MATERIALS AND METHODS

Plant material: Carnations (Dianthus caryophyllus cv. White) were grown in the greenhouse standard production methods (pakdasht, Tehran, Iran). The experiment was started on February 15, 2010 and chlorophyll content, Membrane stability, MDA content and ACC oxidase activity were measured at 11th day of vase life. The experimental site was in horticulture laboratory of agriculture faculty of University Azad Karaj, Tehran, Iran. Flowers were weighed initially immediately after harvest and used for setting treatments. The factors were three levels of salicylic acid (0, 1.5, 3 mM) and two levels of sucrose (0.3%). The flowers were individually placed in bottles containing 250 mL of preservative solution and were held at ambient temperature (19±5°C).

Determination of anthocyanin leakage and ACC oxidase activity: Anthocyanin leakage was used to assess membrane permeability and measured using spectrophotometer. The procedure used was based on the method of Poovaiah (1979). ACC oxidase activity was determined according to the method described by Moya-Leon and John (1994).

Assays of MDA content (Lipid per oxidation): Oxidative damage to lipids was estimated by measuring the content of MDA in floret segment homogenates, prepared in 10% trichloroacetic acid containing 0.65% 2-Thiobarbituric Acid (TBA) and heated at 95°C for 25 min, as described by Heath and Packer (1968). MDA content was calculated by correcting for compounds other than MDA which absorb at 532 nm by subtracting the absorbance at 532 nm of a solution containing plant extract incubated without TBA from an identical solution containing TBA.

Chlorophyll (a + b) content measurement: Chlorophyll total (a + b) content was measured by Chlorophyll meter SPAD-502, Minolta Co. Japan which represented by SPAD value. The petal was inserted into the meter and measured SPAD value 3 times from different spot of a single leaves.

Microbe population: Test Microbe population were isolated from vase solutions of carnations. When the flowers had senesced (about 11 days), aliquots of the vase solutions were diluted 100-times and 25 μL aliquots of the diluted solution were spread on sterile nutrient agar, in sterile petri plates. The plates were allowed to incubate for 48 h at room temperature and individual colonies of microorganisms, representing the most common colony morphology types, then were picked off the agar media with a sterile loop and streaked on EMB medium for purification. Purified microbe population were maintained axenically on EMB medium and transferred daily to fresh medium.

Vase life: Vase life was considered to be terminated when wilting occurred.

Experimental design and statistical analysis: Experiment was arranged in a factorial test with complete randomized design with four replications. Analysis of variance was performed on the data collected using the General Linear Model (GLM) procedure of the SPSS software) Version 16, IBM Inc.). The mean separation was conducted by Tukey analysis in the same software (p = 0.05).

RESULTS AND DISCUSSION

Anthocyanin leakage and Acc-oxidase activity: Table 1 and 3 showed the effects of SA on the anthocyanin leakage of the petals of carnation under SA and SA+ SU treatment compared to the control. Two treatments (SA 1.5 mM and SA + SU (SA1.5 + SU3%) improved membrane permeability by the decrease of anthocyanin leakage and ACC-oxidase activity (Table 1, 3) while, two treatments (3 mM and 3 mM + SU 3%) impaired membrane permeability by increasing anthocyanin leakage and increase ACC-oxidase activity (Table 1, 3) (p≤0.05). Result showed that addition of 1.5 mM SA maintained membrane permeability, 1.5 mM SA with or without SU could alleviate or decrease cell wall damages (Table 3). It is evident from the data presented in Table 1 that the maximum anthocyanin leakage was recorded in 3 mM SA compare another treatments and control (p≤0.05). Present results showed that adding SA in vase solution can prevent anthocyanin leakage by maintained PH vase solution. Results also showed that adding SA was found to be positively correlated with anthocyanin leakage of the carnation cut flower (Table 2) (p≤0.01).

Table 1:	Mean comparisons of chlorophyll content, vase life, MDA, bacteria populations in vase flower preservative solution, antocyanin leakage and ACC oxidase activity in SA treatment
Means in each column followed by similar letters are not significantly different at 5% level using Duncan multiple rang test

Table 2:	Simple correlation lines between the SA treatment with other variables
*, **: Significant different at 5 and 1% level, respectively

Table 3:	Mean comparisons of chlorophyll content, Vase life, Antocyanin leakage, MDA content, bacteria populations in vase flower preservative solution and ACC Oxidase Activity in SA*SU treatment
Means in each column followed by similar letters are not significantly different at 5% level using Duncan multiple rang test

