Effect of priming techniques and seed storage period on soybean (Glycine max l) germination

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1 Available online at Journal of Dynamics in Agricultural Research Vol. 2(5), pp.46-53, December 2015 Article ID: JDAR/15/020 Author(s) retain the copyright of this article Copyright 2015 Original Research Paper Effect of priming techniques and seed storage period on soybean (Glycine max l) germination G.A.K. Sibande*, V.H. Kabambe, M.F.A Maliro, V. Karoshi Department of Crop and Soil Sciences, Lilongwe University of Agriculture and Natural Resources (LUANAR), Bunda College Campus, P.O Box 219, Lilongwe, Malawi. *Corresponding Author. Received 7 September, 2015; Accepted 9 November, 2015 Open Access article distributed under the terms of the Creative Commons Attribution License permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. ABSTRACT Inadequate supply of improved seed varieties is one of the major constraints to soybean production in Malawi. Consequently, most of the seeds being planted by small scale farmers were obtained from open grain markets or derived from previous harvest some of which were already overage, exposed to poor storage conditions and likely suffered certain degree of deterioration. Use of such aged soybeans for planting, predisposes the seeds to imbibitional injury due to their hygroscopic nature thereby resulting in poor germination. Seed quality could be improved by pre-treatment techniques such as seed priming. An experiment was designed to study the effect of seed priming using water, potassium nitrate (KNO3), Moringa oleifera leaf powder (MLP), custard apple (Annona reticulata L) leaf powder (CALP) and a control based on the rate of germination, germination percentage and other seedling vigour traits in three soybean seed lots of Serenade with different storage durations in June, The trial was conducted in the laboratory in a completely randomized design (CRD) with four replicates to determine the effect of seed priming and storage period on germination traits, root and shoot lengths, seedling dry weight and seed vigour index on Glycine max L. Seed priming methods had significant (P<0.001) effect on soybean germination parameters. However, results showed a significant reduction in rate of germination and germination percentage as a result of seed priming. The unprimed (control) soybean seed had the highest germination results. There was significant reduction in germination percentage with increase in storage period. The highest rate of germination and germination percentage were recorded in the freshly harvested seed (control) while the lowest rates were attained in two years old seed. The results therefore showed that soybean seed priming may not always lead to beneficial effect on the rate of germination, germination percentage and other seedling vigour traits but seed storage duration. Key words: Glycine max L., seed priming, seed storage, germination percentage, germination rate. INTRODUCTION Soybean (Glycine max L.) is a leguminous vegetable of the Fabaceae family that grows in tropical, subtropical, and temperate climates (FAO, 1992). It is believed to have been introduced to Africa in the 19 th Century by Chinese traders along the east coast of Africa (Jones, 2003; FAO, 1992) and in Malawi, in the year 1910 (Malawi Government, 2012; Shurtleff and Aoyagi, 2009). Soybean is an important crop with protein content of 40%, 32%

2 Sibande et al. 47 carbohydrate, 20% unsaturated fat, 5% minerals with 3% fiber, and other trace substances. In Malawi, soybean has an important role to play in establishing household food security. This crop serves as an alternative source of cash in those areas where groundnut pops is a problem (ICRISAT, 2014; Kananji et al., 2013). The crop is well adapted to a range of agro-ecologies unlike beans and pigeonpea. Soybeans are more resistant to insect pests thus significantly increasing their yield (ICRISAT, 2014; Malawi Government, 2012). Availability and accessibility of seeds of good quality is the key for the successful agricultural industry in increasing production yield for soybeans (Sathish and Bhaskaran, 2013). Since seeds are considered as one of the important agricultural inputs, good quality seeds act as a catalyst with all other inputs in agriculture. In Malawi, there are a number of new varieties under commercial production that have been developed like Makwacha, Nasoko, Ocepara-4, Soprano, Solitaire, Squire and Tikolore. Despite this improvement and development of soybean