Effect of seeds pretreatment and storage on improvement of the germination and emergence of Anthyllis cytisoïdes

1 Institut National de Recherche Agronomique, Boukhalef, Hay Hassani, BP 9301, Tanger, Morocco. 2 Faculté des Sciences et Techniques Al Hoceima, Université Mohamed Premier, BP34, Ajdir, Al Hoceima, Morocco. 3 Laboratoire des Sciences de l’Eau, Environnement et Ecologie, Faculté des Sciences, Université Mohamed Premier, BP 717, Oujda, Morocco. 4 Institut National de Recherche Agronomique, 10, Bd Mohammed VI, BP 428, Oujda, Morocco.

along roads (Al Hoceima city [lat. 35°15′05″N,long. 3°56′14″W] to Nador city [lat.35°10'7.02"N, long. 2°55'39.3"W];Al Hoceima to Taza [lat. 34°12'38.02"N,long. 3°59'52.97"W];Oujda [lat. 34°40′53″N,long. 1°54′30″W] to Taza city) or along rivers.In this region, planting of other species like Atriplex nummularia promotes regeneration of A. cytisoïdes L. because the former prevents rain water from running off slopes.The water retained by A. nummularia induces germination of A. cytisoïdes L. seeds after their imbibition.Moreover, A. cytisoïdes L. can grow in different types of soil, preferably in plowed fields and/or in fallow land (Robledo et al., 1990).Anthyllis is a great pastoral interest and is highly nutritious for livestock (Selmi and Ferchichi, 2015).It is also of great ecological importance and plays a special role in improving soil fertility (Boza et al., 1988;Moreno Rios et al., 1985).Legumes are interesting to research on because of their adaptation to arid and semi-arid environments, their capacity to grow in poor soil, their quality as forage crops for livestock, and their ease of multiplication and natural dispersion of seeds.Because soil erosion and degradation processes currently affect much of the Mediterranean coastline, there is an urgent need to take action regarding the conservation of natural resources, regeneration and improving the fragile ecosystems in arid and semi-arid regions.To accomplish this, it would be important to rehabilitate A. cytisoïdes L., which is currently in danger of disappearing because of its extensive use in eastern Morocco.Rehabilitation of certain indigenous legumes, including A. cytisoïdes L. and Retama sphaerocarpa Boiss., has been the focus of several recent research studies.Many authors mentioned that legumes often have impermeable integuments that induce physiological dormancy and make germination of their seeds very difficult (Jayasuriya et al., 2013).For this reason, it is important to identify and investigate the endogenous and exogenous factors that influence the normal emergence of stored legume seeds.Seed lots with high initial viability have a higher germination capacity than seed lots with low initial viability.A. cytisoïdes L. seeds have an anatomical structure typical of legumes, which is characterized by a hard envelope that prevents the imbibition of the seed by water, awakens the embryo and triggers germination and emergence.Under these conditions, germination is still weak, slow and heterogeneous.This implies that natural and artificial decortications of the seed envelope could improve imbibition and germination (Musara et al., 2015;Valente et al., 2016).To test this, experiments were carried out to determine which factors can improve the rate of germination and emergence of this species.It is important to note that germination and emergence are two crucial steps for native and domestic species (Giusti and Grau, 1983).
The main objective of this study was to verify the hypothesis that pretreatment of seeds would have significant effects on the germination of A. cytisoïdes L. and that seeds lose their viability with age.

Date and location of seed collection
The used plant material was seeds of the species, A. cytisoïdes L., which is an indehiscent legume containing a single seed.The seeds have dimensions of 0.2 × 0.3 mm and the average dry weight is 1 to 4 mg.They are naturally disseminated mainly by wind, water and sometimes animals.The seeds were collected in July 2006July , 2008July , 2009July and 2010 in the area of Guenfouda (a province of Jerada) [lat.34°18'22.24"N, long. 2°10'45.89"W].Seeds were stored at room temperature in the laboratory at the Institute National de la Recherche Agronomique, (INRA) of Oujda-Morocco, in plastic bottles.

