Available online at Universal Research Publications. All rights reserved. Original Article

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1 Available online at International Journal of Agricultural and Food Science Universal Research Publications. All rights reserved Original Article ISSN EFFECT OF THE HYDROGEN PEROXIDE TREATMENTS ON GERMINATION OF OIL PALM (Elaeis guineensis Jacq.) SEEDS Beugré Manéhonon Martine 1, *Kouakou Tanoh Hilaire 1, Koné Mongomaké 1, Kouamé Kouamé Roger 2, Konan kouakou Eugène 2 and Kouadio Yatty Justin 1 1 Université d Abobo-Adjamé, UFR des Sciences de la Nature, Laboratoire de Biologie et Amélioration des Productions Végétales, 02 BP 801 Abidjan 02, Côte d Ivoire ; 2 Programme Palmier, Laboratoire de culture in vitro, Centre National de Recherche Agronomique (CNRA) Station la Mé, 13 BP 989 Abidjan 13, La Mé, Côte d Ivoire. *Corresponding author: Tel: (+225) ; tanohilaire@yahoo.fr Abstract Received 14 September 2011; accepted 29 September 2011 Oil palm seed production has an important role in human nutrition and industry. Success in oil palm plant cultivation is related to seed production with high viability and rapid germination, because these seeds rapidly loose their viability by fats oxidation. Thus, in this work we studied the influence of exogenous application of hydrogen peroxide on oil palm seeds germination. The relationship between hydrogen peroxide treatment efficiency, soaking length, heating time and germination ability of oil palm seeds were also established. The results showed that the germination percentage of seeds was highest with genotype C70XX (41.25%) than that of genotype C24XX (34.13%). Indeed, C70XX seeds previously heated during 60 and 80 days and then soaked in hydrogen peroxide at 5 and 10% for 4 days give the greater germination percentage compared to the other treatments. However, it is worth noting that hydrogen peroxide treatment of oil palm seeds in our experimental conditions does not improve the percentage of germination compared to controls. Thus, we suggest the use of a wider range of concentrations of hydrogen peroxide in the seeds treatment Universal Research Publications. All rights reserved Key words: Elaeis guineensis, heating, hydrogen peroxide, oil palm, Oxidative stress, seed germination. 1. INTRODUCTION Oil palm seeds pose difficulties in terms of commercial germination due to a strong dormancy. Bewley [1] indicated that this dormancy could last several years, depending on the environmental conditions before and after harvesting. According to Ferwerda [2], it takes 6 months after planting to reach 30% germination and 9 months to reach 50%. Germination and dormancy are an important mechanism in seed physiology and is a complex trait, influenced by a myriad of genetic and environmental factors that interact to maximize the long term chance of survival of the seed [3,4]. Seed germination is the most crucial and sensitive stage of a plants life cycle. Oil seeds have a special place in food security and industry throughout the world. One of the most important factors for oil seed cultivation is seed product with high vigor and rapid seed germination, because these factors are critical to crop production under different conditions. In oil palm, seed germination is a sensitive stage; particularly oil palm seeds are very sensitive to storage and store conditions such as temperature [5]. Oil palms are dependent on seed propagation. In natural conditions, oil palm seed germination is very slow (1 to 3 years) with a weak percentage. These seeds are thus ranged among recalcitrant seeds, according to Braun [6] and Chin et al. [7]. This slow and weak capacity of oil palm seed germination represents a major constraint for the establishment of plantings [8] and contributes to the high cost of producing established plants. The slow germination can be attributed to the extremely thick, hard mesocarp and endocarp of oil palm fruits and the primitive structure of the embryo. Rees [8] detailed the dry 58