varieties, the adoption of small scale farmers towards this recent development remains slow due to poor seed supply (ICRISAT, 2014; Malawi Government, 2012). Consequently, most of the seeds planted by small scale farmers are obtained from open air grain markets or derived from previous harvest (ICRISAT, 2014). Some of these seeds being sourced from the open air grain markets were overage in air-dried conditions which had underwent degree of deterioration (Butler et.al, 2009). Use of such aged seed especially pulses like soybean for planting predisposes the seed to imbibitional injury due to their hygroscopic nature which resulted to poor germination (Tilden and West, 1984). Seed quality could be improved by pre-treating the seeds with water, osmotic solutions and matrix materials (Bradford, 1986). Several priming approaches have been reported to enhance germination percentage, uniformity in emergence, germination in a wide range of temperatures and seedling establishment for many field crops such as rice, maize, soybean, wheat, black gram, lentils among others. Rice seeds showed the highest germination (98.67%), germination energy (88.00%), germination speed (96.83%) when osmo-primed with NaCl (Islam et al., 2012). Osmo-priming seeds with polyethylene glycol (PEG 8000) and KNO3 solutions for 12 hours improved germination and seedling vigour of wheat (Salehzade et al., 2009). In maize, priming methods and duration enhanced germination percentage, germination rate, numbers of seed row, ear length, ear diameter, 1000 seed weight, biological yield and seed yield. This was especially when maize seed was primed with water for a period of 18 hours (Soleimanzadeh, 2013). Again in maize, Dezfuli et al., (2008), found out that when the seeds had been primed for a period of 36 hours there was maximum invigoration in the seedlings as indicated by higher germination rate and radical length. Tavilliet al. (2010) reported that seed priming had significant effect on the seed germination and early growth characteristics of Bromus tomentellus L. Seed that was osmoprimed with poly ethylene glycol (-0.6 MPa for 12 hours) had its final germination increased. When accelerated aged seed of black gram (Vigna mungo L.) were dry dressed (primed) with 3g/kg of fenugreek seed powder with others treated with 4g/kg -1 of custard apple leaf powder and shake for 1 hour, the seeds registered an increase in physiological performance in terms of germination percentage, root length, shoot length, dry matter production and vigour index (Sathish and Bhaskaran, 2013).Sathish and Bhaskaran, (2013) reported that the pronounced increase in the aforementioned physiological parameters in the aged seeds was an indication of the efficacy of the botanicals in alleviating the deleterious effects of accelerated aging in black gram seeds. Oyekale et al., (2012) reported that during storage, when a kilogram of sesame seed was separately treated with 75g of neem leaf powder (NLP) and dry pepper powder (DPP), seed germination was enhanced by 89.53% and 82.35%, respectively compared to synthetic chemical powder (46.47%). Throughout the storage period, it was observed that NLP improved seedling vigour index (339.24) compared to the synthetic chemicals that had registered a vigour index of only Despite all these beneficial effects of priming, few studies had been conducted to understand the efficiency of priming techniques. This study was conducted to determine priming method using water, potassium nitrate, Moringa oleifera leaf powder and custard apple (Annona reticulata L) leaf powder that would lead to improved germination and seedling emergence in three soybean seed lots. The objective of the research was to assess the effect of priming techniques and seed storage durations on germination and emergence. MATERIALS AND METHODS General Study characteristics The experiment was conducted in a laboratory at Lilongwe University of Agriculture and Natural Resources (LUANAR), Bunda Campus, Malawi. The experiment was laid out as two factor factorial experiment in a completely randomized design (CRD) with four replications. The first treatment factor was seed storage age of soybean consisting of fresh soybean seed harvested in 2014; one year old carry over seed harvested in 2013; and two yearold carry over soybean harvested in 2012 designated as S1, S2 and S3, respectively. Soybean seed of Serenade variety was sourced from Chitedze Agricultural Research Station, SeedCo Malawi Limited and Bunda College of Agriculture. The second experimental factor was five levels of priming techniques that is, non-priming (control), hydro priming, salt priming using KNO3 and botanical priming using Moringa leaf powder (MLP) and Custard apple leaf powder (CALP) designated as P1, P2, P3,P4

