مهندسی متابولیت ثانویه در کشت سوسپانسیون سلولی گیاه دارویی هندوانه ابوجهل (Citrullus colocynthis (L). Schrad.) با حضور محرک زیستی و غیر زیستی

نوع مقاله : مقالات پژوهشی

نویسندگان

• معصومه سهرابی ¹
• داود صمصام پور ¹
• عبدالنبی باقری ²

¹ گروه علوم باغبانی، دانشکده کشاورزی و منابع طبیعی، دانشگاه هرمزگان، ایران

² عضو هیات علمی، استادیار گروه تحقیقات گیاه پزشکی، مرکز تحقیقات کشاورزی و منابع طبیعی هرمزگان، سازمان تحقیقات، آموزش و ترویج کشاورزی

10.22067/jhs.2022.75356.1142

چکیده

گیاهان دارویی در طول تاریخ جزء منابع اصلی پزشکی و داروسازی در اکثر نقاط جهان بوده­اند. از جمله این گیاهان می­توان به هندوانه ابوجهل با نام علمیCitrullus  colocynthis (L). Schrad.  اشاره کرد. کشت سوسپانسیون سلولی یکی از موثرترین و پرکاربردترین روش­ها برای بررسی میزان تغییرات متابولیت­های ثانویه است. همچنین با توجه به نقش الیسیتورهای قارچی در افزایش میزان متابولیت­های ثانویه، از قارچ‌های اندوفیت Alternaria solani، Fusarium sp. و Setosphaeria rostrata استخراج شده از گیاه هندوانه ابوجهل (جمع آوری شده از استان هرمزگان) به عنوان الیسیتور زیستی در محیط کشت سوسپانسیون سلولی استفاده شد. این مطالعه بر اساس آزمایش فاکتوریل در قالب طرح کاملاً تصادفی با دو عامل (عامل اول شامل دو سطح ترکیب هورمونی مختلف و عامل دوم نیز هشت سطح از عصاره قارچ­های اندوفیت) با سه تکرار در آزمایشگاه بیوتکنولوژی دانشگاه هرمزگان انجام شد. نتایج مقایسه میانگین اثر متقابل تیمار تنظیم کننده رشد × عصاره قارچی نشان داد که در شرایط ترکیب تیماری هورمون 1mg 2,4-D + 1mg BA و تلقیح توام گیاه با سه قارچ، میزان فنول و فلاونوئید به ترتیب 11/62 و 18/49 درصد نسبت به شاهد افزایش یافت و در سطح احتمال یک درصد و پنج درصد معنی دار بود، اما فعالیت آنتی­اکسیدانی به میزان 78/62 درصد در ترکیب تیمار هورمونی 1mg 2,4-D + 1mg Kin و تلقیح توام سه قارچ نسبت به شاهد افزایش نشان داد و در سطح احتمال یک درصد معنی دار بود. بطورکلی نتایج نشان داد که برهمکنش قارچ و هورمون می‌تواند نقش موثری در افزایش میزان متابولیت­های ثانویه گیاه دارویی هندوانه ابوجهل داشته باشد. این کار از طریق فعال کردن مسیرهای هورمونی انجام می­شود که در تولید متابولیت­های ثانویه نقش دارند.

کلیدواژه‌ها

• الیسیتور
• اندوفیت
• سوسپانسیون سلولی
• متابولیت‌ ثانویه
• هندوانه ابوجهل

20.1001.1.20084730.1402.37.1.16.0

موضوعات

• گیاهان دارویی

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عنوان مقاله [English]

Engineering of Secondary Metabolites in Cell Suspension Culture of Citrullus colocynthis (L). Schrad. with the Presence of Biological and Non-biological Stimulants

نویسندگان [English]

• M. Sohrabei ¹
• D. Samsampoor ¹
• A. Bagheri ²

¹ Horticulture Sciences Department, Faculty of Agriculture and Natural Resource, University of Hormozgan, Bandar Abbas, Iran

² Associate Professor of Plant Protection Research Department, Hormozgan Agricultural and Natural Resources Research and Education Center, Agricultural Research Education and Extension Organiziation (AREEO), Bandar Abbas, Iran

