Hormonal effect on okra seeds germination (Abelmoschus esculentus L.) under salt stressYAKOUBI Fatima, BABOU Fatima Z, BELKHODJA MoulayLaboratory of Plant PhysiologyUniversity of Oran 1 Ahmed Ben Bella Oran Algeria email: [email protected] study aims to evaluate the effect of gibberellic acid (GA3) and abscisic acid (ABA) on the germination of okra seeds (Abelmoschus esculentus L.) exposed under salinity constraint.In the first part, seeds are germinated in hormonal solutions of GA3 (5 ?M and 10 ?M) and ABA (5 ?M and 10 ?M), in the presence of NaCl (100 mM) or not. Results show that NaCl reduces significantly the precocity of seeds germination without influencing its final rate; contrarily, it has a negative effect on hypocotylar growth, fresh weight and seedlings water content. The application of GA3 treatment attenuates the depressive effect of NaCl on germination by stimulating it from the first day of sowing (after 24 h) with 20% and 26.66% under the effect of 5 ?M and 10 ?M of GA3 respectively. In the same way, this phytohormone seems positively influencing on the hypocotylar length, fresh and dry weight as well as the water content of the okra seedlings. On the contrary, ABA does not act effectively in inducing tolerance to salinity.Key words: Abelmoschus esculentus L., ABA, GA3, germination, okra, salinity, NaCl.1. INTRODUCTION In their natural environment, plants are subjected to a large number of environmental constraints of a biotic and an abiotic nature that will influence their growth and development (MAAROUF and RAYNAUD, 2007). Soil salinity is one of the major abiotic factors limiting agricultural production in arid and semi-arid regions (Koca and al., 2007, DONG and al., 2009) affecting more than 80 million ha of arable land worldwide (FAO, 2008). This salinity is due to the presence of high amounts of minerals such as Na + and Cl- in soils and water (TAVAKKOLI and al.,2010,XXXXX) .Algeria is among the threatened countries with 3.2 million ha that are affected by salinity (Szablocs 1989, Belkhodja and Bidai 2004).Germination is considered as a critical step in plants development cycle. Indeed, it conditions the seedling’s installation, its adaptation to the environment and its subsequent productivity (Kaur and al., 1998, Bahrani 2015, ZHU, 2016). Salinity generally reduces germination in both glycophytes (Siddiqui and al., 2006, Bia?ecka and K?pczy?ski, 2009, Ndiaye and al., 2014) and halophytes (Debez and al., 2004; XXXXXX;Gul and al., 2013). For that, the search for treatments that can reduce the effect of salinity during this phasis will be essential. In the last two decades, physiological treatments such as hormone inputs have been intensively studied, assuming that the depressive effect of salinity on germination could be related to either a decrease or an increase in endogenous hormone levels (Debez and al. , 2001, Fusun and al., 2004; XXXXXX).Among the main phytohormones regulating the process of seeds germination is abscissic acid and gibberellins (XXXX.Graeber et al., 2012, Shu et al., 2016b, Nee and al., 2016) .Wang and al. (2001) and Zhang and al. (2006) have clearly defined that abscisic acid (ABA) increases in response to salinity while it has been found in parallel that salt stress induces a reduction of GA3 levels thereby preventing embryonic expansion and lifting the dormancy (Atia and al., 2009, Cao and al., 2014) .As a result, the application of GA3 can play a central role in plant responses to salt stress especially in the early stages of development (Khan and al. Gul., 2006; XXXXXX;Iqbal and Ashraf, 2013).Liopa-Tsakalidia and al. (2012) showed that hormone supplementation of GA3 increases germination rate and seedlings growth, and provides an interesting approach to counter the effects of salt stress.In our knowledge very little work is conducted on the behavior of okra (Abelmoschus esculentus L.) and therefore the objective of this work is to analyze the effect of gibberellic acid (GA3) and abscissic acid (ABA) on the process of seeds germination of this specy subjected to salinity constraint. 2. MATERIEL ET METHODES 2.1. Vegetal material:Seeds are harvested in July 2012 from the fruits of okra (Abelmoschus esculentusL.) that is grown on a parcel of agricultural land.2.2. Seed preparation for germination tests:Seeds are disinfected with 2% of sodium hypochlorite during 3 minutes, rinsed thoroughly with distilled water and then dried on filter paper and placed in Petri dishes lined with two layers of sterile filter paper. Each test is carried out on 30 seeds which makes 3 repetitions of 10 seeds per Petri dish. Control seeds receive 10 ml of distilled water and the same volume of the different saline and hormonal solutions: NaCl (100 mM), ABA (5 and 10 ?M ), GA3 (5 and 10 ?M), ABA / NaCl, GA3 / NaCl is added to the treated seeds.The Petri dishes are put in an incubator that is set at 28 ° C. Daily, a count of germinated seeds is carried out for a week. The germination is marked by the exit of the radicle out of the seed’s coat which its length is at least 2 mm (Côme XXX). During this test the parameters are studied:-The precocity of germination: represented by the rate of the first germinated seeds, so be after 24 hours of sowing.