3 48 J. Dyn. Agric. Res. and P5, respectively. Seed that was used in the trial was first subjected to germination tests to ascertain their level of viability before being primed for the trial. Five hundred seeds each freshly harvested, two and one year old was planted in plastic trays using sand as the germination media. The seeds registered 90.20%, 76.60% and 37.40% for fresh, one year and two year old seeds, respectively. Seed priming method Moringa and custard apple leaves were sourced locally in Karonga district. These leaves were air dried on the mat and grounded to obtain fine powder before mixing them with water. One kilogram of soybean seed from each of the three seed lots was weighed and each batch was soaked in one litre of water; second batch was soaked in water mixed with 6g of KNO3 for 6 h; third batch was soaked in water mixed with 4g of Moringa leaf powder while the fourth batch was soaked in water mixed with 4g of custard apple leaf powder for 2 h. The other lot was not treated and served as control. After priming, seed was washed with tap water and then air-dried for 48 h at room temperature before subjecting them to laboratory germination conditions. Germination Test The germination test was conducted in the laboratory using the sand germination method. The germination test was carried out in quadruplicate using 100 seeds. Moist sand was used as the germinating substrate for the seeds. Clean sand was filled into plastic trays with adequate drainage. The sand was then watered with distilled water until moist but not soaked. Equidistant holes were then drilled in the sand. Twenty five seeds of both fresh and old soybean were placed in each of the plastic trays by placing one seed in each hole. After planting, the holes were covered with sand and the entire trays were gently watered so as not to dislocate the seeds. Germination was counted in 48 hours interval and was continued until no further germination occurred. Calculations and data analysis: Germination percentage; speed of germination; mean germination time; root length; shoot height; seedling dry weight and vigour index data were determined using the following formulae as described below: Speed of germination was calculated following the method of Adebisi and Oyekale, (2005) No. of seeds at first count + No. of seeds at second count Day of first count Day of second count +... No. of seeds at final count Day of final count Germination percentage (%) Germination (%) = Number of seeds germinated x100 Total number of seeds planted Mean Germination Time (MGT) MGT = Σ Dn/Σn (Ellis and Roberts, 1981) Where n is the number of seeds that had germinated on day D and D is the number of days counted from the beginning of germination. Root length (cm) Root length was determined by randomly selecting five normal seedlings in each treatment from all the replications at the end of the germination count and the mean root length in cm was calculated. The root length was measured from the tip of the primary root to the base of the hypocotyls using a ruler. Shoot length (cm) The same seedlings that were previously used to determine root length were also utilized to measure the shoot length in cm. Shoot length was measured from the tip of the primary leaf to the base of the hypocotyls. Seedling dry weight (g) The five seedlings used were placed in the oven at 65±10 o C for 48 h. The mean seedling dry weight was expressed in g. Vigour Index The vigour index was calculated using the formula as described by Abdul-Baki and Anderson, (1973). VI = GP X SDW Where VI = Vigour Index GP = Germination Percentage SDW = Seedling Dry Weight (g per 10 seedlings) Data were subjected to Analysis of Variance (ANOVA) using Genstat 16 th Edition computer package. Treatment means were compared using Tukey's 95% confidence intervals. RESULTS AND DISCUSSION The analysis of variance (Table 1) indicated that the rate of germination, germination percentage, root and shoot lengths were significantly (P 0.001) affected by the seed priming methods, seed storage age and their interaction. The effect of soybean seed priming on mean germination time, seedling dry weight and vigour index (Table 1)