The produce ethylene is in petal cut flower harmful to cut flowers and their consequent accelerate flower senescence, anthocyanin leakage and cell death. Adding a suitable inhibit produce ethylene in vase solution can prevent accelerate flower senescence, anthocyanin leakage and cell death. Present results are in agreement with previous scientists who have reported decreased accelerate flower senescence, anthocyanin leakage and cell death of cut flowers when placed in solutions of a suitable inhibit produce ethylene (Fan et al., 2008). The protective function of SA includes the regulation of ROS and antioxidant enzymes (Khan et al., 2003; El-Tayeb et al., 2006; Shi and Zhu, 2008; Joseph et al., 2010). Fan et al. (2008) showed that SA can extending the vase life of cut flowers with increase Membrane stability. The data reported here provide good evidence that SA had an additive effect in decreased accelerate flower senescence, anthocyanin leakage and cell death. It seems that in high concentrations of SA, pH decreased and affected vacuoles pH and resulted Anthocyanin leakage (Table 1). Table 1 and 3 demonstrate that Acc-oxidase activity stimulates anthocyanin leakage from petals. Results indicating that Acc-oxidase activity increases membrane permeability in an unspecific fashion. Therefore, since anthocyanins are localized within the vacuole of all cells, result our showed that the action of ethylene in enhancing the rate of leakage of this pigment can be interpreted as an effect of the gas on senescence and the membrane permeability of cut flower. SA in suitable concentration (1.5 mM) with sucrose can prevent negative effects low PH that cause Anthocyanin leakage. Unfortunately, High Salicylic acid concentrations rapidly increase Anthocyanin leakage, cell death and accelerate flower senescence.

Lipid per oxidation, MDA content and chlorophyll content: The effects of SA on the data for Malondialdehyde (MDA) content are presented in Table 1 and 3 (p≤0.05). Under the effect of 3 mM SA treatment MDA content increased significantly in compared to control (Table 1) (p≤0.05). Salicylic acid 1.5 mM and salicylic acid 1.5 mM+sucrose 3% significantly reduced MDA content of cut flower in all treatments except than 3 mM SA and SA+SU (SA 3 mM + SU 3%) treatment compare to other treatment (Table 3) (p≤0.05), addition of SA at level 1.5 mM decrease produces this factor and could show better results compare to alone sucrose treatment and high SA concentration. Results showed adding SA was found to be positively correlated with Lipid per oxidation of the carnation cut flower (Table 2) (p≤0.01). This indicates that with SA concentration increased, the Lipid per oxidation was increased. Cut flower under senescence is in good correlation with increased lipid per oxidation levels and this result is in good correlation with the increase in MDA content under senescence (Shakirova, 2007). A lower lipid per oxidation resulting from elevated activities of antioxidants was also reported on of cut gerbera jamesonii flower (Yuping, 2009). The results of the present experiment were similar with the findings of Fan et al. (2008) in Gerbera which showed that added SA decreased the permeability of the plasma membrane of petal cells and decreased MDA level. It was reported that SA enhanced the stability of lipids in cell membranes of Gerbera when cut flower exposed to vase solution having SA. The evidence suggests that SA decreases the permeability of plasma membranes and membrane lipid per oxidation and maintains the membrane integrity. In this study, Two treatments (3 mM SA and 3 mM SA + SU 3%) decreased chlorophyll content in carnation cut flower but SA at 1.5 mM could prevent decrease chlorophyll content in carnation cut flower (Table 1). Canakci (2008) reported that treatment with salicylic acid significantly extends the vase life with increases total chlorophyll content.

Microbe population: The Microbe population vase solution of carnation cut flowers was decreased by the concentration of salicylic acid 1.5 and 3 mM used (Table 1). The Microbe population was lower in salicylic acid at 1.5 and 3 mM compared to salicylic combined with sucrose treatment and control (Table 3) (p≤0.05). The higher Microbe population was attained when sucrose was use comparison to control (Table 3). Means of Microbe population on vase solution of cut flowers in various salicylic acid + sucrose containing vase solutions was slightly significantly than control while means of microbe population on vase solution of cut flowers in various salicylic acid containing vase solutions was higher significantly than control. Results showed adding SA was found to be negatively correlated with microbe population vase solution of the carnation cut flower (Table 2) (p≤0.01). This indicates that with SA concentration increased, the microbe population vase solution was decreased.

Vase life: Table 1 showed that SA alone was capable of increasing longer vase life of carnation flowers than sucrose alone or together with SA (Table 1, 3) (p≤0.05). It is evident from the data presented in Table 3 that the maximum vase-life (10, 11 days ) were recorded in SA at 1.5 mM and Salicylic acid 1.5 mM+sucrose 3% respective compare other treatments and control. The minimum vase-life was noted in SA at 3mM compare other treatments and control (Table 3) (p≤0.05). Fan et al. (2008) showed that the treatment of salicylic acid extended the vase life and improved flower quality with reduced respiration rate delay senescence and decrease Lipid per oxidation, MDA content. Fan et al. (2008) showed that the treatment of salicylic acid extended the vase life and improved flower quality with reduced respiration rate delay senescence and decrease Lipid per oxidation, MDA content. Ichimura and Hiraya (1999) reported that treatment with sucrose extends the vase life of florets harvested at a bud stage. Keeping the flowers in vase solutions containing sucrose has been shown to extend their vase-life (Han, 2003; Yamane et al., 2005). Present results showed Adding SA was found to be positively correlated with vase life of the carnation cut flower (Table 2) (p≤0.01). The correlation was significant. This indicates that with SA concentration increased, the vase-life was decreased.

CONCLUSION

This study shows that Salicylic acid and sucrose treatment did show significant effect on quality parameters and rose flower longevity. Salicylic acid and sucrose proved more effective in delaying petal senescence and/or flower wilting. However, our result showed that Salicylic acid and sucrose treatments maintained the vase life of flowers for a longer period. Further studies are required, especially to validate some antioxidant enzyme activity and antioxidant capacity.