Effect of Seed Priming Methods on Seed Quality of Okra ( Abelmoschus esculentus (L.) Moench) Genotypes

Seed priming is an efective way of promoting seed germination and vigor of okra by alleviating seed dormancy in fresh or stored okra seeds. An experiment was carried out to evaluate the efect of seed priming treatment on seed physiological quality of okra genotypes. Tis experiment was conducted in a laboratory at Haramaya University in a completely randomized design with 4 replications. It comprised of 5 seed priming treatments (untreated seeds, tape water, 200ppm GA 3 , 0.5% KH 2 PO 4 , and 50% cow urine) and 5 okra genotypes (Clemson, Arka Anamika, SOH701, 240207, and 240586). Te results showed that the main and interaction efects of seed priming treatment and genotypes signifcantly afected physiological seed quality attributes and hard seed percentage. GA 3 -treated genotype Clemson showed the highest germination (78.28%) and germination speed (25.29). Similarly, GA 3 -treated genotypes SHO701 and 240586 had the highest seed vigor index I (13.161) and vigor index II (34.14), respectively. Tere were no hard seeds in genotype Clemson treated with GA 3 and cow urine, genotype SOH701 treated with GA 3, besides genotype 240207 treated with KH 2 PO 4 and tap water. All seed priming treatments had a signifcant positive efect on physiological quality and seed overcame seed hardness in all 5 okra genotypes compared to controls. Terefore, in this study concluded that GA 3 seed priming treatment improved physiological seed quality, and alleviated seeds hardness in okra genotypes. As an alternative to GA 3 seed priming treatments, Ethiopian farmers can also use tape water, cow urine, and KH 2 PO 4 seed priming treatments.


Introduction
Okra (Abelmoschus esculentus (L.) Moench) is one of the most commonly used species in the Malvaceae family [1]. Okra is native to Ethiopia and Sudan in Northeastern Africa [2]. It is a self-pollinated, seed-propagated annual herbaceous plant [3,4]. Okra is grown in tropical, subtropical, and warm temperate regions of the world [5,6]. It is highly nutritious [7] and has economic and industrial value [8]. Te nutritional values of okra include carbohydrates, protein, fber, thiamine, ribofavin, and ascorbic acid [9]. Immature green fruits and fresh leaves of okra are used in salads, soups, and stews [10], and dried seeds are used to make vegetable curd or roasted and ground and used as an additive or substitute for cofee [11]. Okra is mainly grown in the western part of Ethiopia, but there is no complete data on okra production and productivity within the country [12].
Te hard seed coat of okra inhibits water uptake and is an important physiological constraint for uniform stand establishment and performance [13,14]. Genotypes, pod harvest time, and seed priming have been reported to infuence on okra seed germination and vigor [15,16]. Although there is no information on genetic variation in Ethiopia okra germplasm for seed hardness, the presence of genetic diversity in other traits of the crop has been reported [12,17]. Hence, it is possible to predict the presence of variations in okra germplasm in the Ethiopian okra germplasm for many agromorphological traits, including seed hardness. However, there is no information on the seed quality of okra germplasm in Ethiopia.
Seed priming is a simple, inexpensive, and efective method to synchronized germination and early seedling growth by increasing the speed of germination, seed vigor and overcoming dormancy, and allowing better establishment and high-quality yields of plants in stressful and stressfree environmental conditions [18][19][20]. However, there is no recommended seed priming method to overcome the seed hardness of the okra genotypes and no information on seed quality diferences between okra germplasm from Ethiopia and commercial varieties from other countries. Terefore, this study was initiated to assess the impact of seed priming on seed physiological quality of okra genotypes.

Experimental Materials.
A total of fve okra genotypes; two genotypes were collected from Ethiopia (240586 and 240207), one variety was from the USA (Clemson), and the other two varieties were introduced from India (SOH701 and Arka Anamika) were used for this experiment. Seeds of these fve genotypes were harvested in November 2019 at Tony Farm, a research site of Haramaya University in Dire Dawa, Ethiopia (9°36′N, 41°52′E and longitude 60°N, 41.867°E) [21]. Te harvested seeds from each genotype were kept in canvas bags and kept at room temperature at the horticultural laboratory at Haramaya University, and various seed primings, including tap water, 50% cow urine, 0.5% KH 2 PO 4 , and 200 ppm GA 3 , were used for this experiment.

