ارزیابی شرایط برای نگهداری طولانی مدت کالوس حاصل از بنه زعفران

زیرکشت کالوس‌های گیاهی، نه تنها فرایندی پرهزینه و وقت گیر است بلکه احتمال آلودگی سلول ها، تغییرات ژنتیکی و امکان تغییر در ماهیت متابولیت ها نیز وجود دارد. در این تحقیق روش‌های افزایش مدت زمان نگهداری کالوس‌ حاصل از بنه زعفران به نحوی که قابلیت باززایی سلول‌ها حفظ شود، بررسی شد. این روش‌ها شامل نگهداری در یخچال، دمای اتاق و در زیر لایه ای از پارافین بودند. در تیمار کنترل، کالوس ها در دمای اتاق بدون حضور پارافین نگهداری شده و هر 20 روز یکبار زیرکشت شدند. بعد از اتمام دوره کشت، کالوس ها به محیط sh مایع با ترکیب هورمونی naa(2mg/l)+ba(1mg/l) انتقال یافتند. سپس در طی 6 هفته پارامترهای شاخص رشد سلولی، تغییرات پی اچ محیط کشت و تعداد سلول های زنده و غیرزنده اندازه گیری شدند. در انتهای دوره کشت میزان کروسین سلول‌ها با استفاده از روش اسپکتروفتومتری اندازه‌گیری شد. نتایج نشان داد بیشترین میزان رشد سلول از کالوس هایی بدست آمد که در دمای 4 درجه سانتیگراد نگهداری شده بودند. از طرفی کالوس هایی که در زیر لایه ای از پارافین نگهداری شدند، قابلیت تکثیر در کشت تعلیقی را از دست دادند. بر اساس این نتایج، کالوس های حاصل از بنه زعفران را می‌توان بدون نیاز به زیرکشت، تقریباً سه ماه در یخچال نگهداری کرد. از بین تیمارهای بررسی شده فقط تیمار کنترل حاوی 1/1 میلی‌گرم کروسین در هر گرم سلول خشک بود.  با توجه به اینکه این مطالعه به‌عنوان اولین بررسی در روش‌های نگهداری طولانی مدت کالوس زعفران انجام شد بدیهی است در این راستا به تحقیقات بیشتری در زمینه افزایش متابولیت‌های آن پس از نگهداری طولانی مدت نیاز است.

assessment of conditions for long storage of saffron corm-derived callus

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.