2 heat process, a commercial method for large-scale plant production, for interrupting seed dormancy in Deli dura palms. This method consists of subjecting the seed to temperatures of between 38ºC and 40ºC for 40 to 60 days, although the authors originally indicated periods of 70 to 80 days [5, 9, 10]. The possibility of using a faster method in place of dry heat would reduce the high cost of this treatment, as well as the time necessary to stimulate germination. Attempts to achieve improvements in the oil palm seed germination process through the use of growth-regulating substances have not yielded satisfactory results [1, 11-14]. However, a better understanding of germination metabolism in general and the workings of the growth-regulating substances has allowed for the consideration of using chemical treatments to stimulate the germination of dormant seeds [4, 15, 16]. It is well known that many phenolic compounds in plant tissues are potential antioxidants: flavonoids, tannins and lignin precursors may work as reactive oxygen species (ROS) scavenging compounds. Under different conditions, reactive oxygen species (ROS), such as superoxide anion radicals, hydrogen peroxide, and hydroxyl radicals, are generated [17]. ROS in seed physiology are usually considered as toxic molecules [18]. However, exogenously applied H 2 O 2 ameliorates seed germination in many plants [19,20]. This has been explained by the fact that the scavenging activity for H 2 O 2 is sufficiently high, resulting in the production of O 2 for mitochondrial respiration. In contrast, H 2 O 2 promoted seed germination in a dose dependent manner as did respiratory inhibitors, indicating that H 2 O 2 itself possibly promotes seed germination rather than O 2. Jann and Amen [21] show that pre-treatment of seeds with oxidants such as H 2 O 2 leads to breaking seed dormancy. Some authors have shown that the production of ROS during seed germination may be a beneficial biological reaction, linked with high germination capacity and seedling development [22-24]. The present study was, therefore, designed to characterize the effects of hydrogen peroxide treatments on germination of oil palm seeds. The aims of this work were to study the influence of exogenous application of hydrogen peroxide on germination of recently-harvested oil palm seeds, and to understand in more detail the relationship between hydrogen peroxide efficiency, soaking length, heating time and germination ability of maturing oil palm seeds. 2. MATERIAL AND METHODS 2.1. Plant material The seeds of two lineages of Elaeis guineensis Jacq. Variety Dura provided by the National Center for Agricultural Research (NCAR) of Côte d Ivoire (West Africa) were used for this study. The linear LM provides the seeds category C70XX and the linear LM provides the seeds category C24XX. LM is the product of a cross of female parent Dura (DA DA3D x 115D) x (115D x DA3D DA) with an unknown male parent and LM is from a cross of female parent Dura (LM404D) x (DA 115D) with a unknown male parent. These oil palm linears are the products of a genetic selection program Seed storage Six month old seeds are harvested, fermented in water for one week, and pulped. Seeds were then washed, dried, and disinfected with a fungicide (Benlate 0.5 to1.0 g.l Dithane M45) in 10 L of water to prevent the formation of mould. The seeds were kept in a room at 21 ± 1 C and a 60% relative humidity during 6 months. After storage, two large groups of seeds were established. The first group of seeds was constituted to be soaked in hydrogen peroxide at different concentrations and soak time prior to heat. Second group of seeds was formed to be soaked in tap water before heating. Controls were realized, where the hydrogen peroxide was replaced by the tap water before or after heating. For each genotype and treatment, three independent tests of 500 to 700 seeds were used Water content of seeds The water content of seeds was determined before germination test. Twenty seeds were weighed in order to determine their fresh weight and then, dried in an oven at 105 C for 24 h to obtain a constant dry weight. The water content of seeds was calculated according to the method described by Beugré et al. [5] Seed germination treatments Germination experiments were carried out in room at 25 C. For the first group, seeds were soaked in hydrogen peroxide (H 2 O 2, 5 or 10%), during 2 and 4 days and were then transferred to tap water for respectively 5 and 3 days. The reagent solution was changed once a day for freshly prepared solutions to insure exogenous exposure of the seeds to a uniform level of hydrogen peroxide, which is a prerequisite, to study detailed effect of oxidative stress imposed by H 2 O 2 on germination of oil palm seed. In second seeds group, seeds were soaked instead in water for 7 days with daily renewal water. After soaking, all seeds were treated with a fungicide for 10 to 15 min, to prevent the formation of mould as previously, described, and then spread on jute bags for their wiping. Each group of seeds was divided into 3 