Pretreatment of seeds
The seeds of A. cytisoïdes L. are covered by hard and cutinized envelopes, which completely prevent the imbibition of water and, sometimes, even prevent gas exchange.However, without imbibition and gas exchange, initiation of embryonic growth and germination is impossible.The physical dormancy of this kind of seed is often observed in several leguminous species.
Therefore, to break dormancy, seeds of A. cytisoïdes L. were pretreated as follows: (T1) seeds were soaked in running water for 48 h; (T2) light scarification was made with abrasive paper; (T3) strong scarification was made with abrasive paper; (T4) seeds were soaked in running water for 48 h; (T5) seeds were soaked in hot water for 10 min; (T6) seeds were totally decorticated.These pretreated seeds were compared to controls seeds lacking any pretreatment (T0).

Seed scarification
Physical pretreatment methods that facilitate transition from dormancy consist of piercing, or splitting the integument in order to make it more permeable without damaging the embryo or the endosperm of the grain (Bonner, 1984).To test the effectiveness of mechanical scarification, seeds were placed between two sheets of abrasive paper (10 cm in diameter) and rubbed several times upstream and moving forward under the pressure of hands for light scarification in experiment T2.This method was used for T3 with increased friction until the yellow layer appeared.After mechanical scarification of the pericarp, seeds were placed in pots filled with potting soil.

Soaking in running water
Some pretreatments involve soaking seeds in water.These treatments make it possible to combine the effects of softening integuments and leaching of chemical inhibitors from the seeds (Vázquez et al., 2014).seeds were soaked in water for 24 and 48 h in a beaker filled with running water.Then, they were used in experiments T1 and T4, respectively.

Soaking in hot water
Pretreatments in hot water were tested for seeds.Seeds were immersed in boiling water at 100°C for 10 min, removed from the heat source and cooled slowly for about 1 h.This pre-treatment is Each pot contained 20 seeds of A. cytisoids L. and each pretreatment condition was repeated five times.δ R2 = 0.928 (R2adjusted = 0.911).γ α = 0.05; * Significant.
usually used to reduce the mechanical restriction of the cover layer of the seed, make it softer and help in germination (Hajlaoui et al., 2016).The seeds that had imbibed water and swelled were only subsequently planted in pots filled with potting soil.

Decorticating seeds
Seed extraction is easily accomplished by spreading seeds on a platform, a straw mat or other suitable support and beating them with a flail or a thin pole.If the quantities of decorticated seeds extracted are high, it is possible to adapt mechanical methods used in agriculture (Habit et al., 1981).Extraction of the seeds was carried out by threshing and using abrasive paper to take off the coat and release the seed (Appendix 1).The threshing of 100 seeds by hand took about 30 min, and separation was done manually.

Seed viability
The effects of seed viability on the germination rate were evaluated and tested using four seed lots collected in the years, 2006, 2008, 2009 and 2010.

Experimental procedures
Before seeding, 140 pots (2 L) were filled with soil (compost) rich in organic matter.Each pot contained 20 seeds and each pretreatment condition was repeated five times.The pots were irrigated and after 24 h, pretreated grains were seeded into the pots.During the emergence period, the pots were irrigated every 2 days.The pots were placed in a glasshouse located at the INRA-Oujda for optimal germination and growth conditions.The duration of the experiments was 35 days, from January 30 th until March 5 th , 2015.Observations were recorded daily.The appearance of a cotyledon was the criterion for seed emergence (Appendix 2).

Statistical analysis
The experimental design was a complete random block with two factors.The first factor was the pretreatment condition, which included seven variations, and the second factor was age of the seeds, with four age groups.The pretreatment and viability data were analyzed using an ANOVA (analysis of variance) via IBM

Effects of decortication
Regardless of seed age, results shown in Table 1 revealed a significant effect of the pretreatment and seeds age on the rate of emergence.The pretreatment T6 had a significant higher value of germination relative to all other pretreatments, with emergence reaching 69% at the end of the experiment (Table 2).In terms of the time to emergence, the seeds germinated quickly, within 15 to 20 days, and accounted for 60 to 70% of the germination rate (data not shown).

