
Gopitha, K, Lakshmi. B, A & Senthilmanickam, J. (2010). Effect of the different auxins and cytokinins in callus induction, shoot root regeneration in sugarcane. International Journal of Pharma and Bio Sciences, 1(3).
Ilahi, I., Jabeen, M. & Firdous, N. (1987). Morphogenesis with saffron tissue cultures. J. Plant Physiol., 128: 227-232.
Iranian, S., Masoumian, M., & Masoudsinki., J. (2014). The effect of NAA and IBA hormones on rooting of the medicinal plant Aloe vera. Iranian Congress of Agricultural Sciences and Plant Breeding.
Izanloo, A., Derakhshan, A., Alizadeh, Z., & Behdani, M. (2019). Cormlet production of saffron (Crocus sativus L.) using in vitro Culture Techniques. Journal of Saffron Research, 6(2), 179-189.
Khodakovaskaya, M., Silva, K., Biris, A. & Villagarcia, H. (2012). Carbon nanotubes induce growth enhancement of tobacco cells. ACS Nano, 6(3): 2128-2135.
Khodakovaskaya, M., Kim, B. S., Kim, J. N. & Cernigla, C. (2013). Carbon nanotubes as plant growth regulators: effects on tomato growth, reproductive system, and soil microbial community. Small, 9(1): 115-123.
Manouchehri, P., A.Milani, S., & Abolghasemi, H. (2021). Use of response surface methodology for optimizing process parameters of thorium adsorption on amino-functionalized titanosilicate nanoparticles. Journal of Nuclear Science, Engineering and Technology (JONSAT), 42(1), 57-66.
Moral, S., Rao, R. & Chakrapani, R. (2011). Factors affecting seed germination and seedling growth of tomato plants cultured in vitro conditions. Chemical, Biological and Physical Sciences, 1(2): 328-334.
Moradi, S., Fallahi, H. R., Behdani, M. A., & Mahmoodi, S. (2024). The effect of corm storage conditions during the summer dormancy stage on reproductive growth and yield of saffron. Journal of Saffron Research, 12(1), 1-14. doi: 10.22077/jsr.2020.3747.1141
Plessner, O., Ziv, M. & Negbi, M. (1990). In vitro corm production in the saffron (Crocus sativus L.). Plant Cell Tissue Organ Cult, 20: 89-94.
Ramandi, A., Gholizadegan, A., & Seifi, A. (2022). Optimization of callogenesis and cell suspension culture in saffron. Journal of Saffron Research, 10(2), 276-284. doi: 10.22077/jsr.2022.5718.1198
Raja, W., Zaffer, G. & Wani, S. (2007). In vitro microcorm formation in saffron (Crocus sativus L.). Acta Hortic, 739: 291-296.
Sajjadi, M., & Pazhouhandeh, M., (2015). Study on effect of type of explant and hormone on callus induction and regeneration in saffron (Crocus sativus L.), Saffron Agronomy and Technology, 3(3), 195-202. magiran.com/p1445584
Sharifi, G., Ebrahimzadeh, H., Ghareyazie, B. & Karimi, M. (2010). Globular embryo-like structures and highly efficient thidiazuron-induced multiple shoot formation in saffron (Crocus sativus L.). In Vitro Cell. Dev. Biol. Plant, 46: 274-280.
Simona, L., Petolescu, C., Florina, F., Lazar, A., Giancarla, V., Danci, M. & Maria, B. (2013). In vitro regeneration of Crocus sativus L. The Journal of Horticultural Science and Biotechnology, 17, 244-247.
Tadayon, M., Falah, S., Fadaei, A., & Norouzim, S. (2013). Effects of multi wall carbon nanotube and nanosilver on some physiological and morphological traits of faba bean (Vicia faba L.). Journal of Plant Process and Function, 2(3), 61-72.
Tang, J., Chen, X., Katuyoshi, S. (2002). The influences of culture conditions on the callus induction, tissue culture and regulation of secondary metabolism of eucommia ulmoides oliver, Journal of Zhejiang University (Engineering Science), 2(36), 193-198.