Investigation of qualitative traits and evaluation of flower yield of pot marigold (Calendula officinalis L.) during its growth period under drought stress

Introduction
Insufficient-water induced stress, causes morphological, physiological and biochemical changes in plants and has the potential to reduce leaf area, height and dry weight and cause stomatal closure, chlorophyll and photosynthesis reduction, amino acids accumulation, enzyme (Hassani and Omid Beighi, 2002) and protein destruction and changes the biosynthesis of the proteins (Jiang and Huang, 2000). Despite there are extensive studies on the effects of environmental stresses on the growth and yield of crops, there is very few information about medicinal herbs responses to these stresses (Amiri Deh Ahmadi et al., 2014). While water stress decreases growth of some medicinal plants such as Hypericum brasiliense (Nacif de Abreu and Mazzafera, 2005) and Bupleurum chinense (Zhu et al., 2009), many studies have shown that drought enhances the amount of secondary metabolites in wide veriety of plant species, such as Rehmannia glutinosa (Chung et al., 2006). Conversely drought caused a significant reduction in all growth parameters and essential oil yield and percentage in some medicinal plants such as peppermint (Mentha piperita L.) (Khorasaninejad et al., 2011).Considering the importance of the medicinal plant pot marigold (Calendula officinalis L.) in some industries such as pharmaceutical industry (Bousselsela et al., 2012), drought stress effects on flower yield and some quality-related traits of two marigold types was studied.
 
Materials and methods
Effect of drought stress on quantity and quality of harvested flowers of pot marigold was studied, using a complete block design as split plot with four replications in faculty of agriculture, Birjnand University, in 2015. Two factors including drought stress with three levels consisting of watering as 75, 50 and 25 percent of the soil field capacity (non-stressed, moderate and severe stress, respectively) and plant type (medicinal and ornamental type) were considered. Flowers were harvested and oven dried, 22 times during plant growth period. Logistic and linear models were compared using SAS software to choose the best model describing the rate of cumulative flower yield changes during growing season of pot marigold. Plant dry weight was the average dried weight of three randomized chosen plants at the end of growing season. Aluminum chloride colorimetric method with some modifications was used for flavonoid content determination (Yi et al., 2007). Essential oil of fresh flowers was extracted using Clevenger apparatus. As the essential oil content of pot marigold was very low, diethyl ether solvent was used to favor the extraction procedure.
 
Results and discussion
Evaluation of flower yield during the growth period (22 harvests) showed that this trait was significantly reduced by drought stress. The trend of cumulative flower yield was better described using non-linear logistic model compared with linear one. Comparing parameters of logistic model revealed that in non-stressed level flower yield increasing per plant was 0.059 g.day^-1 that was approximately 69% higher than severe stress level (0.035 g.day^-1). The highest and lowest cumulative flower yield were recorded in non-stressed (6.86 g.plant^-1) and severe stress (3.46 g.plant^-1) treatments, respectively. In addition, two marigold types were not significantly different in terms of flower production during the whole growth period. Under drought stress conditions, cell elongation in higher plants is inhibited by reduced turgor pressure. Reduced water uptake results in a decrease in tissue water contents and turgor is lost. Likewise, drought stress also trims down the photoassimilation and metabolites required for cell division (Farooq et al., 2009). As a consequence, impaired mitosis, cell elongation and expansion result in reduced growth (Kaya et al., 2006). In addition, reduced leaf size under drought stress leads to reduced light trapping capacity and as a result, total photosynthesis declines (Hsiao, 1973). The significant correlation between flower yield and plant dry weight (0.51**) indicates that reduced flower yield is a consequence of impaired dry weight under water deficit condition. 
Measured flavonoid content of flowers was primarily increased with increasing drought stress intensity from non-stressed (25.14 mg rutin equivalent.g^-1 extract) to moderate stress (38.97 mg rutin equivalent.g^-1 extract) level, however it was considerably decreased afterwards and reached the lowest amount (22.96 mg rutin equivalent.g^-1 extract) at severe stress level. Increased flavonoid content at moderate stress level could be attributed to antioxidant function of these compounds (Franco et al., 2008). Circumstances, in which antioxidant enzymes are inactivated, stimulate biosynthesis of flavonoids. This indicates that flavonoids could act as a secondary antioxidant system for scavenging ROSs in plants under prolonged drought stress (Fini et al., 2011). Reduced flavonoid content at higher levels of drought however could be attributed to reduced activity of enzymes involved in flavonoid biosynthesis (Yang et al., 2007). Medicinal type of pot marigold showed a higher potential of flavonoid production. Flavonoid content of medicinal type of pot marigold was approximately 28% higher than ornamental type. Increasing drought stress also decreased the essential oil of flowers, so that the essential oil from 0. 120 in non-stressed level reduced to 0.062 mg. g^-1 fresh flower in severe stress level. Observed decline in flower essential oil of pot marigold might be a result of disturbed photosynthesis and carbohydrate production and suppressed growth of stressed plants (Flexas and Medrano, 2002). However flowers of two marigold types had the same content of essential oil.
 
Conclusion
Cumulative flower yield of pot marigold as described by logistic model, was significantly reduced by water deficit, where the highest and lowest cumulative flower yield belonged to non-stressed and severe stress treatments, respectively. Flavonoid content was primarily increased and then decreased with increasing drought stress intensity. Increasing drought stress also decreased the essential oil of flowers, so that the essential oil in non-stressed treatment was approximately twofold higher compared with severe stress level.