lots. Seeds were placed in transparent polyethylene bags, tightly closed and then, transferred for heating in a room on wire mesh shelves which allow a full flow of hot air. The heating temperature is maintained at 40 C. The heating time was respectively of 40, 60 and 80 days. After heating, seed of the first group were soaked in water for 5 days (with soaking water renewed daily) and cleaned. The second group of seeds was soaked separately into some hydrogen peroxide (H 2 O 2, 5 or 10%), at room temperature during 2 and 4 days and were then transferred to tap water for respectively 5 and 3 days. The soaking hydrogen peroxide and water were renewed daily. Seeds were treated with a fungicide for 10 to 15 min as above, and spread on jute bags for their wiping. After each soaking treatment, seeds were wiped thoroughly again and 59

3 Figurei1. Effect of heating length on seed oil palm germination A: Seeds soaked in H 2 O 2 before heating; B : Seeds soaked in H 2 O 2 after heating. Values are the means of three replications. Values with the same letter are not significantly different at the level Bars represent the standards errors. placed in polyethylene bags and germination was monitored for 8 weeks. The seeds were accepted as germinated when the radicle reached 5 mm [25]. The germination percentages were recorded for each week up to the 8th week Statistical analysis The experiment was arranged in a completely randomized design. Each treatment was analyzed in three replications. When analysis of variance (ANOVA) showed significant treatment effects, Duncan s multiple range test was applied to compare the means at P<0.05. Figure 2. Effect of hydrogen peroxide concentration and soaking length on germination of oil palm seeds. A: Seeds soaked in H 2 O 2 before heating ; B : Seeds soaked in H 2 O 2 after heating ; O5 and O7: Seeds soaked in water during 5 and 7 days; 5.2 : Seeds soaked in H 2 O 2 5% for 2 days ; 5.4 : Seeds soaked in H 2 O 2 5% for 4 days ;10.2 : Seeds soaked in H 2 O 2 10% for 2 days ; 10.4 : Seeds soaked in H 2 O 2 10% for 4 days. Values are the means of three replications. Values with the same letter are not significantly different at the level Bars represent the standards errors. 3. RESULTS 3.1. Water content of seeds The water content in the oil palm seeds of both genotypes is recorded in Table 1. Water content shows a significant difference between the two genotypes of oil palm seeds. Thus, water content of genotype C70XX seeds (9.78%) was highest than seeds of genotype C24XX (8.93%) ones Effect of hydrogen peroxide concentration and soaking times on germination percentage Seeds treated before heating The germination of seeds observed after 8 weeks, showed a varied percentage, ranged from 3.06 to 22.25%, 60

4 Table 2. Effect of H 2 O 2 treatment before heating on seed germination at the various levels of both oil palm genotypes Percentage of germination Heating Day 2 Day 4 time Treatment (days) C24XX C70XX C24XX C70XX Control ± 3.35 f ± 2.40 d ± 3.20 f ± 2.33 d 40 H 2 O 2 5% ± 1.27 j ± 1.46 i ± 0.92 k ± 1.38 j H 2 O 2 10% ± 1.45 j ± 1.49 k ± 0.76 k ± 1.01 k Control ± 4.27 c ± 3.46 a ± 3.55 c ± 3.22 a 60 H 2 O 2 5% ± 1.87 i ± 1.26 g ± 1.35 i ± 2.05 i H 2 O 2 10% ± 1.63 j ± 1.74 i ± 1.50 i ± 1.80 i Control ± 2.08 e ± 2.93 b ± 2.23 e ± 2.77 b 80 H 2 O 2 5% ± 1.48 i ± 1.88 g ± 1.21 i ± 1.64 h H 2 O 2 10% ± 1.42 h ± 1.66 g ± 1.60 h ± 1.50 i F = 5.1 P = Values are the means of three replications. Values with the same letter are not significantly different at the level 0.05 (±SD). Table 2. Effect of H 2 O 2 treatment before heating on seed germination at the various levels of both oil palm genotypes Percentage of germination Heating Treatment Day 2 Day 4 time (days) C24XX C70XX C24XX C70XX Control ± 3.35 f ± 2.40 d ± 3.20 f ± 2.33 d 40 H 2 O 2 5% ± 1.27 j ± 1.46 i ± 0.92 k ± 1.38 j H 2 O 2 10% ± 1.45 j ± 1.49 k ± 0.76 k ± 1.01 k Control ± 4.27 c ± 3.46 a ± 3.55 c ± 3.22 a 60 H 2 O 2 5% ± 1.87 i ± 1.26 g ± 1.35 i ± 2.05 i H 2 O 2 10% ± 1.63 j ± 1.74 i ± 1.50 i ± 1.80 i Control ± 2.08 e ± 2.93 b ± 2.23 e ± 2.77 b 80 H 2 O 2 5% ± 1.48 i ± 1.88 g ± 1.21 i ± 1.64 h H 2 O 2 10% ± 1.42 h ± 1.66 g ± 1.60 h ± 1.50 i F = 5.1 P = Values are the means of three replications. Values with the same letter are not significantly different at the level 0.05 (±SD). depending to seeds genotype, hydrogen peroxide the other hand, the lowest germination percentage (09.16%) concentration, soaking and heating times. Significantly was observed with the same genotype seeds soaked in H 2 O 2 difference was found in germination percentage of the both 10 % for 2 days at 40 days heating. genotype seeds (Table 2). Highest germination percentage 3.3. Genotype effect (22.25%) was obtained with C70XX seeds soaked in 5% of Significant difference was found between H 2 O 2 during 2 days at 60 days heating, while the lowest germination percentages of the both oil palm genotype seeds germination was observed with the C24XX seeds soaked in treated with hydrogen peroxide before and after heating. 