Int'l Journal of Advances in Agricultural & Environmental Engg. (IJAAEE) Vol. 2, Issue 2 (2015) ISSN 2349-1523 EISSN 2349-1531 Evaluation of Chemical Scarification and Priming Treatments to Break Physical Dormancy of Crotalaria senegalensis seeds Atif Hassan Naim*  In Sudan, there are two species, Crotalaria senegalensis and Crotalaria retusa, locally called "Safari", both grow in areas degraded by erosion on infertile soils and of bad physical properties [3]. The plant is a popular forage shrub due to its high crude protein content (about 17%) and an excellent palatability for grazing animals such as camels, cows and goats in natural ranges of Sudan. C. senegalensis (Safari) has an adaptive advantage of having an annual cycle combined with a "seed escape" habit [3]. The plant as a selfreseeding legume has developed specific strategies to ensure adaptation and reproduction under harsh climatic conditions [3]. Abstract— The present study was conducted to evaluate various chemical scarification methods for breaking physical dormancy of Crotalaria senegalensis seeds. The seeds were subjected to the following treatments: (1) soaking in hot distilled water (80°C) for 15 and 30 min, (2) immersion in H2SO4 (98%) for 20, 25 and 30 min, (3) immersion in 0.5 %, 1% and 1.5 % KNO3 and (4) soaking in 50, 100 and 150 µM H2O2. All scarification treatments improved the germination capacity of C. senegalensis seeds, the highest final germination rate and germinate rate index, were recorded after soaking in 150 µM H2O2, followed by soaking intact seeds in H2SO4 (98%) for 30 min. and immersion seeds in 1.5 % KNO3. The results showed that using concentrated H2SO4 (98%) for 30 min to break seed physical dormancy of C. senegalensis was the second most effective treatments ( after 150 µM H2O2 treatment) but it is commonly not preferred due to its cost, safety risk and environmental precautions involved. In conclusion, to break both physical and/or physiological dormancy of C. senegalensis seeds, soaking in 150 µM H2O2 and 1.5 % KNO3 represent the most recommended treatments. Generally, legumes seeds exhibit hardseededness resulting in dormancy [3] [4]. Several studies have been conducted on legume germination using different seed coat pre-sowing treatments [5] [6] [7]. The function of the seed coat is to protect the embryo and endosperm from desiccation, mechanical injury, unfavorable temperatures and attacks by bacteria, fungi and insects [8]. Similarly to many other taxa of legumes, the seeds of Crotalaria plants remain in a state of physical dormancy until the seed coat is made permeable by some environmental factors in natural conditions [9]. Keywords— Chemical Scarification, Crotalaria senegalensis, Germination rate, Hydrogen peroxide, Physical dormancy. I. INTRODUCTION Rapid and uniform field emergence is essential to achieve high yield with respect to quantity and quality of crops [10]. Seed priming currently is widely used to accelerate seed germination rate and improve seedling uniformity in many crops [11]. According to [11], in priming, seeds are exposed to limited water availability under controlled conditions which allows some of physiological processes of germination to occur and also accumulates certain oxidant compounds which activate special enzymes which ultimately break dormancy and accelerate germination speed [11]. It has been known for a long time that an oxidant compound called hydrogen peroxide (H2 O2 ) is synthesized at very high rates in the plant cells and involved in virtually all major areas of aerobic biochemistry. Hydrogen peroxide (H2 O2 ) is involved in copious quantities by several enzymes (e.g. plasmalemma-bound NADPH-dependent superoxide synthase and other anti-oxidant enzymes. Recently it was reported that exogenously pretreatment with hydrogen peroxide (H2 O2 ) leads to breaking seed dormancy and promoted germination of Zinnia elegans L. seeds which had hard seed coat [12]. O RTHODOX seeds are shed from their mother plants at low water contents and are tolerant to desiccation. On the other hand, recalcitrant seeds are shed at high water contents and are sensitive to desiccation [1]. Most of forage legumes in rangelands and pastures are orthodox seeds. A variety of mechanisms of desiccation tolerance has been