Treatments and Experimental
Design. Te experiment consisted of fve seed priming methods (untreated seeds, tape water, 200 ppm GA 3 , 0.5% KH 2 PO 4 , and 50% cow urine) and fve okra genotypes, two of which were collected from Ethiopia (240586 and 240207), one from the United States (Clemson), and the other two (SOH701 and Arka Anamika) were brought over from India. Terefore, there were 25 treatments in the factorial arrangement. Except for the control treatment, all fve okra genotypes were treated with each seed priming method for 24 hours and shade dried for 6 hours before sowing. Te experiment was conducted in January 2020 in a laboratory at Haramaya University, Ethiopia, in a completely randomized design (CRD) with 4 replicates. For each replicate, hundred seeds were used per treatment.

Seed Quality Parameters
(1) Hundred Seed Weight (g). A hundred seeds of each genotype were randomly selected from four replications, weighed, adjusted to have 12% moisture content, and recorded as the hundred seed weight.
(2) Seed Moisture Content (%). Five grams of seeds from each genotype with four replications were taken from the sample seeds, and the seeds were ground, weighed, poured into small covered containers, and kept in an oven maintained at a temperature of 103°C for 17 hours. Te moisture content of the seeds was then determined by the following formula (1): Note: M 1 , weight in grams of the container and its cover, M 2 , weight in grams of the container, its cover + ground material before drying, and M 3 , weight in grams of the container, its cover + ground material after drying.
(3) Normal Seeds (%). Hundred randomly taken seeds of each genotype with four replications were classifed as normal and abnormal seeds. Seeds were considered normal if they were well flled (not wrinkled), were of average seed size in the sample, and had not developed any symptoms of rot. Seeds wrinkled, cracked, broken, partially or completely missing seed coat, very small or large seeds, extensive rot, and symptoms of other diseases were considered as abnormal seeds. Te normal seeds were counted and calculated the seed percentage as follows: Normal seeds(%) � number of normal seeds total number of seeds x100%.
(2) (4) Seed Germination (%). A total of four hundred seeds (four replications of 100 seeds) were used in the germination test for each treatment. In sterile petri dishes, hundred seeds from each treatment were placed on flter paper that had been moistened with distilled water. Finally, the petri dishes were placed in a growth chamber at 25°C for 21 days, and the seeds were then kept moist with distilled water for 21 days in the laboratory. Germinated seedlings were counted starting with the frst seedling that germinated and ending 21 days after placing the seeds in the petri dish. At the end of this test, the seeds were classifed as germinated, fresh ungerminated, hard, or dead seeds. Germinated seeds were used to calculate the seed germination percentage [22].

Germination(%) �
Number of germinated seeds Total number of seeds sown x100.
(3) (5) (7) Speed of Germination. Te number of germinated seeds was counted every two days until the end of the germination test. An index was calculated by dividing the number of seeds that germinated each day by the number of days that were in the petri dish [25].

Speed of Germination
Note: N1 (number of germinated seeds at frst count), N2 (Number of germinated seeds at the second count), Nf (number of germinated seeds at the fnal count), C1 (days to frst count), C2 (days to the second count), and Cf (days to the fnal count) (8) Seedling Length (cm). Normal seedling length was measured (roots and shoots separately) according to International Seed Testing Association (ISTA) [22] at the end of the germination experiment.
Seed vigor index I is calculated as follows: Seed vigor index I � Germination percentage(%)x seedl ing length(cm).
(9) Seedling Dry Weight (mg). At the end of the germination experiment, the normal seedlings without cotyledons were dried in an oven maintained at a temperature of 103°C for 48 hours, after which the seedlings were transferred from the oven to an activated silica gel desiccator and left for 30 minutes. Seedling dry weight was measured in grams using a sensitive balance, according to ISTA [22]. Seed Vigor Index II is calculated as Seed vigor index II � Germination percentage x seedl ing dr y weight(mg).