چکیده [English]

Introduction
 Medicinal plants have historically been one of the main sources of medicine and pharmacy in most parts of the world, that among these plants, we can mention the species Citrullus colocynthis L. Schrad. Cell suspension culture is a widely used method to increase the rate of secondary metabolites. The secondary metabolites of plants, are species compounds often produced during a certain period of growth and development and have important ecological functions in plants. They induce the ability of plants to cope with herbivores, microbial pathogens, adsorbents, and seed-spreading organisms. Also, due to the role of fungal elicitors to increase the rate of secondary metabolites in plants, in this study, we studied the role of the endophytes Alternaria solani. Fusarium sp. and Setosphaeria rostrata extracted from Citrullus colocynthis L. Schrad were collected from different regions of Hormozgan province as bio-elicitors in a cell suspension culture medium.
Materials and Methods
 The study was performed based on a factorial experiment in a completely randomized design with two factors (the first factor had two levels of different hormonal composition and the second factor had eight levels of endophytic fungal extracts) with three replications in the biotechnology laboratory of Hormozgan University and the results were analyzed statistically using SAS 9.4 software. To produce the callus and culture of cell suspension under hormone treatment, watermelon seeds were first disinfected for a period of time and then the seeds were transferred to a culture medium containing MS and placed in a suitable incubator for seed germination. After germination and leaflet production, pieces with an area of approximately 1 mm² were separated from the primary leaves and for callus formation were transferred to Petri dishes containing MS medium (3% sucrose, 0.8% agar), with two levels of 1mg 2,4-D + 1mg BA and 1mg 2,4-D + 1mg kin and placed in a suitable incubator for three weeks. Three fungal endophytes Alternaria solani, Setosphaeria rostrata, and Fusarium sp. were transferred separately to PDA (Potato Dextrose Agar) culture medium to prepare the bio elicitor and placed at 30 ° C for 7 days. From 7-day cultures, 1 cm² of mycelium was isolated and inoculated into 150 ml of PDB (Potato Dextrose Broth) culture medium. The cultured cells were stored at 30 ° C in a 500 ml Erlenmeyer flask and placed on a shaker for 7 days at 120 rpm. The fungal cells were isolated and dried at 65 ° C for 24 hours. The powder from the dried cells was dissolved in water (10 g / l) and autoclaved for 20 minutes at 121 ° C. The extracts of these cells were finally used as a bio elicitorto study the change in the number of secondary metabolites. After that, the growth rate of cells in cell suspension culture was measured before and after the application of fungal extract. Parameters such as total phenol content, antioxidant activity, and flavonoids were also studied.
Results and Discussion
The results showed that the treatment combinations of 1mg 2,4-D + 1mg BA and inoculation of the plant with three fungi, the amount of phenol and flavonoids increased by 62.11% and 49.18%, respectively, compared to the control and at the levels of 1% and 5% probability were significant  and was observed in the combination of hormonal treatment 1mg 2.4-D + 1mg Kin and inoculation of three fungi, the amount of antioxidant production increased by 62.78% compared to the control and at the levels of 1% probability was significant. The results indicated that the cell extraction of the fungal endophyte Alternaria solani, Fusarium sp., and Setosphaeria rostrata under condition hormonal treatment can be used as an effective stimulant in increasing the amount of secondary metabolites (phenol, flavonoids and  antioxidant) of Citrullus colocynthis L. Schrad.
 
Conclusion
 It was revealed that by adding the elicitor to the culture medium, cell growth was increased. The results showed that the combination of three types of endophytic fungi Alteynaria solani, Setosphaeria rostrata and Fusarium sp. led to a significant increase in cell dry weight compared with the control treatment. Also, an increase in cell growth was observed even when a fungal extract was used alone. The amount of metabolites in cells treated with fungal extracts (fungal elicitors) was significantly higher than metabolites produced in the control. According to the results of this experiment, using a combination of three fungal extracts was the best treatment to increase the metabolite production in the culture of cell suspension of Citrullus colocynthis L. Schrad.

کلیدواژه‌ها [English]

• Cell suspension
• Citrullus colocynthis L. Schrad
• Elicitor
• Endophyte
• Secondary metabolite

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