ROLE OF PRIMING TECHNIQUE IN GERMINATION PARAMETERS OF CALENDULA (CALENDULA OFFICINALIS L.) SEEDS

Seed priming is known for its promoting effects on seed and seedling properties under stress conditions such as salinity, temperature and drought stresses. Poor plant stand establishment is a common problem for marigold production. Therefore, an experiment was conducted to investigate the effect of different seed priming techniques on calendula seed germination and seedling enzyme activity. For this purpose, different seed priming techniques: hydropriming (distilled water, 24 and 72 h), osmopriming (0.02% or 0.04% KNO3 for 24 and 72 h) and hormonal priming (100 or 500 mg L GA3 for 24 and 72 h) were assessed. Seeds without any treatment were considered as control. Compared with control, 100 mg L GA3 and distilled water for 72 h showed the highest promoting effects on germination percentage. The highest vigor index (525.75) was related to hydropriming (for 24 h) treatment. Also, the maximum root length was found with 0.02% KNO3 for 72 h. Catalase significantly increased in the primed seeds. The highest CAT activity and total soluble sugars were found in the seeds primed with 100 mg L GA3 for 72 h. Moreover, the enzyme activity was significantly higher in the distilled water and KNO3 treatments compared with the untreated seeds.


Introduction
Marigold (Calendula officinalis L.) is typically recognized as an annual, vertical or prone and branched, glandular, with a strong root.The marigold flowers are ornamental and are used for food flavor and color (Lim, 2014).Its medicinal uses are well known in Europe, China, India and several other places in the world (Muley et al., 2009) and it is one of the most used domestic herbal medicines in Iran (Asadi-Samani et al., 2015).Leaves and flowers of marigold are used as antiinflammatory, antiseptic, anti-spasms, relaxant, styptic, cholagogue, sudorific, blood coagulants and wound ameliorators (Grieve, 1971;Chevallier, 1996).Thus, the cultivation of marigold as an alternative crop is gaining popularity day by day in the agriculture business.Poor plant stand establishment is a common problem for marigold production (Bosma et al., 2003).Exposure to seed priming is one of the strategies being used to accelerate the activation of plant growth, especially seed germination in many crops.Seed priming is defined as a pretreatment of seeds by various methods in order to improve seed germination rate, germination percentage, and improve uniformity of seedling emergence by controlling the water available in the seed.The pretreatment initiates the early stages of germination, but does not permit radicle protrusion, and then the seeds are dried until needed (Farooq et al., 2006).Guzman and Olave (2006) showed that nitrate solutions used as seed priming technique in melon improved the germination rate, radicle growth and germination index.Seeds are flooded in water during the hydropriming method.Consequently, water is independently available to seeds, then its uptake is controlled by the vicinity of the seed tissue for water (Taylor et al., 1998).Seed germination of onion was investigated by Caseiro et al. (2004), and they found that hydropriming of seeds was the most effective method in improving seed emergence, particularly when the seeds were exposed to water for a longer time (96 h) compared to control.In hormonal priming, seeds are submerged in plant growth regulators.Haigh et al. (1986) investigated the primed seeds of carrot, onion and tomato showing that priming these seeds with GA3 (Gibberellic acid) increased the germination percentage and rate.This study was aimed to investigate the effect of seed priming techniques on germination parameters of calendula and its seedling enzyme activities.

Seed viability test
In order to determine the viability of calendula seeds, 50 seeds (with three replications) were exposed to the standard germination test.Two layers of filter paper were placed under the seeds in 90-mm Petri dishes.Petri dishes were wrapped with plastic bags to avoid moisture loss after adding 5 mL of water to each Petri dish.For better seed germination, the Petri dishes were placed under dark conditions at room temperature (20±1•C) for 7 days.Seeds were considered germinated when the radicle lengths were 2 mm long.The number of germinated seeds were noted every 12 h until the 7 th day.

Seed priming
After determination of seed viability, 20 seeds of calendula were treated with distilled water (hydropriming), KNO 3 (0.02% or 0.04%) or GA 3 (100 or 500 mg L -1 ).The seed priming duration times were 24 and 72 h.Treated seeds were kept in an incubator at 10 and 15±1°C.Also, untreated seeds were termed as unprimed.

Germination percent (GP)
The number of normal seedlings at the end of the standard germination test indicated the germination percent.