Data Analysis.
Data were subjected to analysis of variance (ANOVA) using GenStat software (16 th edition), following the statistical procedures outlined in Gomez and Gomez [26]. Prior to analysis, the percentage data was transformed using the arcsine transformation technique. Te mean separation was performed at the 1% or 5% level of the least signifcant diference (LSD). Te correlations between seed quality attributes were estimated using STA-TISTICA 7 (U.S.A., 2002) statistical software.

Efect of Genotype on Seed Moisture, Normal Seed Number, and Seed Weight.
Te physical seed quality of the fve okra genotypes was tested before the application of the priming treatments. Analysis of variance revealed signifcant diferences in seed moisture content, number of normal seeds, and hundred seed weight among the fve okra genotypes (p < 0.01) ( Table 1). Te highest seed moisture content (11.97%) was measured with the genotype Clemson, with no signifcant diference from the moisture content of genotype Arka Anamika seeds. Genotype Clemson also had the lowest number of normal seeds (85.75%) and 100 seed weights (3.05 g). In contrast, genotype 240586 had signifcantly lower seed moisture content (8.18%) and a signifcantly higher weight of 100 seeds (5.55 g), statically equal to genotype 240207. Genotype Arka Anamika had a signifcantly higher number of normal seeds (93.25%) with no signifcant diference from the number of normal seeds of genotype 240586 (Table 2). Te results showed that the genotypes had inherent diferences with respect to seed physical quality parameters. Similar results were reported by Balla [27].

Efect of Seed Priming on Seed Physiological Quality of Okra Genotypes.
All physiological seed quality parameters of okra genotypes, including germination percentage, speed of germination, seed viability, seedling length, seedling dry weight, seed vigor index I and II, and number of hard seeds were signifcantly infuenced by genotype diferences and seed priming. Except for seed viability, the interaction of genotype and seed priming also afected all of the abovementioned physiological seed quality parameters and the number of hard seeds (Table 3).

Efects of Genotype and Seed Priming on Seed Viability.
Te main efects of genotype and diferent seed priming treatments signifcantly (p < 0.01) afected the seed viability (Table 3). In contrast, the interaction efects of genotype and diferent seed priming treatments were not signifcantly (p < 0.05) afected the seed viability (Table 3). Genotype Arka Anamika showed signifcantly higher (72.12%) seed viability with no signifcant diference from genotypes Clemson and SOH701. In contrast, genotype 240586 had signifcantly lower (67.48%) seed viability without signifcant diference to genotype 240207 (Table 4). Te observed variability in results may be due to the genetic factors of the okra genotypes. In general, the results showed that the seed viability of the Ethiopian genotypes were lower than that of the introduced genotypes. Tis result is in agreement with other researchers, marking notable signifcantly varied in seed viability percentage across ten okra genotypes [28].
Okra seeds primed with GA 3 resulted in signifcantly higher seed viability (74.59%) than other treatments. In contrast, the seed viability of untreated seeds was signifcantly lower (66.87%) and not signifcantly diferent from KH 2 PO 4 and cow urine-treated seeds (Table 4). Te results showed that diferent seed priming treatments afected the viability of okra seeds in diferent ranges. Te highest seed viability with GA 3 treatment was due to the fact that GA 3 promotes early DNA replication, recovered seed damage and deterioration, increases RNA and protein synthesis, and increases ATP availability in seeds [29]. Te present result is consistent with the studied by [20,30].

Efects of Genotype and Seed Priming on Germination
Percentage. Te main efects of genotype and diferent seed priming treatments signifcantly (p < 0.01) afected the germination percentage (Table 3). Similarly, the two factors interact signifcantly to infuence germination percentage (p < 0.05) ( Table 3). Genotype Clemson seeds treated with GA 3 showed a signifcant increase in the germination percentage (78.28%) with no signifcant diference from the germination percentage of genotypes SOH701 and Arka Anamika seeds treated with GA 3, and genotypes Arka Anamika and Clemson treated with tap water. In contrast, the germination percentage of untreated seeds of genotype 240586 was signifcantly lower (59.16%) and not signifcantly diferent from that of untreated seeds of genotype 240207 (Table 5).
In general, primed seeds of okra genotypes in GA 3 increased the germination percentage, ranging from 7.5% (240207) to 14.5% (Clemson) compared to untreated seeds of the respective genotypes. Seeds treated with tap   water and KH 2 PO 4 increased the germination percentages of all fve okra genotype seeds by 2.5 to 9%. Seeds treated with cow urine for genotype 240207 had a 4.5% reduction in germination percentage, while seeds of other genotypes treated with cow urine had increase a germination percentage from 1 (240586) to 12.5% (Clemson). Te results showed that the germination of seeds of all genotypes increased better when treated with GA 3 , but the magnitude of the increase difered by genotype. Seeds treated with other priming treatments also showed a diferent amount of increase in germination percentages across genotypes compared to untreated seeds. Te enhanced germination observed in seed priming treatments might be due to the fact that the seeds were completing the pregermination process during seed priming, and GA 3 might afect the synthesis of hydrolyzing enzymes, particularly amylase and protease and the hydrolyzed food was utilized by the growing embryo and thereby enhancing the germination of seeds [31,32]. Consistent with others, the present results also showed a signifcant increase in germination percentage with different priming treatments [33]. Seeds primed with cow urine had a 15.6% higher germination percentage than untreated seeds of cotton [34].

