Effects of Osmopriming on Germination and Vigour Traits of Bersim Clover ( Trifolium alexandrinum L . )

Previous studies suggested that fast and uniform germination is important for good crop establishment. The present study was conducted to investigate the possibility of increasing germination characteristics by seed priming techniques. An experiment was conducted with three replicates and two treatments including: 2 different priming duration (8 and 12 hours) and 6 osmotic potential of PEG 6000 solutions (-0.8, -1,-1.2,-1.4,-1.6 Mpa) and distilled water as a control group). The priming solutions were prepared using polyethylene glycol 6000 (PEG). Our results showed that the most effective osmotic potential in improving germination characteristics of Trifolium alexandrium is -0.8 MPa for 16 hours.


Introduction
Bersim clover (Trifolium alexandrinum L.) is highly used as a forage crop during recent decades.Fast growth and the great final production of this crop make it a good option for farmers to use it as a fresh forage products (Shrestha et al., 1996;Musavi Aghdam, 1985;Khoshgoftar, 1992).The results of researches shows that good production highly depends on suitable sowing date, climate condition, soil fertility, shrub height, the number of harvests and variety.For example in North part of Iran, farmers usually planting this crop after harvesting rice and the rate of production exceeded to 55-70 t/ha (Zarrineh et al., 1985;Khoshgoftar, 1992).Regarding to the important rule of this plant in production cycle of dairy and protein substance, the rules in fertility preservation and the plant coverage of soil is one the another important roles of this crop in agricultural production (1 st national forage crops congress of Iran).So one of the most important research priorities in forage production is to choose suitable forage performance and production strategies.Seed germination could be known as important and consequent factors in seedling establishment via different conditions.Seed enhancement techniques, such as seed priming, can be useful in increasing the forage yield.In osmopriming treatment which is also known as osmoconditioning, seeds incubated in polyethylene glycol (PEG), sorbitol, mannitol solution and let to uptake water in order to primary metabolic activities of germination process is started and radicle emergence happen (Ashraf and Foolad, 2005).This tech-nique has been used in some vegetable seeds to increase the germination rate, total germination and seedling uniformity, mainly under unfavorable environmental conditions (Bradford et al., 1990;Ozbingol et al., 1998;Nascimento and Arago, 2004;Korkmaz, 2006).When the seeds were primed and sowing them in the field, they usually show faster and more uniform germination comparing to nonprimed seeds (Bourgne et al., 2000).Priming can increase the germination and growth of seedling under stressed conditions in sunflower seeds (Demir et al., 2006).The aim of this study is to increase germination percentage and seedling vigor of bersim seeds by osmopriming with PEG, and to define the optimal combination of PEG concentration and treatment time.tential of -1.2 MPa comparing to other potentials and control group (Fig. 1).And regarding to the 16 hours time period of priming the highest rate was observed in the potential of -0.8 MPa in this time.It was reported that, osmopriming of watermelon seeds caused the decrease mean germination time and in contrast, increase percentage of germinated seeds (Demir and Van de Vanter, 1999).Most of researchers reported the positive effects of seed priming on seed germination percentage (Misra and Dwivedi, 1980;Chiu et al., 2002).

Germination Rate
Highest germination rate was observed in primed seed of 8 h at -1.2MPa osmotic potentials (Fig. 2).Subedi and Ma (2005) reported that one of the most important conditions for crop potential performance is fast and uniform germination which is a resultant of priming seeds in the farm.Harris et al. (2001), Giri and Schilinger (2003) and Finch-Savage et al. (2004) reported that the seed priming have a positive effects on seed germination rate comparing to control.

Seedling Length (SL)
The highest length of seedling was observed in the same mentioned composition of priming treatments (8 h and -1.2MPa; 16h and -0.8MPa) (Fig. 3).Basra et al. (1994) reported the increase of radicle and plumule growth of primed onion seeds in 7 days after sowing and the primary seedling growth.It was observed that seeds number and performance of each plant in prime seeds of chickpea (water and mannitol 4%) was higher in comparison with nonprime seeds.This is the resultant of the increase of acid invertase activity in the apical of the main stem after sowing, because the availability of apical to hexsose, increased as the result of enzyme activity (Subedi and Ma, 2005).