Mean germination time (MGT)
MGT was calculated by the following equation (Moradi Dezfuliet al., 2008): MGT = ΣDn/Σn (1) where n is the final germinated seeds on day D, and D is the total days from the germination time.
Vigor index (VI) VI was calculated through the following equation: VI = seedling length × germination percentage (2)

Enzyme assay
The seedling samples were homogenized in 3 ml of tris (PH 7.8), and the samples were centrifuged at 20000 g for 20 min.The supernatants were used for enzyme activity assays.Catalase (CAT; EC 1.11.1.6)activity was determined with changes in the absorbance range at 250 nm by the spectrophotometric method (Kato and Shimizu, 1987).

Total soluble sugars
Total soluble sugars were quantified in the marigold sample (0.1 g) after grinding with the help of a mortar and pestle followed by hydrolysis with 2.5N HCl and then neutralized by sodium carbonate.The distilled water was used to make the final volume of 10 ml, centrifuged at 10000 g and the supernatant was used for measurement of total sugars following the phenol-sulphuric acid method (Thimmaiah, 2004).

Experimental design and statistical analysis
A completely randomized factorial design with four replications was used in this study.The SAS software was used for analysing the results, and also the significance of variation between means was tested by Least Significant Difference (LSD).

Germination percentage
The interaction of time duration × priming treatment was significant for the germination percentage.Seed priming had a significant positive effect on different parameters of seed germination.The maximum germination percent was observed in the treatment with distilled water and 100 mg L -1 GA 3 for 72 h (Figure 1).Also, the duration of treatment significantly affected the final germination percentage.In the best treatment (distilled water and 100 mg L -1 GA 3 for 72 h), seed priming increased the germination percentage by around 30% comparing to controls.An increase in GA 3 and KNO 3 concentrations from 100 to 500 mg L -1 and from 0.02% to 0.04%, respectively, led to the reduction of the germination percentage.distilled water for 24 or 72 h; G100 or 500: GA 3 100 or 500 mg L -1 for 24 and 72 h; K2 or 4: KNO 3 0.02% or 0.04% for 24 and 72 h).

Shoot and root length
Results indicated that the highest shoot length was 4.15 cm for 100 mg L -1 GA 3 and the lowest length of 3.7 cm was related to control (Table 1).The highest root length was 2.63 cm for KNO3 0.02% (Table 1).

Vigor index
Comparison of the interaction time duration × priming treatment indicated that regarding vigor index, there was a significant difference among various experimental treatments.The highest and lowest vigor indices were estimated as 525.75 and 311.50 related to hydropriming (24 h) and control treatments, respectively (Figure 2).According to the variance analysis, significant differences were obtained through 24 and 72 h of treatments.Vigor index was reduced by increasing GA 3 and KNO 3 concentrations from 100 to 500 mg L -1 and from 0.02% to 0.04%, respectively.The regression relationship between seed germination and vigor index indicated that with increasing the germination rate, the vigor index increased (Figure 3).

Mean germination time (MGT)
The effects of seed priming solutions on mean germination time of calendula seeds are presented in Table 1.The difference between treatments was statistically significant.The control took the maximum time of 7 days for germination.The minimum germination time (6 days) was related to distilled water treatment.

Catalase activity
According to our results, seed priming increased the catalase activity of seedlings.CAT significantly increased in the primed seeds (Figure 4).The highest CAT activity was found in the seeds primed with 100 mg L -1 (72 h) GA 3 .Moreover, the enzyme activity was significantly higher in the distilled water and KNO 3 treatments compared with the untreated seeds.