- Final rate of germination (Tg): this parameter is the best way to identify the germination ability of the seeds under the different treatments used. It is expressed as the ratio of the percentage of germinating seeds (Ni) to the total number of seeds recorded at the end of the test (Nt).Tg = Ni × 100 / Nt (Tg: germination rate)-The length of hypocotyls: this parameter is a good step to evaluate the salt stress effect and the hormonal treatments effect on the hypocotyl’s growth, this length is estimated using a caliper at the end of the germination test (after 7 days of sowing).- water content (MC?): The water content of the seedlings is determined by the calculation of the fresh weight (FW) of the seedlings before drying them in the oven at 80 ° C during 48 hours. The dry weight is then determined (DW) and the water content is calculated by the formula of (MONNEVEUX, 1991):WC (%) = (FW-DW / FW) * 100The results are statistically analyzed using the Fisher test at the significance level P = 0.05 using SPSS software version 17.00.3. RESULTATS1. Precocity of germination1.1. In the absence of NaCl, the first germinations occur from the first day of sowing for the control seeds and those treated with AG3. It should be noted that the germination rate increases considerably for the seeds receiving the hormonal solution at 5 and 10 ?M of GA3 with respective levels 76.66% and 83.33%. On the other hand, any germination is observed on seeds under the ABA effect during the first 48 hours; beyond that, the seeds germination appears in a slow way compared to those treated with GA3.2.2. In the presence of 100 mM NaCl, the seeds germinate only from the second day of sowing. When the seeds receive the GA3 at 5 and 10 ?M associated with the saline solution, the germination starts the first day of sowing with respective rates of 20% and 26.66%. On the other hand, a very pronounced germination delay is recorded in the seeds treated with ABA. Figure 1. Effect of hormone/salinity interaction on germination precocity (%). 2. Final rate of germination:2.1. In the absence of NaCl, a final rate of 100% is recorded in the control seeds and those irrigated with GA3. This rate increases to 93.33% in the presence of ABA at 5 ?M while the lowest rate is recorded in seeds receiving 10 ?M ABA with 76.66% sprouted seeds.2.2. In the presence of 100 mM NaCl, the final rate of germinated seeds reaches 93.33% then it increases until all of the seeds are germinated under the combination of GA3 with NaCl solution, the lowest final rates are recorded in the seeds subjected to the combined ABA / NaCl treatments (5 and 10 ?M of ABA) with respective rates of 23.33% and 6.66%.Figure 2. Effect of hormone/salinity interaction on the final rate of germination (%)3. Length of hypocotyls3.1. In the absence of NaCl, the hypocotylar length is positively influenced by the presence of GA3 in the imbibing medium; the highest length is recorded in seeds receiving 10 ?M of GA3 (12 cm) followed by control seeds and those receiving 5 ?M of GA3. In addition, it should be noted that treatment with ABA acts by slowing the hypocotylar growth.3.2. In the presence of 100 mM NaCl, the hypocotyl’s growth is slowed down, whereas the treatment with GA3 (10 and 5 ?M) associated with NaCl has positively affected this parameter so that the respective lengths vary between 10.4 cm and 8.47 cm. On the other hand, it should be noted that the hypocotylar growth is none in seeds subjected to ABA / NaCl treatment. Figure 3. Effect of hormone/salinity interaction on the hypocotyl length (cm)4. Fresh and dry weight of seedlings4.1. In the absence of NaCl:The FW seems positively influenced by the GA3 effect. Thus, the highest FW is noted in treated seeds at 10 ?M GA3 (0.58 g). In contrast, the seeds exposed to ABA record a relatively low FW (0, 07g).On the other hand, it should be noted that the DW does not seem to be influenced by the different treatments. Indeed, the DW of seedlings oscillates between 0.03g and 0.035g.4.2. In the presence of 100 mM NaCl, the fresh weight of the seedlings is low, so be 0.14 g. On the other hand, it amounts to 0.45 g in seeds receiving GA3 at 5 ?M combined with NaCl and reaches up to 0.58 g in the presence of 10 ?M. The treatment with ABA at 5 ?M and 10 ?M combined with NaCl induced a significant reduction in seed’s FW (0.064 g and 0.061 g). It should be noted that the DW does not seem to be influenced by the different treatments. In fact, the dry weight of seedlings varies between 0.03 g and 0.036 g (fig.X).Figure 4. Effect of hormone/salinity interaction Figure 5. Effect of hormone/salinity interaction on seedlings fresh weight (g) on seedlings dry weight (g)5. Water content of seedlings5.1. In the absence of NaCl, the highest water contents are recorded in control and gibberellic acid treated seeds with a maximum value of 94.03% in seeds receiving GA3 at 10 ?M. The application of the ABA treatments (5 ?M and 10 ?M) is accompanied by a decrease in water content of 55% and 55.68%, respectively.5.2. In the presence of 100 mM NaCl, the water content of the seedlings reached only 77.93%, the latter amounts to 92.22% and 92.96% in the presence of GA3 in saline solution. However, the application of NaCl in combination with ABA greatly reduces the seedlings’ water content. Figure 5. Effect of hormone/salinity interaction on seedlings water content (%)TAB 01. Mean values of parameters measured during seeds germination and okra seedlings growth under combined hormone / salinity treatment.Treatments Germination precocity(%) Final rate of germination(%) Hypocotyls length (cm) Fresh weight of seedlings (g) Dry weight of seedlings (g) Water content (%)Control 60±10 100 8±1,11 0,46±0,04 0,03±0,003 93,265µM GA3 76,66±5,77 S 100 8,1±0,95 0,48±0,03 0,032±0,002 93,3310µM GA3 83,33±5,77 S 100 12±0,98 S 0,58±0,06 S 0,035±0,002 94,035µM ABA 0 S 93,33±5,77 0,28±0,03 S 0,07±0,005S 0,03±0,004 55 S10µM ABA 0 S 76,66±5,77 S 0 S 0,07±0,01S 0,03±0,003 55,68 S100 mM NaCl 0S 96,66±5,77 1,25±0,5 S 0,14±0,04 S 0,03±0,006 77,93 S5µM GA3/NaCl 20 S 100 8,47±0,73 0,45±0,06 0,035±0,005 92,2210µMGA3/NaCl 26,6±5,77 S 100 10,4±1,39 S 0,58±0,06 0,036±0,003 92,965µM ABA/NaCl 0 S 23,33±11,54 S 0 S 0,06±0,01 S 0,03±0,002 52,34 S 10µMABA/NaCl 0 S 6,66±11,54 S 0 S 0,06±0,005S 0,03±0,002 44,92 SS: Significant effect compared to the control.4. DISCUSSIONThe results of this study showed that the application of salt stress at 100 mM significantly reduced the precocity of okra seeds germination without affecting its final rate. This delay of germination caused by this level of salinity is due to a difficulty of seeds’ hydration because of a high osmotic potential and can be explained by the time necessary for the seeds to trigger the mechanisms allowing them to adjust their osmotic pressure (Jaouadi and al., 2010, Diallo and al., 2013), which is already reported by Kaya and al. (2006) and Zemani (2009). Our results show that the NaCl acts negatively on the hypocotylar length and seedlings water content, indicating that NaCl affects not only germination rate but also the seedlings’ growth. Similar results have been highlighted by Bahrani and Pourreza (2012) on wheat and Kumar and al. (2014) on oats. According to Werner and Finkelstein (1995), high salinity can inhibit the lengthening of roots and hypocotyls by slowing water absorption by the plant.The application of GA3 at 5 ?M and 10 ?M in the 100 mM saline solution modified the responses of the seeds in particular the precocity and the final rate of germination which corroborates with the work of Chouhim (2011) on the same species then by Samad and Karmoker (2012) on triticale seeds; similarly, this phytohormone has a positive effect on hypocotylar growth, fresh and dry weights and water content of seedlings. The same results are reported by Turkyilmaz (2012) on soft wheat and by Abdel-Hamid and Mohamed (2014) on barley.Under salt stress, gibberellins induce the lifting of physiological seeds’ dormancy, increase water absorption and stimulate the synthesis and the activation of hydrolytic enzymes mainly ?-amylase, thus releasing the reducing sugars and amino acids that are essential for embryo development (AJMAL KHAN and UNGAR, 1998, VIERIA and al., 2002, AJMAL KHAN and al., 2004). Similarly, gibberellins improve seeds germination by inhibiting ABA activity by either activating the enzymes involved in its catabolism or by blocking the pathway of its biosynthesis (ATIA et al., 2009;XXXXX; MIRANSARI and SMITH, 2014).In parallel, the application of ABA associated with NaCl on okra seeds does not seem to attenuate the depressive effect of salinity on the studied parameters. This observation expresses the inhibitory effect of ABA on germination already confirmed by the work of THAKUR and SHARMA ( 2005) and Atia and al., (2009). However, this ABA acts by limiting water absorption and inhibiting the synthesis of germination-specific enzymes such as ?-amylase by counteracting the stimulatory effect of GA3 on them (Kondhare and al., 2014). The decrease in germination rate observed under ABA treatment can be attributed to the induction of secondary dormancy and the inhibition of seeds germination by limiting the availability of energy and metabolism (Garciarrubio and al., 2003, Leymarie and al., 2008) 5.CONCLUSIONDuring seeds germination stage, the presence of NaCl at 100 mM negatively affects the precocity, but does not appear to influence the final rate of sprouted seeds. However, at the seedlings growth phase, this saline concentration significantly reduces seedlings growth and their development. 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