Alleviate Seed Ageing Effects in Silybum marianum by Application of Hormone Seed Priming

Most of the medicinal plants have seed dormancy or poor seed germination. This is due to their natural compounds or their morphological structure. Existence of such mechanisms makes the seeds able to endure harsh environments, stressful conditions or natural ageing. Different seed enhancement treatments were proposed in order to improve seed germination. In this study, it was examined Silybum marianum seed longevity (0, 48 and 72 hour of ageing) and the response to seed priming when using different concentrations of cytokinin (50, 200, 350 and 500 ppm) and different duration of treatment (8, 12 and 24 hour). Results revealed that ageing could be successfully alleviated using seed priming. Seed ageing significantly affected seed germination and vigour. Application of cytokinin considerably improved seed vigour in aged and non aged seeds. The most effective seed priming treatment was with 500 ppm cytokine, with the duration of 24 hours. In conclusion, it can be recommend the used of plant growth regulators like cytokine, as a good priming agent, to recover losses of seed quality and improve germination characteristics.


Introduction
Seed priming has been widely used by many researchers to improve germination parameters in different crops.Most of medical plants are known to have hard germinating seeds.In order to improve plant emergence and crop establishment it is critical for seedling for be as vigorous as possible and to achieve this goal, scientists try different seed enhancement techniques, like seed priming (Bewley, 1997).Seed priming is started by imbibition and ended before testa rupture.It means that in seed priming, seed imbibition is completed; lag phase of seed germination, which is the most important phase, is completed, but seeds are prohibited to enter into the next phase.During the second phase of seed germination most of metabolic pathways and recovering processes start, so that the embryo is assured that everything is prepared for successful germination (Varierl et al., 2010).
Silybum marianum is a medicinal plant which is under high consideration nowadays due to its medicinal properties and metabolites.The plant extract is widely used for treating liver disorders, protecting liver from hypotoxic drugs and chronic hepatitis.Flavonoids which are extracted from its seeds are high in antioxidants and could protect cell damage by oxidative stress.Among all different environmental factors which affects seed vigour and viability, seed ageing is a common stress, especially in soil seed bank or seed storage rooms.Seed ageing decreases quality of seedling growth and germination traits of many plants (Eisvand, 2010).Different physiological processes are active during ageing.Changes in anti-oxidative activity, reverse mobilization and embryo weakening are severe effects of seed ageing.Application of accelerated ageing test was firstly used by Delouche and Baskin to measure seed vigor for different plant species.Lipid peroxidation may be the main key factor of decreasing seed quality by seed ageing.It is initiated with generation of free oxygen radicals (Gille and Joenje, 1991;Larson, 1997;McDonald, 1999).There are studies concluding that the application of plant growth regulators like GA3, ethephone, infused ether, kinetin could improve seed storage and germination characteristics like germination rate in different plant species.
In this study it was investigated Silybum marianum seed germination and seedling growth parameters.It was studied if hormone seed priming may recover side effects of seed ageing in regard to germination and growth parameters.

Materials and Methods
Milk thistle (Silybum marianum L.) seeds were obtained from Pakan Bazr Company, Isfahan, Iran.Three accelerated aging regimes were performed by placing the seeds in the incubator at a temperature of 40 °C and relative humidity of 90 to 95% for 0, 48 and 72 hours.
For each aging treatment, about 400 g of pure Milk thistle seeds were scattered within a vacuum container on wire screens; the container was filled with distilled water (70% of the total container volume).The containers were placed in an incubator at the fixed temperature of 40 °C.
Seeds were subjected to phytohormone seed priming using different concentrations of cytokinin (50, 200, 350 and 500 ppm); duration of seed priming was another experimental factor that was considered for 8, 12 and 24 hours.The experiment was performed using a completely randomized design with four replications.The standard germination test was performed by placing 25 seeds on top of two Whatman no. 1 filter papers in 10 cm petri dishes.All petri dishes were moistened with 10 ml of distilled water and covered with plastic bags in order to reduce the water evaporation and then all petri dishes moved to germinator with 25 °C temperature, in dark condition.
Seeds were observed daily until day 7 th ; seeds were considered germinated when the radicle length reached 2 mm.Investigated parameters were the final germination percentage, mean daily germination (MDG) (Scott et al., 1984), coefficient of velocity of germination (CVG) (Maguire, 1962), root length, stem length and seedling vigour.
Where: G1-GN: Numbers of germinated seed from the first day of experiment to the day n.
Statistical data analysis was performed by using Minitab, 16, MSTAT-C and Microsoft Excel 2010 software.