5% of H 2 O 2 during 4 days at 40 days heating. Thus, C70XX show higher germination percentage compared Seeds treated after heating to that of C24XX whatever the method of heat treatment The germination percentage of seeds was ranged (Table 4). from 9.16 to %. Significant difference was found 3.4. Effect of heating length on germination between the germination of both genotype seeds The germination percentage evolves similarly for underitreatments (Table 3). Highest germination percentage both genotypes, whatever the type of heat treatment. No (41.75 %) was obtained with seeds of genotype C70XX significant difference was observed. However, germination soaked in 10 % of H 2 O 2 during 4 days at 60 days heating. On percentage increased with heating length (Figure 1). 61

5 Table 3. Effect of H 2 O 2 treatment after heating on seed germination at the various levels of both oil palm genotypes Percentage of germination Length Day 2 Day 4 heating Treatment (days) C24XX C70XX C24XX C70XX Control ± 3.35 f ± 2.40 d ± 3.12 f ± 2.22 d 40 H 2 O 2 5% ± 1.51 i ± 1.90 i ± 2.47 g ± 1.76 g H 2 O 2 10% ± 1.80 i ± 1.46 i ± 2.43 f ± 2.78 f Control ± 4.27 c ± 3.46 a ± 4.27 c ± 3.46 a 60 H 2 O 2 5% ± 2.61 i ± 2.68 h ± 2.44 f ± 2.72 f H 2 O 2 10% 14.8±3.11 h 11,83 ± 1,72 i ± 2.48 f ± 4.05 d Control 39.06±2.08 e ± 2.93 b ± 2.23 e ± 2.12 b 80 H 2 O 2 5% ± 2.42 g ± 2.37 f ± 2.48 f ± 2.98 d H 2 O 2 10% 17.86±1.92 h ± 2.24 g ± 2.50 f ± 2.60 e F = 2.71 P = Values are the means of three replications. Values with the same letter are not significantly different at the level 0.05 (±SD). Table 4. Genotype effect on seed germination under H 2 O 2 treatments Percentage of germination Before heating After heating C24XX ± 2.00 a ± 1.68 c C70XX ± 2.95 b ± 2.56 d F P Values are the means of three replications. Values with the same letter are not significantly different at the level 0.05 (±SD) Effect of hydrogen peroxide concentration and length of soaking seeds of oil palm on germination No significant difference was found between germination percentages of the both genotypes palm oil seeds soaked in hydrogen peroxide before heating. However, there is a very highly significant difference when comparing control and treated (Figure 2A). With seeds treated by hydrogen peroxide after the heating, significant difference was found between germination percentages of the both genotypes palm oil seeds. Similarly, germination percentage of seeds soaked in water (control) was significantly higher than that of seeds soaked in hydrogen peroxide (Figure 2B). 4. DISCUSSION Water contents in oil palm seeds show a significant difference between the two genotypes. The seeds of C70XX were significantly more germination ability than those of C24XX whatever the method of heat treatment. These results indicated that the two genotypes of oil palm have a physiological difference in the germination characteristics during seed maturation. The influence of oxidative stress induced by hydrogen peroxide on germination in oil palm seed has not yet been studied nevertheless it has been documented [26] recently that oxidative stress produce at cellar level by low 62

6 dose irradiation of seeds can stimulates seed germination and other stages of plant development. For example, oxidative stress induced by ionizing radiation and hydrogen peroxide can stimulates growth of sprouts and roots in barley, wheat, pea, maize and melon [26]. In present study, it was observed significantly higher germination percentage when seeds are beforehand warmed before their treatment in the hydrogen peroxide. This is not the case when seeds are treated before their heating. The hydrogen peroxide seems to inhibit the seeds before the heating. The germination percentage, the various concentrations or soaking length of seeds treatment, is always lower than untreated seeds (control). Our results are opposite to those of Yang et al. [4] and of Kouakou et al. [14] who find similar germination percentages between treated and control. In the same way, these results are contrary to those of several