suggested to confer protection against the consequence of water loss at different hydration levels, and the effective expression of one or more of these could determine the relative degree of desiccation tolerance [2]. Naturally shed seeds of Crotalaria senegalensis are both desiccation tolerance and dormant, requiring warm weather and priming in water or scarification with H2SO4 to ensure high levels of germination at 20-25oC [3]. Atif H. Naim* is with Department of Crop Sciences, Faculty of Agricultural and Environmental Sciences, University of Gadarif, Gadarif State, Sudan (phone:+249112300344; e-mail: atif882002@yahoo.com). http://dx.doi.org/10.15242/IJAAEE.ER1215035 67 Int'l Journal of Advances in Agricultural & Environmental Engg. (IJAAEE) Vol. 2, Issue 2 (2015) ISSN 2349-1523 EISSN 2349-1531 This study aims at evaluating the impact of various chemical treatments on the germination response of Crotalaria senegalensis in order to develop an effective method of breaking seed physical dormancy. 4) Germination rate index (GRI) According to [14] germination rate index can be calculated as follows: GRI = [G1/1 + G2/2 + ………..+ Gx/x] Where, II. MATERIALS AND METHODS G = the germination on each alternative day after placement. 1, 2, x = the corresponding day of germination A. Plant materials Fresh seeds of Crotalaria senegalensis seeds were collected at maturity from shrubs growing in Gadarif state in Sudan (rainfall 700 mm per year, mean temperature 25-27oC with a mean winter minimum about 15-2oC and a mean summer maximum about 35oC) in October and November, 2014. The fresh weight of 1000 seeds was 7.0 g and the water content was 4.6% on dry weigh basis, which was estimated after drying the seeds at 70oC for 24h. All seeds were surface sterilized in a solution of 1% sodium hypochlorite (NaOCl) for 3 minutes and then rinsed three times in sterilized water prior to an experimental procedure to prevent fungal contamination. 5) Corrected germination rate index (CGRI) CGRI = (GRI/FGP) X 100 [14] D. Statistical analysis All experiments were arranged in a completely randomized design. There were 11 treatments replicated 4 times, and each replication consisted of 20 seeds. Data were subjected to one way analysis of variance (ANOVA) and mean separation among treatments was carried out by Least Significant Difference (LSD) using SPSS program (version 15). Excel computer software was used for making graphs. III. RESULTS B. Physical scarification and Priming treatments Seeds were subjected to different physical scarification and chemical treatments. Physical scarification was carried out by soaking intact seeds in hot distilled water (80°C) for 15 and 30 min. After completion of hot water treatments, seeds were removed from the water and left to cool for 10 min. Chemical scarification (priming) was accomplished by using three different chemical compounds. First, samples of intact seeds were soaked separately in concentrated sulphuric acid (98% H2SO4) for 20, 25 and 30 min. Secondly, samples of intact seeds soaked separately in potassium nitrate (KNO3) at 0.5, 1, 1.5 and 2 % for 24 h. Thirdly, soaking intact seeds in 50, 100 and 150 µM of (H2O2) for 30 seconds. The effects of various pre-sowing seed treatments on the time-course changes in germination percentage of Crotalaria senegalensis are shown in (Fig.1). In the hot water treatments; germination percentage at both exposure time was identical (Fig. 1A). Intact seeds exposed to hot water for 15 and 30 min hot were capable to break seed dormancy of C. senegalensis. On the other hand, in the H2SO4 treatments, germination percentage was improved with increasing exposure time of seeds to the acid (Fig.1B). Also, increasing concentration of KNO3 significantly increased germination percentage of soaked seeds (Fig. 1C). The Results also showed that intact seeds soaked in hydrogen peroxide (150 µM H2O2) significantly increased germination rate compared with other treatments (Fig. 1 D). C. Germination assessment Effects of pre-sowing treatments on final germination percentage (FGP), germination rate index (GRI), corrected germination rate index (CGRI) and time to 50% of germination (GT50) were shown in Table 1. All breaking dormancy treatments significantly (P < 0.05) affected germination attributes of