The purpose of this work was to stimulate drought stress using polyethylene glycol 6000 and modulate it by nano silicon dioxide in two Iranian damask rose genotypes under invitro conditions. In this case, according to our preliminary experiments, two genotypes of damask rose were choosed for more experiments according to their reaction to drought stress (data not published yet). Explants from the mid-stem region of one-year stemes (0.4-0.6 cm in diameter) of two local Damask rose (R. damascena Mill) from University of Maragheh in west north of Iran (37.3892° N, 46.2534° E) and Kashan (33.9850° N, 51.4100° E) in central region of Iran were chosed. The plant material and seeds for wild collections were obtained under the supervision and permission of Maragheh University guidelines and according to national guidelinesand all authors comply with all the local and national guidelines.The central part of vegetative shoots of damask rose having axillary buds were chosed for the experiment. At the first Each of 1.5 to 2 cm shoot explants were washed with a commercial disinfectant solution for 20 min and then rinsed with running tap water. The mentiond explants were sterilized by 10% chlorox solution for 15 min and then washed 3 times in ddH2O. Finally they were planted in culture bottles having 25 ml of MS culture medium salts68 and vitamins plus 30 g L-1 agar and 360 µg-1 L benzyl adenine and 30 µg L-1 NAA. pH of the media was adjusted to 5.7 using NaOH or HCl. All jars containing explants were placed in a germinator with a temperature of 25 °C and 8 hours of darkness, 16 hours of light and 60-70% humidity. After transferring the explants to the germinator, screen them daily and, if there is fungal or bacterial contamination, remove the glasses and autoclave to remove the contamination. Approximately seven days after the establishment of the explants, the first traces of bud growth appeared, and finally, after four to five weeks, when the explants had grown sufficiently, they were taken out of the germinator to be filled and placed in a proliferating environment. For the experiment we used of regenerated plants (~4 cm) after 35 days as an experiment explants (Figure 9) and transferred to sterile bottles including 25 ml of medium (five shoots per bottle), as experiment materials.
Preparing the treatment medium including PEG and nSiO2
Polyethylene glycol was used to induce drought stress. For this purpose, treatments applied at five levels (0, 25, 50, 75 and 100 g L-1) or with osmotic pressure of 0, -0.2, -0.5, -0.7 and -0.9 MPa on two genotypes was applied. After preparing the propagation medium, the shoots were placed in culture medium. After preparing the mentioned concentrations and complete dissolution of PEG in water and adjusting the pH, it was added to the culture medium so that it is one centimeter higher than the medium, then five shoots were placed in each bottle and transferred to the germinator and the level of proliferation of explants, was evaluated fter 4 weeks. Nano particles of silicon (size˂50 nm) were used in our experiment were bought from the NANOSANY Corporation (Mashhad, Iran) the same as our last work20, and prepared at three levels (0, 50 and 100 mg L-1) and then suplemented to the culture medium in phase suspension in MS medium68. Then five shoots were placed in each glass and transferred to the germinator, after About 14 days collect them and measured the traits. All invitro culturs were maitaned at 23 ± 2ºC under a 16/8 h day/night photoperiod provided by coolwhite fluorescent lamps at 40 µmol m-2 s-1 (Philips TLD 36W/95). After About 14 days collect them and measured the traits.
Mesurment of physiological traits of Rosa damascena
Leaf relative water contents. The amount of RWC of leaf was determined in the fully expanded topmost leaf of explants. At the first the fresh weight of the leaves was recorded and then they were plunged in ddH2O in a Petri dish. After 2 h and removing the surface water of samples, their turgid weight were recorded. The sample leaves were then placed in an oven at 70°C and deried to reach a stable weight. The amount of relative water content of leaves was calculated as the methods described by Turner69.
Membrane Stability Index. The leaves were cut into the small samples with the same size. Then leaf discs were weighed and transferd to the test tubes having 10 mL of ddH2O. The mentined tubes were transferd to a water bath with 40°C for 30 min and then the EC of the samples was recorded. The samples were placed in to the other test tubes and incubated at 100°C in the boiling water bath for 15 min, and their EC was recorded as mentined before. The amount of MSI was evaluated by means of the following formula70:
EL%= [ EC1/EC2)] x100
Measurement of photosynthetic pigments and chlorophyll fuorescence of leaf. Chlorophyll a, Chlorophyll b, total Chlorophyll and carotenoids were evaluated in leaves of explants according to the method of Arnon 71 by means of spectrophotometer (Shimadzu, Model UV 1800, Kyoto, Japan) at 470, 663 and 645 nm, respectively and calculated as mg g Fw-1. The Chlorophyll parameters of Rosa damascena explants were measured using a portable photosynthesis meter (Walz GmbH Eichenring, 691090 Efeltrich, Germany) at the end of experiment. Minimal fuorescence, F0, was evaluated in leaves after 30 min dark-incubation and then for measuring the maximal fuorescence, Fm, we used of the mentined leaf samples under full light conditions. Maximal variable fuorescence (Fv) and the photochemical efficiency of PSII (Fv/Fm) were then evaluated from the recorded parameters72.
