Response of seeds and pollen of Onobrychis viciifolia and Onobrychis oxyodonta var . armena to NaCl stress

Sainfoin (Onobrychis viciifolia Scop.) is an important forage legume crop with 52 species adapted to dry and poor soils in Turkey, but little is known about the effects of salinity on germination and seedling growth in arid and semiarid regions suffering from salinity problem. The seeds and pollen of two species of sainfoin O. viciifolia and O. oxyodonta var. armena (Syn: O. armena) were exposed to 0, 5, 10, 20 and 30 dS m of NaCl under in vivo and in vitro conditions and evaluated for germination under salt stress by comparing germination percentage, mean germination time, root and shoot length, fresh and dry seedling weight and dry matter. Increased salinity levels generally resulted in decrease in all traits except time to germination, dry seedling weight and dry matter, which increased at high salinity levels. O. viciifolia seeds germinated and grew more rapidly compared to O. armena seeds under NaCl stress. No decrease in germination and seedling growth up to 10 dS m was recorded. On the other hand, there was a clear difference for germination and seedling growth between in vivo and in vitro conditions. Lower values were obtained from in vitro experiments; suggesting that mineral salts, sucrose and agar may have resulted in higher osmotic potential inhibiting germination and seedling growth of species compared in vivo conditions. Decrease in pollen germination with increasing salinities was very sharp, indicating that pollen germination had higher sensitive to salinity. But, pollen grains of O. armena germinated rapidly compared to O. viciifolia. The results emphasize that in vivo experiments could be used for screening of NaCl tolerance in sainfoin cultivars without expensive chemicals and sophisticated equipments, but pollen germination is more appropriate for its wild relatives.


Introduction
A total of 160 Onobrychis species exists throughout the world and are spread from Baltic States to Mediterranean region passing through all region lying in the Asia and Iran-Siberian element.In total 27 Onobrychis species are endemic to Turkey.Sainfoin (Onobrychis viciifolia Scop.) is a perennial forage legume crop which is widely adapted to calcareous, well drained, poor and dry soils (Açiköz, 2001;Cavallarin et al., 2005).It has been grown extensively in Eastern Europe and West Asia (Sancak et al., 2003).It is highly tolerant to salinity and drought and improves soil fertility by fixing atmospheric nitrogen (Özaslan-Parlak and Parlak, 2008;Imanparast and Hassanpanah, 2009).It has erect stems and deep tap roots holding soil firmly to the deeper layer and prevents soil erosion.It is preferred to other forage plants on dry soils of Anatolia because it produces satisfactorily hay under adverse conditions (Cavallarin et al., 2005).O. oxyodonta var.armena (Syn: O. armena Boiss.and Huet) is a winter-resistant wild sainfoin species and is found extensively in meadows of Central Anatolia and Eastern Anatolia regions (Elçi, 2005).
Sainfoin is largely grown on low moisture soils of Anatolia where evaporation exceeds precipitation, resulting in salt accumulation on the soil surface (Kaya et al., 2003;Pessarakli, 1999).There are limited reports on the effects of salinity on seed germination and growth in Onobrychis species.Elçi (2005) indicated that it was suitable for salt infected soils and favorable yield could be obtained at moderate level of salinity.Greub et al. (1985) reported that sainfoin (Onobrychis viciifolia Scop.) was tolerant to NaCl, but it ranked lower than the grasses.Özaslan-Parlak and Parlak (2008) has indicated a decrease in plant height, dry hay yield and crude protein ratio due to increased salinity in irrigation water.However, it could not be determined clearly that differences between species against salt tolerance are mainly due to genetic or morphologic factors.Therefore, this study investigated the seed and pollen germination and the seedling growth of O. viciifolia and O. oxyodonta var.armena under various NaCl stresses using in vivo and in vitro techniques. of O. viciifolia Scop. and O. oxyodonta var. armena (Syn: O. armena Boiss. and Huet) were collected from plants grown at same field, under similar cultural practices.Both species were tested against NaCl under in vivo and in vitro conditions.NaCl levels were adjusted as 5, 10, 20 and 30 dS m -1 (electrical conductivities of the solutions) of NaCl using a conductivity meter (Model WTW Cond.314i, Germany).Distilled water served as a control (0 dS m -1 ).

