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

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.

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 gl-1) in order to induce salinity stress. Various physiological aspects and stress related mechanisms such as growth, osmo-protection 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, glycine-betaine content, and the activity of betaine aldehyde dehydrogenase (BAD) were studied as a part of osmo-protection 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. Glycine-betaine content was measured using spectroscopy method by KI-I2 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 nitro-blue tetrazolium reduction activity. Malondialdehyde content was also measured by using spectroscopy method.

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 gl-1. This slow growth was also observed at high stress levels such as 12 gl-1. 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 glycine-betaine content also increased significantly. The highest level of glycine-betaine content and also the activity of BAD activity were observed in 12gl-1 NaCl treated calli. A significant correlation between glycine-betaine 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 gl-1 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 12gl-1 NaCl treated calli. A significant correlation between malondialdehyde content and the activity of free radical scavenger enzymes was observed.

In general, the calli of Dianthus barbatus cultured in vitro are sensitive to salinity stress induced by more than 1 gl-1 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 gl-1 NaCl and survived without chlorosis.