Efects of Genotype and Seed Priming on Speed of
Germination. Analysis of variance results showed that both the main and interaction efects of genotype and diferent seed priming treatments signifcantly (p < 0.01) afected the speed of germination (Table 3). Genotype Clemson, followed by SOH701 and 240207, treated with GA 3, showed signifcantly higher speed of germination than other treatment combinations, with no statically signifcant diference among themselves. Untreated seeds of genotypes 240586 and 240207 showed a lower speed of germination, but there was no statistically signifcant diference in the speed of germination in untreated seeds of all other genotypes (Table 5). Increased speed of germination by seed priming treatment might be due to the completion of pregermination metabolic activity of seeds and the seed ready for radical protrusion [31]. Te result is in agreement with the fndings of other researchers [35].

Efects of Genotype and Seed Priming on Number of
Hard Seed. Analysis of variance results showed that both the main and interaction efects of genotype and diferent seed priming treatments signifcantly afected the number of hard seeds (p < 0.01) ( Table 3). Tere were no hard seeds in genotype Clemson treated in GA 3 and cow urine, genotype Note. Mean value within columns followed by the same letter (s) of trait (s) were not signifcantly diferent at 5% probability level; LSD, least signifcant diference; CV (%), is coefcient of variation in percent; GA 3, gibberellic acid; KH 2 PO 4 , potassium dihydrogen phosphate; and the value in the bracket is the original (nontransformed) data.
Advances in Agriculture SOH701 treated in GA 3 , and genotype 240207 treated in tap water and KH 2 PO 4 . In contrast, untreated seeds of genotype 240586 had a signifcantly higher number (24.34%) of hard seeds than all other treatment combinations. Moreover, this genotype showed a persistently high percentage of hard seeds across diferent seed priming treatments (Table 5). In general, genotypes showed diferent responses to diferent seed priming treatments in terms of seed hardiness percentage. Te results showed that the seed priming treatment signifcantly reduced the percentage of hard seeds in all tested okra genotype seeds. Tis may be due to the softening efect of the hard seed coat, the leaching of inhibitors, and the stimulation of hydrolytic enzymes required for the degradation of the cells surrounding the radicle, thereby suppressing the radicle [31]. Similar observations have been reported by other researchers [16,23].

Efects of Genotype and Seed Priming on Seedling
Length. Analysis of variance results showed that both the main and interaction efect of genotype and diferent seed priming treatments signifcantly (p < 0.01) afected seedling growth (Table 3). Te seeds of genotype 240207, followed by genotypes 240586 and SOH701 treated with GA 3 had signifcantly longer seedlings without signifcance diference among their mean values. In contrast, the untreated seeds of genotype 240207 had signifcantly shortest seedling length without signifcant diference with the untreated seeds of genotype Arka Anamika and Clemson (Table 6). In general, primed seeds of okra genotypes by GA 3 increased the seedling length in the range between 36.5% (Clemson) and 61.6% (240207) compared to untreated seeds of the respective genotypes. Seeds of genotypes SOH701 and 240586 treated in tap water showed a slight reduction in seedling length, while in the other genotypes 3.8-27.8% increments was observed. Similarly, KH 2 PO 4 and cow urine also increased the seedling length, except in genotype SOH701 in the range from 7.7%, genotype 240586 treated with KH 2 PO 4 to 20.6% of genotype Arka Anamika treated with cow urine. Results showed an increase in seedling growth, but the extent of the increase varied by genotype. Tis pointed to diferent genotypes responding diferently to diferent seed priming treatments for seedling growth. A possible reason for the longer seedling length of genotypic seeds from seed priming was earlier germination. Tis could be because seedling growth was improved and GA 3 increased cell division, causing elongation and an increase of seedling roots and shoots within the apical meristem of seedlings [36]. Terefore, seed priming using GA 3 can be successfully used to promote okra seed germination and seedling growth. Cow urine and KH 2 PO 4 may also afect longer seedlings by providing nutrients and growth-promoting bioactives [37]. Te present results correspond to those of Sanodiya et al. [35].

