تاثیر تنش شوری درون شیشه‌ای بر شاخص‌های رشدی، محافظت اسمزی و تنش اکسیداتیو در پینه گیاه قرنفل

قرنفل (dianthus barbatus l.) یکی از مهمترین گیا‌هان فضای‌ سبز در مناطق معتدل است که به‌عنوان گیاه باغچه‌ای، گلدانی و همچنین گل‌بریدنی کشت می‌گردد. شوری خاک و آب در بخش‌های وسیعی از کشور مهمترین مشکل پیش‌رو در گسترش فضای ‌سبز شهری آن می‌باشد. باتوجه به اهمیت قرنفل در فضای ‌سبز شهری و درک بهتر مبانی فیزیولوژیکی پاسخ گیاه به تنش در محیط درون شیشه‌ای، در این پژوهش تاثیر تنش شوری بر گیاه قرنفل در شرایط درون شیشه‌ای مورد بررسی قرار گرفت. بدین منظور پینه‌های حاصل از ریزنمونه‌های برگ که در محیط‌کشت ms حاوی 2 میکرومولار bap + 6 میکرومولار naa پرآوری گردیده بودند، تحت تاثیر سطوح مختلف کلریدسدیم (0، 1، 3، 6، 9 و 12 گرم‌درلیتر) در محیط کشت ms قرار گرفتند. سپس شاخص‌های مختلف فیزیولوژیکی (همچون رشد، نرخ رشد نهایی، وزن‌تر و خشک) و بیوشیمیایی (همچون میزان تغییرات پروتئین‌کل، اسمولیت گلاسینبتائین، فعالیت آنزیم بتائین آلدهایددهیدروژناز و همچنین میزان فعالیت آنزیم‌های کاتالاز، پراکسیداز، سوپراکسیددسموتاز و میزان بیومارکرتخریبی مالون‌دی‌آلدهاید) در پینه‌های تنش‌یافته مورد بررسی قرار گرفت. نتایج نشان داد با افزایش سطح تنش، رشد پینه‌ها کاهش و درنتیجه از وزن‌تر و خشک آن‌ها نیز کاسته شد. از سوی دیگر میزان پروتئین کل، فعالیت آنزیم بتائین آلدهایددهیدروژناز و درنتیجه اسمولیت گلاسینبتائین با افزایش سطح تنش، افزایش یافت. فعالیت آنزیم‌های شکارکننده رادیکال آزاد همچون کاتالاز، پراکسیداز و سوپراکسیددسموتاز نیز با افزایش میزان تنش به‌طور‌معنی‌داری افزایش یافت. در بین سه آنزیم شکارکننده رادیکال‌آزاد، پراکسیداز کمترین میزان فعالیت و کاتالاز بیشترین میزان فعالیت را درنتیجه تنش داشتند. پراکسیداز همچنین کمترین میزان افزایش فعالیت در نتیجه تنش را به خود اختصاص داد. این درحالی بود که افزایش میزان فعالیت در دو آنزیم کاتالاز و سوپراکسیددسموتاز تقریباً به یک اندازه مشاهده گردید. بیومارکر تخریبی مالون‌دی‌آلدهاید نیز علیرغم افزایش فعالیت آنزیم‌های شکارکننده رادیکال‌آزاد با افزایش تنش شوری، افزایش یافت. به طور کلی نتایج این تحقیق نشان داد که گیاه قرنفل در محیط درون شیشه‌ای به تنش شوری حساس بوده و آستانه تحمل آن به شوری محیط غلظت 1 گرم در لیتر کلریدسدیم می‌باشد. این درحالی است که، پینه‌های این گیاه تا شوری 12 گرم در لیتر کلریدسدیم مقاوم بوده و ضمن رشد بطئی به حیات خود ادامه دادند.;

Effect of in Vitro Salinity Stress on Growth Parameters, Osmoprotection and Oxidative Stress of Dianthus barbatus L. Calli