C. senegalensis. Soaking C. senegalensis seeds in hot water significantly (P < 0.05) increased FGP (Table 1). In this regard, soaking seeds in hot water for 30 minutes recorder higher FGP compared with Hot soak for short time “15min.” (Table 1). Immersing seeds in H2SO4 also significantly (P < 0.05) broke the dormancy of C. senegalensis seeds at all application times; but exposing seeds to H2SO4 for 30 minutes significantly (P < 0.05) recorded the highest FGP among the other two H2SO4 treatments (Table 1). Interestingly, increasing the time of immersion seeds in concentrated H2SO4 significantly increased FGP of C. senegalensis seeds (Table 1). The results also revealed that soaking the seeds in hot water at both times (15 minutes and 30 minutes caused identical and insignificant After completion of pre-sowing physical and priming treatments, batches of 20 seeds from each treatment were germinated on moist filter paper in closed Petri dishes (12.5 cm) for 20 days. Germinated seeds were counted every 48 h. All Petri dishes were incubated at 25°C and 16 h photoperiod by a fluorescent light at 40 µmol m-2 s-1. According to [9], seeds were considered germinated upon emergence of radicals ( length ≥ 2 mm). The following germination parameters were recorded: 1) Final germination percentage (FGP) = (number of number of germinated seeds/number of total seeds) X100 2) Mean time to germination (MTG or G50) was calculated according to the following equation [13]. 3) MTG or G50 = ∑ Dn / ∑ n Where, n = number of seeds which were germinated on day D. D = the number of days counted from the beginning of germination. http://dx.doi.org/10.15242/IJAAEE.ER1215035 68 Int'l Journal of Advances in Agricultural & Environmental Engg. (IJAAEE) Vol. 2, Issue 2 (2015) ISSN 2349-1523 EISSN 2349-1531 this regard, soaking the seeds in 150 µM H2O2 significantly scored the highest GRI and CGRI among all pre-sowing studied treatments followed by 1.5 % KNO3 (Table 1). The significant lowest GRI and CGRI were recorded in the immersion treatment in H2SO4 for 20 minutes (Table 1). The half time of germination (GT50) was also significantly (P < 0.05) affected by all studied treatments (Table 1). Soaking the seeds in H2O2 was significantly (P < 0.05) curtailed the GT50 (Table 1). The highest GT50 was recorded by soaking the seeds in 2 % KNO3 (Table 1). Soaking seeds in hot water for 15 min significantly increased GT50 and scored the highest days to reach 50% germination (6.5 d). The Hydrogen peroxide (H2O2) treatments significantly increased FGP, GRI and CGRI and decreased GT50 (Table 1). difference in the FGP (Table 1). TABLE I: EFFECT OF PRE-SOWING TREATMENTS ON FINAL GERMINATION PERCENTAGE (FGP) GERMINATION RATE INDEX (GRI), CORRECTED GERMINATION RATE INDEX (CGRI) AND TIME TAKEN TO REACH 50% OF FINAL GERMINATION PERCENTAGE (GT50) FOR CROTALARIA SENEGALENSIS AFTER 20 DAYS IN CULTURE Treatments Hot water soak (15 min) Hot water soak (30 min) H2SO4 (20 min) H2SO4 (25 min) H2SO4 (30 min) 0.5 % KNO3 1 % KNO3 1.5 % KNO3 50 µM H2O2 100 µM H2O2 150 µM H2O2 LSD 0.05 FGP GRI CGRI GT50 67.3 (55.1)* e 0.20 e 0.36 b 6.5 a 67.5 (55.3) e 0.20 e 0.36 b 5.8 b 45.0 (40.5) g 0.12 f 0.19 e 0.30 c 0.35 b 4.0 cd 3.8 d 0.32 b 0.39 ab 4.5 c 0.37 a 0.38 a 3.0 e 0.26 c 0.36 b 5.6 b 0.31 b 0.39 ab 6.0 ab 0.25 d 0.40 a 2.5 f 65.0 (53.8) f 95.0 (82..5) b 37.0 (37..5) h 90.0 (72..3) c 94.0 (80.5) bc 80 (62.1) d 90 (72..3) c 100 (90)a 4.2 0.28 c 0.39 a 2.2f 0.37 a 0.41a 2.8ef 0.02 0.04 0.6 *Values between two bracts represent arcsine transformation of FGP. IV. DISCUSSION The present study evaluates chemical treatments to break physical dormancy of Crotalaria senegalensis seeds. The results of the this study revealed that all chemical (priming) and hot water treatments significantly (P< 0.05) broke the physical dormancy of C. senegalensis. This response provides evidence that the seed coat of the plant is the main inhibitor of germination. In treatments using intact seeds, soaking in hot water increased GP and GRI. Many researchers [15] found that soaking seeds in hot water for specific period break exogenous seed dormancy due to making scratch in hard seed coat which facilitate the imbibition. Moreover this treatment enhance seed germination in many plant species.. This results are in harmony