Seedling Dry Weight (SDW)
There was no significant effects of priming on seedling dry.
Vigour Index (1) Vigour index was higher in the primed seed with -0.8MPa of PEG 6000 for 16 h was higher comparing with other treatments(Fig.4).h under dark conditions.Thereafter, the seeds were rinsed with distilled water three times.The treated seeds were surface-dried and dried back to their original moisture content via experience at room temperature (about 21°C, 45% relative humidity) determined by changes in seed weight.Moisture content of untreated seeds (control, 5.5% moisture content), osmoprimed treated seeds was equilibrated at room temperature for 24 hours.

Germination tests
Three replicates of 50 seeds were subjected to germination between double layered papers with 5 ml of water in 9 cm Petri dishes.These Petri dishes contained seeds were put into sealed plastic bags to avoid moisture loss.Seeds were allowed to germinate at 20±1°C in the dark condition for 7 days (ISTA, 1996).Germination was considered finished when the radicles were 2 mm long.Germination percentage was recorded every 12 h for 7 days.Germination rate was calculated to assess the rate of germination (Ellis and Roberts, 1980).Seedling length, seedling dry weight and vigor indexes 1 and 2 (sign of multiplication germination percentage×seedling length and germination percentage×seedling dry weight respectively) were measured after the 7 th day.

Statistical analysis
The statistical analysis was performed in factorial design were the treatment (6×2) arranged in a randomized completely block design; with three replications and 50 seeds per replicate.The first factor was osmotic potential levels (-0.8, -1.0, -1.2, -1.4,-1.6 and 0 MPa as a control group) (PEG) and the second, duration of seed priming (8 and 16 hours).Data for germination and abnormal germination percentage were subjected to arcsine transformation before analysis of variance was made using MSTAT-C program (Michigan State University).Mean separation was performed by Fisher's least significant difference (LSD) test if F-test was significant at (P < 0.05).

Results and discussions
The results of the experiment showed that, the priming duration, affects significantly the final germination percentage, the mean germination time and also the vigor index 2 (Tab.1).In osmotic potential of osmopriming solution on investigated traits only seedling dry weight was insignificant (Tab.1).Since the interaction effect of time in osmotic potential in osmopriming solution was significant, it was decided to avoid explaining and discussing about main effects so only interaction effects of time * potential on above mentioned traits was discussed and investigated.

Final Germination Percentage (FGP)
As it is shown in the Fig. 1, the highest germination percentage was seen in primed seeds for 8 hours with po- Vigor Index (2) The highest value for this index was observed in primed seed for 8 h in -1MPa osmotic potential (

Discussion and conclusions
This study showed that the seed priming at -0.8MPa for 16h of duration and -1.2MPa for 8h of duration exhibited the highest value of the investigated traits in comparison with other treatments and control group.But the comparison of these two elite treatments shows that seed priming period (16 h) at -0.8MPa potential of PEG solution could be selected as the final choose for seed priming of bersim clover.Akinola et al. (2000) reported that higher duration of exposure to seed treatment resulted in higher cumulative germination in wild sunflower and Caseiro et al. (2004) found that hydropriming was the most effective method for improving seed germination of onion, especially when the seeds were hydrated for 96 h compared to 48 h.The present study revealed that PEG had no toxic effect since all seeds germinated.Mehra et al. (2003) and Michel (1983) indicated that PEG molecules do not enter to seed and Khajeh-Hosseini et al. (2003) found that there was no toxicity of PEG.Seed priming exhibited positive effects on germination characteristic of amaranth cultivars such as speed of germination and root length and antioxidant enzymes activities were increased due to seed prim-ing which could resulted in increasing tolerance of primed seeds to environmental stresses such as salinity (Moosavi et al., 2009).Finally it could be recommended that, the results of this study to be investigate in the farm condition in order to confirm the fulfilled experiments of this project.
Fig 5).Priming treatments significantly affected germination vigour of Bromus and hydropriming for 12 hour could increase seed vigour of Bromus(Tavili et al., 2010)