Introduction: Dianthus barbatus L. from Caryophyllaceae is an important herbaceous landscape ornamental plant in the temperate regions throughout the world. Although this ornamental plant is used as bedding plant, it also has other extensive applications such as cut flower and pot plant. Besides, in some countries the leaves of Dianthus barbatus are used for treatment of digestive disorders in their traditional medicine. Landscape and its quality, plays an essential role in human life. Nowadays, maintenance and development of urban landscape in many regions such as Iran is facing major challenging issues. Soil and water salinity are the most important challenges of urban landscape. Considering the challenges which landscape development faces in many parts of the country and the importance of this plant in landscape, the effect of salinity on D. barbatus during in vitro condition was studied in order to obtain some basic physiological information for future stress studies on this plant.;Materials and Methods: In vitro sterile plants were initially obtained from seeds by culturing them on basal MS medium. Calli were obtained by culturing leaf, stem and root explants on previously reported callogenesis optimization medium which was MS medium supplemented with 2µM BAP + 6µM NAA. Calli proliferation was then conducted on the same media. Proliferated calli with same size were subjected to MS medium supplemented with the mentioned plant growth regulators and various NaCl concentrations (0, 1, 3, 6, 9, 12 gl1) in order to induce salinity stress. Various physiological aspects and stress related mechanisms such as growth, osmoprotection and oxidative stress mechanisms were consequently studied in the stressed Dianthus barbatus calli. Studied growth features were calli growth, final growth rate, fresh and dry weight. Total protein content, glycinebetaine content, and the activity of betaine aldehyde dehydrogenase (BAD) were studied as a part of osmoprotection mechanism. On the other hand, the activity of free radical scavenger enzymes such as sccccuperoxide dismutase (SOD), catalase (CAT), peroxidase (POX) and malondialdehyde damage biomarker content were studied in stressed calli as a part of oxidative stress mechanism. Total protein was measured using Bradford method. Glycinebetaine content was measured using spectroscopy method by KII2 reaction agent. The activity of betaine aldehyde dehydrogenase was evaluated using spectroscopy method by reduction with NAD+. SOD activity was measured using spectroscopy method and formation as an indicator. CAT activity was measured using spectroscopy method by measuring hydrogen peroxide breakdown. POX activity was measured using spectroscopy method by nitroblue tetrazolium reduction activity. Malondialdehyde content was also measured by using spectroscopy method.;Results and Discussion: Results indicated that by increasing salinity level, calli growth and consequently fresh and dry weight reduced. Calli growth was dramatically reduced as salinity level reached above 1 gl1. This slow growth was also observed at high stress levels such as 12 gl1. By salinity increment, total protein content of the stressed calli significantly reduced. On the other hand, BAD activity increased as a result of salinity stress and consequently glycinebetaine content also increased significantly. The highest level of glycinebetaine content and also the activity of BAD activity were observed in 12gl1 NaCl treated calli. A significant correlation between glycinebetaine content and BAD activity was observed. The activity of free radical scavenger enzymes such as SOD, CAT, POX also significantly increased by salinity stress increment. The highest activity of the mentioned free radical scavenger enzymes were observed in 12 gl1 NaCl treated calli. CAT had the most activity among the mentioned studied free radical scavenger enzymes. The highest increase in POX activity was half of the control and also half of the activity increment of other studied free radical scavenger enzymes. Although the activity of free radical scavenger enzymes increased, the level of malondialdehyde damage biomarker also increased as the result of salinity stress increment. The highest amount of malondialdehyde content was seen in the 12gl1 NaCl treated calli. A significant correlation between malondialdehyde content and the activity of free radical scavenger enzymes was observed.;Conclusion: In general, the calli of Dianthus barbatus cultured in vitro are sensitive to salinity stress induced by more than 1 gl1 NaCl; indicating the stress tolerance threshold. The tolerance and resistance mechanisms performed well under stress condition and the stress calli resisted to salinity up to 12 gl1 NaCl and survived without chlorosis.