Mesurment of biochemical traits of Rosa damascena
Hydrogen peroxide (H2O2) determination. The amount of hydrogen peroxide in explants was carried out following a previously established method by Liu et al.73. In this case, 0.5 g of leaf tissues were ground in liquid nitrogen and a potassium phosphate buffer (KPB) (pH 6.8). the grounded leaf sampls were centrifuged at 7000 rpm for 25 min at 4°C. A 100-µL aliquot of the supernatant was added to 1 mL of xylenol solution, completley mixed and let the solution to rest for 30 min. the amount of hydrogen peroxide which is related directly to the intensity of the color and represents ts amount in the samples, was evaluated by spectrophotometer (Shimadzu, Japan) at 560 nm and recorded as µmol gFw-1.
Malondialdehyde (MDA) determination. MDA was determined as 2-thiobarbituric acid (TBA) reactive metabolites74. About 1.5 mL extract of each samples were homogenized in 2.5 mL of 5% TBA made in 5% trichloroacetic acid (TCA). The solution was warmed at 95°C for 15 min, and then cooled on ice, quickly. After centrifugation at 5000 rpm for 10 min, the amount of the supernatant absorbance was recorded at 532 nm. The level of malondialdehyde was measured as nmol gFw-1 according to the following equation.
MDA=1000× [(532nm – 600nm) × 1/049]/155
Proline determination. The amount of proline was measured by homogenizing 0.2 g fresh weight of leavs in 2 mL of 3% aqueous sulfo salicylic acid and then centrifuged at 10000 rpm for 30 min. The supernatant was removed and the pellet was washed with 3% aqueous sulfo salicylic acid for two times. The supernatant was pooled and the amount of proline was evaluated using ninhydrin reagent and toluene extraction75 and the protocol for each determination was calibrated with standard curve of proline solution within the detection range of the method (0-39 μg mL-1).
Protein determination. The amount of protein was measurd following the Bradford method76 and the method was calibrated for each determination with bovine serum albumin standard curve. In this case, 100 mg treated explants were placed in a test tube with 2 mL of 50 mM potassium phosphate buffer at pH 7.0. The solution were centrifuged at 7000-12000 rpm. Then supernatant was recovered and centrifuged at 3000 rpm for 15 min at 4°C. Samples were praeperd with 1:100 dilution ratio and measured at 595 nm and recorded in terms of mg g Fw-1.
Analysis of Antioxidant enzyme activities. One gram of leaf samples was weighted and quickly homogenized in 5 mL of 50 mM K–phosphate buffer (pH 7.0), brought to 5 mM Na–ascorbate and 0.2 mM EDTA by the addition of concentrated stocks. The homogenated samples were centrifuged at 10000 rpm for 15 min at 4°C. Then the supernatants were used for enzyms assays and were carried out at 4°C. The activity of SOD, POD and CAT was measured, as previously established by Li et al.77 Fresh leaf samples (0.5 g FW) were chosed from 2-week-old treated explants, harvested and ground in liquid nitrogen and extracted with following described method: 100 mM potassium phosphate buffer (pH 7.8) including 0.1 mM EDTA, 1% (w/v) PVP and 0.1% (v/v) Triton x100. The extracted solution was centrifuged at 10,000 rpm for 15 min at 4°C. The supernatant was collected and used for measuring the activity of enzyms. Guaiacol peroxidase activity was assayed by monitoring the increase in absorbance at 470 nm (ε = 26.6 mM -1cm -1) during polymerization of guaiacol. One unit of activity was defined as the amount of enzyme producing 1 µmol of tetraguaiacol per min at 25°C.
The experiment was conducted as a completely randomized design with 3 replications and five explants in each bottle. Data were statistically analyzed by MSTAT-C software and the means were compared using LSD method and at the level of five percent error probability