Results
The analyses of variance showed that there was a significant difference among the different ageing treatments and this was even more enhanced at higher duration of ageing.Using seed priming reduced some negative effects of ageing, compared to non-primed seeds.

Germination percentage
Analysis of variance showed that all main effects of different ageing treatments, cytokinin concentrations and duration of seed priming respectively, were significant.Among interaction effects, priming (cytokinin*time) was highly significant.Increased duration of the ageing treatment resulted in lower germination properties.Ageing condition negatively affected cell membrane,, therefore oxidative stress severely damaged the cells.Cytokinin promoted cell division and probably it activated some responsible recovery genes, and all eviate ageing side effects (Fig. 1).Increased ageing duration decreased the germination percentage (Table 1).Results showed that the most effective priming treatment for increasing germination percentage was 12 hour soaking seeds in 500 ppm cytokinin, while the combination of 50 ppm cytokinin with 8 hour of seed priming did not show significant difference (Table 2).

Coefficient of velocity of germination (CVG)
Analysis of variance showed that all priming treatments produced significant differences among all groups.Ageing severely decreased CVG, which indicated that germination rate was negatively affected.The longer the ageing period, the lower germination rate index CVG was (Fig. 2).Generally, lower durations of seed priming with cytokinin produced rapid germination and higher CVG value (Table 1).The highest CVG value was observed in priming treatment with 50 ppm cytokinin for 8 hours, which was slightly higher than that obtained for primed seeds with 500 ppm cytokinin for 12 hour (Table 2).
Seed ageing decreased mean daily germination.Seed priming increased mean daily germination in aged and non-aged seeds (Fig. 3).
The highest MDG was observed in priming treatment of 12 h soaking in 500 ppm cytokinin, while the lowest priming  2).
Seed priming significantly affected root elongation in Silybum marianum.Results revealed that longer roots were produced after seed priming treatment of soaking seeds with 500 ppm cytokinin for 24 hour (Table 2).Cytokinin promoted root elongation and positively alleviated ageing side effects on root growth (Fig. 4).
Threeway interaction was significant for stem growth of Silybum marianum.Performance of lower concentration of cytokinin combined with lower priming durations was better, while higher cytokinin concentrations did not show the same trend (Fig. 5).The increase in ageing duration resulted in a reduction of the stem growth, thus to alleviation this side effect of ageing, more cytokinin concentration should be applied to      seeds (Table 6).The results are in agreement with other data, as seed priming significantly improved final germination percentage (GP), mean germination time (MGT), mean daily germination (MDG), daily germination speed (DGS) and germination rate index (GI) of Canola under water stress (Behrouzyar and Yarnia, 2014).Seed vigour decreased for aged seeds, but seed priming positively improved seed vigour.The higher the cytokinin concentration, the more seed vigour was achieved (Fig. 6).The highest seed vigour was achieved for priming treatment of 500 ppm cytokinin with the duration of 48 hours.Results showed that, not only seed priming alleviated ageing side effects, but also it increased seed vigour compared to nonaged seeds (Table 6).Seed priming enhanced germination percentage (GP), germination index (GI) and mean germination rate (MGR) in aged tomato seeds.Seedling characteristics such as radicle and shoot length were increased compared with unprimed aged tomato seeds (Zhang et al., 2014).