Seeds
Four replicates of 50 seeds of each species were germinated between three rolled filter papers with 15 mL of respective test solutions.Seeds were treated with fungicide before planting; papers were replaced every two days to prevent salt accumulation.The rolled paper with seeds was put Sci.Agric.(Piracicaba, Braz.), v.68, n.4, p.477-481, July/August 2011 into sealed transparent plastic bags to avoid moisture loss.Seeds were allowed to germinate at 20 ± 1ºC in the dark for 14 days.
Surface sterilization of the seeds of both Onobrychis species was done using 50% commercial bleach for 15 min followed by rinsing with distilled sterile water.The surface-sterilized seeds were transferred to germination substrate containing MS medium (Murashige and Skoog, 1962), 30 g L -1 sucrose and 8 g L -1 agar supplemented with 2.72, 4.12, 11.30 and 17.22 g NaCl L -1 for obtaining EC values of 5, 10, 20 and 30 dS m -1 , respectively.The pH of medium was adjusted to 5.7 with 1 M NaOH or HCl before autoclaving at 121ºC, 104 KPa for 20 min.Seeds were allowed to germinate at 20 ± 1ºC under white fluorescent light with 16 h photoperiod.
A seed was considered germinated when the emerging radicle elongated to 2 mm.Germination percentage was recorded every 24 h for 14 days (ISTA, 2003).Mean germination time (MGT) was calculated following Ellis and Roberts (1980) to assess the rate of germination.MGT=∑Dn/∑D, where n is the number of the seeds newly germinated on day D, and D is the number of days from the beginning.Root, shoot length, fresh and dry seedling weights were measured on the 14th day.Dry weights were measured after drying samples at 70ºC for 48 h in an oven.
The experimental design was two factors completely randomized design (CRD) with four replications.Data given in percentages were subjected to arcsin transformation before statistical analysis.For all investigated parameters, Analysis of Variance was performed using the MSTAT-C computer software (Michigan State University).Differences among the mean values were compared by Duncan's Multiple Range test (p < 0.05).

