Assessment of Conditions for Long Storage of Saffron Corm-Derived Callus

Amini, Somaye; Ziaratnia, Seyed Mahdi

doi:10.22077/jsr.2025.8554.1258

Document Type : Original Article

Authors

¹ PhD, Department of Horticultural Science, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran &Adjunct Professor of Horticultural Sciences Department, Birjand University, Birjand, Iran.

² Department of Food Biotechnology, Research Institute of Food Science and Technology, Academic member

https://doi.org/10.22077/jsr.2025.8554.1258

Abstract

Introduction: The efficient long-term storage of plant callus tissues plays a crucial role in preserving and capitalizing on plant genetic resources. Traditional subculturing techniques can be laborious and expensive, often resulting in contamination, genetic instability, and alterations in metabolite profiles. Recent researches suggest that lowering storage temperatures, while avoiding freezing conditions, can significantly improve the viability of callus cultures by inhibiting metabolic activity. This strategy may reduce the necessity for frequent subculturing while maintaining the quality of the cultures. This study sought to discover an effective method for preserving saffron (Crocus sativus L.) callus, with a specific focus on sustaining its physiological functions, particularly its regenerative capabilities.

Materials and Methods: This study was conducted at the Research Institute of Food Science and Technology (RIFST) in Mashhad, focusing on the regenerative capability of saffron corm-derived calli. Saffron corms were harvested in mid-May and subjected to surface sterilization through washing with tap water, immersion in 70% ethanol for 1 minute, and treatment with 1% sodium hypochlorite solution. Residual disinfectants were removed by rinsing with sterile water. The sterilized corms were sliced into 1 cm² with 1-2 mm thickness and cultured on solid Gamborg’s medium (B₅) supplemented with 2 mg/L NAA, 1 mg/L Kinetin (Kin), and 3% sucrose. The cultures were incubated in the dark at 22±2 °C, with subculturing occurring every 20 days to promote callus development. Upon reaching sufficient callus volume, the samples underwent various storage treatments: calli were stored at 4 °C and 22±2 °C, both with and without liquid paraffin coverage for three months without subculturing. The control group comprised calli maintained at room temperature with routine subculturing every three weeks. After the storage period, all calli were transferred to a liquid SH medium containing 2 mg/L NAA, 1 mg/L 6-benzyladenine (BA), and 3% sucrose for six weeks. Parameters including cell growth index, medium pH, and cell viability (live versus dead), were assessed every week. At the end of the culture period, the amount of crocin as the main secondary metabolite in the saffron cells was measured using spectrophotometric method.

Results and Discussion: The results demonstrated that the stored calli at 4 °C showed the highest cell growth index after transfer to liquid culture medium. In contrast, calli covered with liquid paraffin at both 4 °C and 22±2 °C exhibited a complete loss of regeneration capacity in suspension culture. Interestingly, low-temperature storage not only maintained but also enhanced cell growth index (1.02) in liquid culture when compared to the control treatment (0.42). It is due to the growth of live cells which was evidenced by live cell counts using trypan blue staining. These findings highlight the critical role of temperature regulation in the successful storage of plant callus tissues. Our study also aligning with prior researches as they suggest that refrigerated storage is suitable for the preservation of calli derived from chilling-tolerant plants, but it is not suitable for chilling-sensitive species.

Conclusion: This study concludes that saffron corm-derived calli can be effectively maintained with preserved regenerative capacity when stored at 4 °C for three months without subculture. Additionally, the regeneration capacity was optimized, reflected by a high ratio of live to dead cells after transferring to a cell suspension culture. Thus, storage of saffron calli at 4 °C is a reliable and efficient method for long-term conservation. Among the studied treatments, only the calli maintained under control conditions showed crocin content (1.1 mg of per gram of dry cells). Considering that this study was the first to investigate long-term storage methods for saffron calli, it is obvious that more research is needed to increase metabolites after the long storage of calli.

Keywords

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