4 Sibande et al. 49 Table 1. Mean squares for germination traits and other traits in soybean (Glycine max L.) under Laboratory conditions. Source DF GI GP (%) MGT RL SL SDW VI Priming Tech ** ** 0.56 ns 5.81 * ** ns ns Seed Storage ** ** 1.03 ns ** ** ns 1162 ** Prim x Seed ns ns 0.75 ns 3.69 * ns ns 27.6 ns Residual 45 Total 59 CV% Note: * and ** indicate significant difference at 5% and 1% probability level respectively while ns indicate none significance. GI Germination index, GP Germination percentage, MGT Mean germination time, RL Root length, SL Shoot length, SDW Seedling dry weight and VI Vigour index. Figure 1. Mean comparisons for germination percentage of soybean under various priming methods in the Laboratory. (Error bars are standard errors of means). were not significant. Rate of germination, germination percentage and emergence percentage In the present study, seed priming methods had significant effect (P 0.001) on the germination percentage, rate of germination and emergence percentage. However, the highest germination percentage, germination rate and emergence percentage were observed in the unprimed seed (control) as compared to those seeds primed with water, KNO3, CALP and MLP as shown in Figure 1 and Table 2. These results are not consistent with the findings of Sathish and Bhaskaran, (2013) in black gram; Amoo-Zad-Khalili et al. (2013) in silybummarianum; Ahmadvand et al. (2012) in

5 50 J. Dyn. Agric. Res. Table 2. Mean comparisons for the rate of germination, germination percent root and shoot lengths of soybean as affected by priming in the Laboratory. Priming Techniques GI GP (%) RL (cm) SL (cm) P4 (MLP) 2.23a 60.00a 4.20a 17.01a P5 (CALP) 2.47a 62.33a b 5.51ab 25.10b P2 (Water) 2.68ab 69.00b 5.86b 24.12b P3 (KNO3) 2.75ab 70.00b 4.48ab 20.29ab P1 (Control) 3.11b 80.00c 4.83ab 19.88ab Grand mean F Pr < <.001 LSD (0.05) CV % Note: Different letters in each column indicate significant differences at 5% probability level. soybean; Khan et al., (2012) in hot paper; Basra et al., (2011) in maize and Phiri, (2010) in sorghum who reported an improved physiological performance in terms of germination percentage, rate of germination and emergence rate due to seed priming. According to Sathish and Bhaskaran, (2013) the improved germination percentage in black gram aged seed might be attributed to the excellent proton radical scavenging property found in the fenugreek seed powder and custard apple leaf powder. Khan et al., (2012) observed in hot pepper that the remarkable improvement in the germination percentage and rate of germination was mainly due to the efficient mobilization and utilization of seed reserves. The reduction in the vigour traits of the primed seed in this study, as compared to the control could be due to nonstressing of the germination conditions. This indicates that the effect of seed priming is more pronounced in unfavorable environmental conditions (Mousavi et al., 2012). In the present study, the least germination rate and germination percentage were recorded in soybean seeds which were primed with CALP and MLP. This could be due to lower nutrients and vitamins which might have been lost during drying of the leaves before treating the seeds (Basra et al., 2011). The reduced germination percentage and germination rate as a result of seed priming could mean that priming had some harmful effects on soybean seeds (Ghassemi-Golezani et al., 2011). Ghassemi-Golezani et al., (2011) reported that seed priming can reduce germination percentage; field emergence percentage and seedling dry weight due to the wet treatment of the seed through priming leading to soaking and imbibition injury. This soaking injury has the potential to reduce soybean germination rate and its germination percentage due to the hygroscopic nature of its seed. Root and shoot lengths Seed priming methods had significant difference (P 0.001) on the seedling root and shoot lengths (Table 2). The highest root length (5.85 cm) was registered in seed primed with water which was not significantly different to those primed with CALP, KNO3 and unprimed seed (control) while the highest shoot length (25.1 cm) was obtained in seed primed with CALP which was statistically similar with seed primed with water, KNO3 and unprimed seed (control). These findings are consistent with those by Sathish and Bhaskaran, (2013) in black gram; Ahmadvand et al. (2012) in soybeans; Zahedi et al., (2012) in sorghum; Shehzad et.al, (2012) in sorghum and Salehzade et al. (2009) in wheat. Salehzade et al. (2012) attributed the increase in shoot and root lengths of sorghum to the increased nuclear replication in shoot and root as a result of seed priming. Rate of germination, germination and emergence percentage as affected by seed age The effect of seed storage duration significantly (P 0.001) affected the germination rate, germination percentage, root and shoot lengths and vigour index (Table 3). Freshly harvested seeds had the fastest germination rate (3.28)