with [16] [17] [18]. The response of H2SO4 as a method for breaking seed dormancy in this study was consistent with other studies in different species [19] [20] [21] [22]. Some researchers [23] [24] reported that the seeds Moreover soaking seeds in hot water for short period as 15 minutes significantly (P < 0.05) recorded higher FGP compared to immersion the seeds in concentrated H2SO4 for 20 minutes (Table 1). In KNO3 treatments using intact seeds of C. senegalensis there were significant (P < 0.05) increase in the FGP (Table 1). The highest FGP was recorded in favor of soaking the seeds in 1.5 % KNO3 and decreased significantly (P < 0.05) by decreasing KNO3 concentration (Table 1). The germination speed (germination rate index (GRI) and corrected germination rate index (CGRI)) were significantly (P< 0.05) affected by all pre-sowing treatments (Table 1). In http://dx.doi.org/10.15242/IJAAEE.ER1215035 69 Int'l Journal of Advances in Agricultural & Environmental Engg. (IJAAEE) Vol. 2, Issue 2 (2015) ISSN 2349-1523 EISSN 2349-1531 of Crotalaria obtained from a natural environment and the H2SO4 scarification treatments simulated pass of the seeds through the digestive tract of animals (birds and rodents), which under natural conditions execute chemical scarification. Although the acid scarification significantly broke the dormancy of crotalaria seeds and enhanced germination , but it is commonly not preferred due to its cost, safety risk and environmental precautions involved, and not reliable or lacking the requisite qualities on seeds of other important plant species [25] [26] [27]. The present study reveals that crotalaria species has dual (physical and physiological) dormancy due to the positive response to chemical scarification treatments such as H2O2 and KNO3. The concentration 1% and 1.5 % of the nitrate treatments in this study enhanced germination compared with the highest concentration 2% of the nitrate in our previous study [3]. These concentrations of nitrate (1 and 1.5%) might simulated the case in the soil after rainfall which dilutes nitrate and make it available for seeds. Supporting evidence was reported by [28]. Nitrate has been stated as being a growth-regulating substance in some plant species such as Salvia [3]. The results of this study also revealed the importance of H2O2 for breaking the physical dormancy of C. senegalensis and the concentration (150 µM) of this oxidant was the best treatment to break dormancy and accelerate germination of C. senegalensis seeds. In accord with this finding, some authors have also observed increased germination of other species by soaking seeds in H2O2[29]. Previously, a researcher in his recent paper [29] showed that O2 and H2O uptake is substantially increased in H2O2 soak treatment for Pseudotsuga menziesii seeds than in the water control, suggesting an enhanced conversion rate of reserve lipids to carbohydrates and, consequently, increased synthesis of cellular components. as a forage in rangeland and pastures. ACKNOWLEDGMENT The authors are grateful for kind financial aid received from the research board of University of Gadarif, Gadarif state in Sudan. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] V. CONCLUSION [10] The present study reveals that crotalaria exhibit physical or exogenous dormancy and is entirely imposed by the hard seed coat. The integument is able to withstand unfavorable conditions such as heat, teeth of dispersing agent and mechanical damage prevailing in the natural habitat. This avoidance of germination is ecologically advantageous to the plant grows in harsh climatic conditions , in that seeds accumulate in the soil to increase the chance that some of these seeds will germinate and create new population to maintain the species. But this is limiting when quick and consistent seed germination is desirable for successful establishment of economically important forage plant species. Our results demonstrate that mechanical scarification and soaking in water for 24 h , soaking in 1.5 % KNO3 solution break dormancy and promote germination of Crotalaria senegalensis. On the other hand, this study clearly found the positive effect of the oxidant agent H2O2 in breaking dormancy of C. senegalensis. 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