Total soluble sugars
All the priming agents significantly affected total soluble sugars.The total sugars were maximally increased in the seeds primed with 100 mg L -1 (72 h) GA 3 (Figure 5).
Figure 5. Effects of the priming treatments on total soluble sugars of calendula seeds (control: unprimed; water: distilled water; GA100 or 500: GA 3 100 or 500 mg L -1 ; KNO: KNO 3 0.02% or 0.04%. For better crop establishment mainly under stressful conditions, the quick and uniform seedling emergence is necessary (Subedi and Ma, 2005;Gupta et al., 2008).Among using techniques to promote seedling emergence under field conditions, seed priming before sowing improves uniform germination and reduce the time duration from planting to emergence (Brocklehurst and Dearman, 1983).The beneficial effects of the seed priming technique on the germination of many plant species are referred to the activity of biochemical mechanisms such as preserving and stability of cell membrane due to synthesis of nucleic acids (DNA and RNA) and proteins (Bewley and Black, 1994) and progressing in the antioxidant defense system capability (Girolamo and Barbanti, 2012).Effects of different seed priming techniques on germination, seedling emergence and growth have been well documented.Ghassemi-Golezani et al. ( 2010) evaluated the effect of different osmopriming treatments (KNO 3 and NaCl) on seed invigoration and field performance of winter rapeseed cultivars and concluded that salt priming, particularly KNO 3 priming, decreased the mean germination time and increased the seedling size, compared with non-primed seeds.Caseiro et al. (2004) reported that hydropriming for 96 h was the best treatment for improving the seed germination in onion plants.When seeds imbibe, the water content partially remains constant at a certain level until germination.Thus, it will be suitable for better stand of seedlings the hydropriming to be used up to the specified period of time.In our study, the maximum germination percentage was observed with 72-h distilled water hydropriming treatment.With increasing in GA 3 and KNO 3 concentrations from 100 to 500 mg L -1 and from 0.02% to 0.04%, respectively, germination percentage was reduced, which agrees with the findings of Eisvand et al. (2011) for carrot.In another study, Moosavi et al. (2009) found high POD activities due to seed priming in Amatanth genotypes.Increasing the antioxidant enzyme activity may be caused by high ROS concentration as a result of stressful conditions.Under these conditions, oxidative stress will reduce cell division and consequently block growth and development, therefore protection from oxidative stress is critical for seed germination.High POD activity due to its protective effects against free radicals could play a key role in cell division and seedling growth.Meanwhile, the activity of CAT plays a significant role in increasing seedling resistance to tough environmental conditions (Ansari et al., 2012).In this regard, Rouhi et al. (2012) found that the activities of catalase and other antioxidant enzymes were significantly increased in osmoprimed seeds of Berseem clover (Trifoliumal exandrinum L.) compared to control.According to our case, seed priming techniques increased catalase activity during the seedling emergence process.CAT significantly increased in the primed seeds.The highest CAT activity was found in the seeds primed with 100 mg L -1 (72 h) GA 3 .The role of gibberellic acid as a growth regulator in seed dormancy, flower formation, root formation, seed germination, cell growth, division and protection is well known.The present study showed that treatment with GA 3 (100 mg L -1 for 72 h) increased the germination percentage in calendula seeds comparing to controls.The present results are in agreement with those of Ghodrat and Rousta (2012) and Jamil and Rha (2007).The hydrolytic enzyme synthesis (especially alpha amylase) process was affected by GA 3 , and this enzyme increased degradation of outer layer cells of radicle and promoted its emergence (Rood et al., 1990).In our study, germination percentage was also enhanced by priming seeds with water.In some cases, the final seedling germination percentage of primed and unprimed seeds is equal, but primed seeds emerge more quickly.Demir and Mavi (2004), while working on germination of primed and unprimed watermelon seeds, observed that unprimed seeds emerged after 4 days of delay compared to primed seeds.The two essential parameters that support the final yield are quick and steady emergences of seedlings under field conditions (Finch-Savage, 1993).For production of the potent plant, the rapid emergence of seedlings is necessary.Probably due to the presence of essential nutrients such as nitrate and potassium, priming with KNO 3 can lead to further growth of primed seedlings (Farooq et al., 2006).In our results, priming with GA 3 , KNO 3 and distilled water significantly extended seedling length compared with the untreated control.

Conclusion
Therefore, it could be concluded that water (hydropriming), GA 3 and KNO 3 may be good candidates for extending germination percentage and germination characteristics in calendula seeds.In our study, germination percentage was also enhanced by priming seeds with water.The most effective treatments in enhancing germination percentage were 100 mg L -1 GA 3 and distilled water for 72 h.The highest and lowest vigor indices estimated as 525.75 and 311.50 were related to hydropriming (for 24) and control treatments, respectively.Therefore, we suggest that the hydropriming is a simple and effective technique for improving seed germination and seedling emergence of calendula.

Figure 3 .
Figure 3. Regression relationship between the vigor index and the germination percentage.

Table 1 .
Effects of different treatments on shoot length, root length and mean germination time (MGT) of calendula seeds.
*Means within each column followed by same letters are not significantly different (p = 0.05).