Results and Discussion
Seed germination percentage decreased by increasing NaCl in both species while the decrease was less in O. viciifolia compared to O. armena (Figure 1).The lowest germination (40.5%) was determined in O. armena at 30 dS m -1 .The lower doses of NaCl did not change the seed germination; however, seed germination drastically declined at doses higher than 20 dS m -1 .
Greater reduction in shoot length due to NaCl was very evident (p < 0.01) (Table 1).The longest shoots were obtained from O. viciifolia under all NaCl stresses.Shoot length was severely influenced by NaCl while the impact was more detrimental on O. armena.Increased NaCl diminished root length of the two species; however, this decrease was more prominent in O. armena.
Depending on the decrease in shoot and root length, fresh and dry seedling weights reduced gradually with the  increasing salinity stress (Table 1).No seedling growth was observed in O. armena subjected to 30 dS m -1 .Higher fresh seedling weights were recorded on O. viciifolia compared to O. armena at all NaCl levels.Dry Seedling weight showed a trend similar to that of fresh weight and showed decline in fresh seedling weight, dry weight enhanced with increasing NaCl levels.Increase in dry weight also resulted in increased dry matter of both Onobrychis species.
NaCl influenced seed germination of the species differently.Seed germination was not reduced at 5 dS m -1 , while 10 dS m -1 caused reduction in germination (Figure 2).Furthermore, higher NaCl concentration resulted in lower vi-   ability of the seeds.The detrimental effect of NaCl on seed germination appeared at 30 dS m -1 .However, mean germination time was delayed by increased NaCl.Apparent difference was recorded among the species.MGT increased with each increase in NaCl stress in the two species.Contrarily, NaCl level of 20 dS m -1 caused drastic delay in germination time of two species in general (Table 2).
For the species, root and shoot length decreased in response to increasing concentration of NaCl; however, this decrease was more prominent in O. armena.No root and shoot growth was recorded at NaCl levels of 30 dS m -1 (Table 2).The longest roots and shoots were obtained from O. viciifolia under all NaCl concentrations.Compared to control, each increase in NaCl concentration resulted in remarkable decrease in fresh and dry seedling weights for the species.Although the species responded variable to different NaCl concentrations, the highest seedling fresh weight (564 mg per plant) and dry weight (64.4 mg per plant) were recorded at 5 dS m -1 for O. viciifolia.Depending on shoot and root length, seedling fresh weight fluctuated.In contrast, dry matter of the species was enhanced with increased NaCl except at 30 dS m -1 NaCl.
Pollen germination in control ranged from 83% for O. viciifolia to 96% for O. armena (Figure 3).Saline conditions induced a decrease in pollen germination.A very different response was detected between species at lower NaCl concentrations while none of the species were able to germinate at 30 dS m -1 .Especially, 20 dS m -1 caused a detrimental effect on pollen germination.
Germination and seedling growth of the investigated sainfoin species were influenced by salt concentrations under in vivo and in vitro conditions.However, the responses of these species to salt concentration were different.Reduction in seed germination is in conformity with Khajeh-Hosseini et al. (2003), Murillo-Amador et al. (2002), who found that decreased germination was due to increased salinity.The results of this study are also in agreement with Prakash et al. (1998), Gadallah and Ramadan (1997), Gadallah (1996), Prakash et al. (1995) and Francois and Bernstein (1964), who emphasized that different varieties showed variable response against various salinity levels.Similarly, Hampson and Simpson (1990), Murillo-Amador et al. (2002), Okçu et al. (2005), Kaya et al. (2006), Karlidag et al. (2009) reported that NaCl had only adverse effect on germination by creating osmotic potential in the medium and caused delayed seed germination.There was a clear difference for germination and seedling growth under in vivo and in vitro conditions.Lower values were obtained from in vitro experiments; suggesting that the sucrose, MS mineral salts and agar and in in vitro conditions might have caused higher osmotic potential inhibiting germination and seedling growth of species than under in vivo conditions.Germination and seedling growth were reduced due to increased salt concentration with varying responses for species while NaCl affected germination of seeds by creating an external osmotic potential preventing water uptake oxyodonta var.armena ( ).The bar represents LSD value which is 7.02.(Pessarakli et al., 1991;Kaya et al., 2006).These results agree with Murillo-Amador et al. (2002) in cowpea and Okçu et al. (2005) in pea but should not be approved as a general rule because opposite results were recorded by Atak et al. (2006) who found reduced seedling growth under NaCl salinity.Pollen germination was also adversely affected by increasing NaCl concentration.O. armena gave higher pollen germination at all NaCl levels; suggesting that there was no overlap in the response of seeds and pollen to NaCl.Martinez-Pelle et al. (1995) found different responses with respect to pollen germination under various salinity stresses in pistacia species.O. viciifolia gave higher seed germination and lower time to germinate under in vivo and in vitro conditions.However, the detrimental effect of NaCl on seedling growth was very evident in O. armena because no shoot and root growth was recorded at 30 dS m -1 .Furthermore, dry seedling weight and dry matter of the species enhanced with increased NaCl except at 30 dS m -1 NaCl for O. armena.Similar findings have been reported in Bothriochloa persuta L., Dichanthium annulatum and Panicum antidotale under salinity (Akhtar and Hussain, 2008).The results are in line with the findings of Rehman et al. (2008) in wheat, Steppuhn et al. (2001) in canola, field bean, dry bean and drum wheat and Li (2008) in Limonium sinense, Sorghum sudanense and soybean.
In conclusion, critical period of crop plant to salinity generally occur on germination and seedling growth stages.The relative time of seed germination and seedling emergence of plants influences the degree of tolerance.Thus, the species or cultivars which rapidly germinate or emerge gain advantages for tolerance to NaCl.NaCl caused an adverse effect on germination and seedling growth with delayed mean germination time in the species.Similar results were detected under in vivo and in vitro conditions while lower values were determined in in vitro.It seems that O. armena germinate and grow more slowly compared to O. viciifolia.Both sainfoin species could keep up with the salinity up to 10 dS m -1 during seed germination and early growth stages, and O. viciifolia was more tolerant to NaCl compared to O. oxyodonta var.armena.

Figure 1 -
Figure 1 -Seed germination percentage of O. viciifolia ( ) and O. oxyodonta var.armena ( ) under in vivo condition.The bar represents LSD value which is 12.5.

Table 1 -
Mean germination time (MGT), shoot length, root length, seedling fresh and dry weight and dry matter ratio of sainfoin species at various NaCl levels under in vivo conditions.
*significant at p < 0.05, **p < 0.01 and ns: non-significant.Different letters at the same column show significant differences at 0.05 level.

Table 2 -
Mean germination time (MGT), shoot length, root length, seedling fresh and dry weight and dry matter ratio of sainfoin species at various NaCl levels under in vitro conditions *significant at p < 0.05, **p < 0.01 and ns: non-significant.Different letters at the same column show significant differences at 0.05 level.