6 Sibande et al. 51 Table 3. Mean comparisons for germination rate, germination percentage, mean germination time, root length, shoot length and vigour index as affected by seed storage duration. Seed storage period GI GP (%) RL (cm) SL (cm) VI S3 (2 Yrs old) 1.71a 42.60a 3.79a 17.20a 6.37a S2 (1 Yr old) 2.95b 75.20b 4.71a 20.44a 18.94b S1 (Fresh seed) 3.28b 87.00c 6.43b 26.20b 20.12b Grand mean F Pr <.001 <.001 <.001 <.001 <.001 LSD (0.05) CV % Note: Different letters in each column indicate significant differences at 5% probability level. Figure 2. Mean comparisons for Root Length and Shoot Height of soybean (Glycine Max L.) as affected by seed storage durations under Laboratory conditions (Error bars are standard errors of means). which was similar to one year old but different from two years old seed. Similarly freshly harvested seed registered the highest germination percentage (87.00%) followed by one year old seed (75.20%) while the least germination percentage (42.60%) was registered in theseed that was stored for two years. The freshly harvested seed achieved longer root (6.430 cm) and shoot (26.20 cm) lengths than the old seed (Figure 2). The decline in the rate of germination and germination percentage in the one and two years old soybean seeds could be due to the ageing effect leading to the depletion of the food reserves in the seeds. The other reason for the decline in germination could be that seed storage duration caused damage to the other seed lots (one and two years old seed) hence negatively affecting their germination rate. One such damage to the

7 52 J. Dyn. Agric. Res. one and two years old seed lots could be loss in membrane competence. Tilebeni and Golpayegani (2011) demonstrated in rice that seed leakage increases with increased amount of aging and seed soaking time. The five days accelerated aged rice seed had more leakage than the control (untreated seed) an indication of more damaged membrane in the aged seed than the fresh seed. This seed resulted into significant reduction in germination percentage than the untreated seed. Balešević-Tubić et al., (2010) reported that soybean seed is more sensitive to damage in storage condition due to their oil content in comparison to other crops like sunflower and this negatively affects its germination rate when planted. Tilden and West, (1984) also attributed the loss of germinability in soybean seed to the cell membrane permeability or rupture in aged seeds. Conclusions and Recommendations Soybean seed priming has shown to have positive results on seed germination. In the current study, there were reductions in germination percentage, emergence percentage and germination rate as compared to the control. However, seed priming methods resulted in the significant increase in the seedling roots and shoot heights. The results also indicated a significant reduction in germination percentage in response to seed storage duration. In this study, therefore, results show that soybean seed priming may not always lead to beneficial effect on rate of germination and germination percentage. From the results of the current study, it has been revealed that the loss in seed germinability could be due to the wet treatment of the seed during priming which might have resulted into imbibition injury. It is therefore recommended that future studies focus on controlled uptake of water through adoption of double or triple filter papers during priming. The present study has shown that custard apple leaf powder and moringa leaf powder could not invigorate soybean seed as fresh substances of these botanicals had done with other crops like maize, black gram and others. The poor performance of CALP and MLP in this study has been attributed to lower nutrients and vitamins in these botanicals which might have been lost during drying of the leaves. It is therefore recommended that future studies should try to use fresh extracts from these botanicals. The current study has also revealed that maximum plant population density and grain yield were obtained in seed that had been in storage for less than six months. It is therefore recommended that to achieve the recommended plant population and yield, such soybean seed should be encouraged to cultivate. 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