Effects of boiling in water
The T5 pretreatment had a significant effect on seed emergence of Anthyllis cytisoïdes L. In that case, the germination rate was 23%, which is higher than the control (Table 2).Figure 1 shows that seeds from the 2009 lot with T5 pretreatment had a germination rate of 40%.This result is also supported in Figure 2 where it shows that T5 pretreatment had the second highest effect on germination rate after T6 pretreatment.This was observed from 17 days after seedling.

Effects of seed scarification
The results from either the light or strong scarification pretreatment showed only a very small increase relative to the control.The low mean germination rates in T3 and T4 pretreatment groups were 12.75 and 6.25%,  respectively (Table 2).

Effects of soaking seeds in regular running water
A Tukey's test (Table 2) demonstrated that there is no significant effect (P<0.05) on the means germination rate among the pretreatment groups T2, T1 and T0: the values were 4.55, 4.50 and 4.00%, respectively.The low levels for T0, T1 and T2 groups were observed in seeds from the 2006, 2008, 2009and 2010 lots (Figures 1 and 2) lots (Figures 1 and 2).

Seed viability following multi-year storage
A comparison of the averages (Table 3) showed that the loss in viability was very low and the three seed lots showed no significant differences.The average germination rates were 18.00, 19.57and 20.57% for seeds from the 2008, 2010 and 2009 lots, respectively.Only seeds from the 2006 lot showed significantly lower percentages of germination as compared to the other lots (12%).

DISCUSSION
The significant effect detected in the T6 pretreatment on the rate of emergence can be explained by the fact that once the mechanical and chemical dormancy was broken by decortication, the imbibition of water and gas exchange allowed the resumption of embryonic growth, germination and emergence of the seeds.As observed, the seeds envelopes constitutes the main barrier to uniform and rapid emergence.In its natural environment, decortication can occur in the mouth of animals as the seeds are crushed by teeth and released.This may explain the high dispersal capacity and abundance of this  This study provides useful information on the preservation and conservation of this native species, which is especially useful now that A. cytisoïdes L. is becoming rarer.This study is of interest to multiple species in these parts of Morocco for pasture rehabilitation, erosion reduction and diminishing the impact of climate change.
For these reasons, it is recommended that future research be done for a better understanding of both sexual and asexual propagation, morphology and anatomy of the seeds and the ecology of this rare species.

Figure 1 .
Figure 1.Effects of pretreatments and storage age of the seeds on A. cytisoides L. germination rate.(T1) Seeds were soaked in running water for 48h; (T2) seeds were lightly scarred; (T3) seeds were strongly scarred; (T4) seeds were soaked in running water for 24 h; (T5) seeds were soaked in hot water for 10 min and (T6) seed coats were removed.Each pot contained 20 seeds of A. cytisoids L. and each pretreatment condition was repeated five times.Seeds from lots collected in four different years: 2006, 2008, 2009 and 2010 were used.

Figure 2 .
Figure 2. Daily emergence rate for different pretreatments and for different seeds lots.(T1) Seeds were soaked in running water for 48 h; (T2) seeds were lightly scarred; (T3) seeds were strongly scarred; (T4) seeds were soaked in running water for 24 h; (T5) seeds were soaked in hot water for 10 min and (T6) seed coats were removed.Each pot contained 20 seeds of A. cytisoids L. and each pretreatment condition was repeated five times.Seeds from lots collected in four different years: 2006, 2008, 2009 and 2010 were used.

Table 1 .
Effects of pretreatments and seeds age on A. cytisoïdes L. germination.
SPSS Statistics 21 software.The univariate general linear model was used.Comparisons of pretreatment averages and seed age were made by Tukey's multiple comparison test.

Table 2 .
Comparison among the averages of each pretreatment (germination rate, %), processed by test of Tukey.T4) seeds were soaked in running water for 24 h; (T5) seeds were soaked in hot water for 10 min and (T6) seed coats were removed.Each pot contained 20 seeds of A. cytisoids L. and each pretreatment condition was repeated five times.The error term is medium square (Error) = 48.9; δ Number of samples of the harmonic means = 20; γ α = 0.05; (n.s): not significant.

Table 3 .
Comparison among the averages of the seeds viability for each lots (germination rate, %), processed by test of Tukey.