Discussion
Seed longevity is the potential of seed survival in the surrounding environment and it will initiate just by the time that seeds have passed to their maturity level.Seed longevity allows plant population to endure long after the disappearance of the parent plants, so it directly influences genetic disturbance and diversity.Seed longevity also is important in designing long-term weed management programs, as well as good knowledge of plant population dynamics (Long et al., 2014).Agreement of the ecophysiological mechanisms of seed longevity is critical for researchers to anticipate how long seeds can survive in the soil seed bank under different climatic conditions.Seed ageing lead to vigour and viability losses and perhaps this is due to losses in cell membrane integrity, because of lipid peroxidation by free radicals, lack of ATP and reduction in protein synthesis (Gidrol et al., 1988;Gille and Joenje, 1991;Larson, 1997).
Seed treatment could help to recover ageing effects on seeds and seed priming is an effective seed treatment to overcome ageing.As it has been cleared in many crops, during imbibition repair process of seeds are initiated and thus seed priming improves germination characteristics (McDonald, 1999;Imran et al., 2013;Prom-u-thai et al., 2012;Yadav et al., 2011).
Upon placing dry seeds in water, the uptake of water will be initiated in three stages (Bewley, 1997).At the first stage, namely imbibition, water uptake is initiated rapidly due to lower water potential of the seeds.During the second stage, germination is started by activation of different physiological activities including protein synthesis, translation of new mRNAs etc.The third has a rapid water trend, when germination will be completed.Therefore, successful seed priming is highly dependent on first and second stages of seed 320 germination, when seeds are starting to absorb water from the surrounding environment (Varierl et al., 2010).
Seed priming activates or synthesis many of enzymes that are involved in remobilization of seed reservoirs like storage proteins.HSP proteins are also synthesized during seed priming.These proteins are stress related proteins, as they could protect seed proteins from natural ageing (Kester et al., 1997;Varierl et al., 2010).
Plant growth regulators have been widely used as priming agent on different plant seeds in order to alleviate the negative effects of stress conditions (Afzal et al., 2005;Egamberdieva, 2009;Eisvand et al., 2010;Gadallah, 1999).Salicylic acid, giberlline, auxin and cytokinin are widely used in seed enhancement experiments.Cytokine is one of the important plant hormones that is involved in controlling cell division, photomorphogenesis, chloroplast development and root growth (Brault and Maldiney, 1999;Davies, 1995).Optimal concentration of cytokinin exhibited favourable effects on germination and seedling growth (Gadallah, 1999;Iqbal and Ashraf, 2006).It has been reported that seed priming with 10 ppm kinetin increased final germination percentage, germination index, shoot length and seedling fresh weight of two tomato cultivars as compared to control (Nawaz et al., 2013).

Conclusions
The current experiment suggested that cytokine plays critical roles in plant responses to ageing and it can be concluded that hormone priming with 500 ppm cytokinin significantly increased seed vigour in both aged and non aged seeds.Seed vigour of primed seeds was not significantly different from non aged seeds.Therefore, hormonal priming with cytokine is recommended as a safe technique to relieve the negative effects of Silybum marianum seed deterioration.

Table 1 .
Mean comparison of ageing duration, priming duration and cytokinin concentrations on germination properties of Silybum marianum Means that do not share the same letter are significantly different.

Table 2 .
Mean comparison of priming duration and cytokinin concentrations on germination properties of Silybum marianum Means that do not share the same letter are significantly different.

Table 3 .
Mean comparison of ageing duration and cytokinin concentrations on germination properties of Silybum marianum Means that do not share the same letter are significantly different.

Table 4 .
Mean comparison of ageing and priming durations on germination properties of Silybum marianum

Table 5 .
Mean comparison of interaction of ageing, priming duration and cytokinin concentrations on germination percentage, CVG and MDG of Silybum marianum

Table 6 .
Mean comparison of interaction of ageing, priming duration and cytokinin concentrations on root length (cm), stem length (cm) and seed vigor of Silybum marianum