others authors who reported a stimulation of germination when seed are treated with hydrogen peroxide [27-29]. Oxidant treatments such as hydrogen peroxide appear to improve germination, providing oxygen to the embryo. In fact hydrogen peroxide acts primarily by oxidizing phenolic compounds suddenly can no longer bind oxygen after treatment [14, 30]. Similar results were obtained with other oxidants such as sodium hypochlorite and sulfuric acid [4, 31, 32]. During germination, there is the massive use of oil reserves. Some of these will be used directly to respiratory oxidation and most of it is converted into carbohydrates [33]. This action appears to be stimulated when the seeds are heated prior to treatment with hydrogen peroxide. The embryo which needs oxygen to germinate, the contribution of the latter would be beneficial for the seed after heating. The results show a significant difference between the two categories of seeds, which is consistent with the work of [34, 35] that shows the capacity of germination that differ from one species to another. The effect of heat to allow an acceleration of seed germination has been demonstrated by authors [36, 37], which showed that it is necessary to break dormancy of seeds. It is known that stress factors such as heat [38] cause increases of oxidants, reactive oxygen species (ROS) as hydrogen peroxide in plant tissues. In other words, environmental stresses result in oxidative stress in plants. However, the fact that hydrogen peroxide markedly alleviated the inhibitions of germination by heating stresses is surprising. Because ROS is generally considered harmful to plant cells [39] due to the fact that they can be extremely reactive and oxidize biological molecules such as DNA, proteins, and lipids Whereas, ROS both appear as intermediates of cellular metabolism under stress less, i.e. normal conditions and are generated in the presence of biotic and abiotic stresses. For example, it has been confirmed that production of ROS by germinating seeds is an active developmentally controlled physiological process [24]. When ROS reach dangerous levels, they can be eliminated by antioxidant enzymes such as catalases and peroxidases [39]. As seen, the seeds have an antioxidant defense system against oxidative stress as a result of environmental stresses. The pretreating of seeds with hydrogen peroxide and heating duration may have mediated "the vigilance" in the seed to an environmental stress, and so, the seed, by moving the mechanisms of defense, might have succeeded germination. Metabolic reactions that occur with hydrogen peroxide and heat have an inhibitory effect. On the other hand, those that occur after heating do not appear to inhibit germination although the germination percentage is lower than the percentage of non treated seeds [40]. Hydrogen peroxide action appears to be inhibitory, probably the soaking time or longer can be a handicap. The action of hydrogen peroxide after heating is shorter (5 days) whereas it is 7 days prior to heating. The embryo needs oxygen to germinate, so the supply of oxygen after heating is required, but not before. 5. CONCLUSION The results of the present study indicate that the seeds of C70XX genotype previously heated for 60 and 80 days and then soaked in hydrogen peroxide at 5 and 10% during 4 days give a better germination percentage compared to the other treatments. Treatment with hydrogen peroxide does not improve significantly the percentage of germination compared to controls. When considering the effect of hydrogen peroxide, concentration of 5% must be accepted. Besides, its action seems more effective after the heating of seeds. However, other concentrations of hydrogen peroxide can be tested with different soaking times. In addition, the evaluation of effects of different chemical compounds which could be involved in the removal of seed dormancy on the seeds germination seems to be necessary. Thus, these compounds could be used alone or in combination under different soaking periods of oil palm seeds. 6. ACKNOWLEDGEMENTS We gratefully acknowledge the financial support from the Winrock International and National Center for Agronomic Research of Côte d Ivoire for supplying oil palm seeds. 7. REFERENCES [1] J.D. Bewley, Seed germination and dormancy, Plant Cell 9 (1997) [2]iJ.D. Ferwerda, Germination of Oil Palm Seeds, Trop. Agric. Trin. 33(1956) [3] M. Koornnef, L. Bentsink, H. Hilhorst, Seed dormancy and germination, Plant Biol. 5 (2002) [4]iQ.H. 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