Efects of Genotype and Seed Priming on Seedling Dry
Weight. Analysis of variance results showed that both the main and interaction efects of genotype and diferent seed priming treatments signifcantly (p < 0.01) afected seedling dry weight (Table 3). Genotype 24027 followed by genotype 240586 seeds treated with GA 3 showed signifcantly higher seedling dry weight, but there was no signifcant diference between the two. In contrast, untreated seeds of genotype SOH701 had signifcantly lower seedling dry weight, followed by the seedling dry weights of other untreated and tap water-treated genotypes, except for genotype 240586 (Table 6). Increased seedling dry weight in seed priming may be due to increased seedling fresh weight, seedling length, and increased dry matter accumulation due to earlier germination and growth [20]. Tis result is consistent with that of other researchers [30].

Efects of Genotype and Seed Priming on Seedling Vigor
Index I. Both the main and interaction efect of genotype and diferent seed priming treatments (p < 0.01) signifcantly afected seedling vigor index I ( Table 3). Seeds of all genotypes treated with GA 3 had signifcantly higher seedling vigor index I than other treatment combinations. In contrast, untreated seeds of all genotypes, except SOH701 had the lowest seedling vigor index I without signifcant differences among them (Table 6). In general, treated seeds of okra genotypes in GA 3 increased seedling vigor index I, ranging from 23.5% (SOH701) to 78.6% (240207) compared to untreated seeds of the respective genotypes. Seeds treated in tap water improved the seedling vigor index I by about 13.7% to 24.2%, except for SOH701. Similarly, KH 2 PO 4 and cow urine also increased the seedling vigor index I of genotypes other than SOH701 by approximately 18.3% to 28.2% and approximately 17.2% to 34.4%, respectively. Te results showed that priming of seeds with GA 3 increased the vigor index I of seeds of all genotypes, seeds treated with other seed priming treatments also showed diferent seed vigor index I, along with increases in most genotypes. In general, the results showed that seeds of diferent okra genotypes showed diferent responses to diferent seed priming treatments for seed vigor index I. Te increase in seed vigor index by seed priming may be due to the fact that the seed priming treatment is appropriate for the metabolic response, improving seed germination performance and seedling growth [36]. Te current result is consistent with the results of Kaura et al. [38].

Efects of Genotype and Seed Priming on Seedling Vigor
Index II. Both the main and interaction efect of genotype and diferent seed priming treatments (p < 0.01) signifcantly afected seedling vigor index II (Table 3). Genotype 240586 seeds treated with GA 3 had a signifcantly higher seed vigor index II, with no signifcant diference from the genotype 240207 seeds treated with GA 3 . Whereas untreated and tap water-treated seeds of all genotypes, GA 3 -treated seeds of genotype SOH701, KH 2 PO 4 -treated seeds of genotypes Clemson and 240207 and cow urine-treated seeds of genotype Clemson and Arka Anamika showed low seed vigor index II, which were statistically at par with each other (Table 6). Te results showed that priming of seeds with GA 3 increased the vigor index II of seeds of most genotypes, but the magnitude of the increase difered by genotype. Te increase in seed vigor index by seed priming may be due to the fact that the priming treatment is appropriate for metabolic response, improving seed germination performance and seedling growth [36]. In general, the results showed that seeds of diferent okra genotypes showed different responses to diferent seed priming treatments for seed vigor index II. Te current result is consistent with the results of Kaura et al. [38].

Correlation of Seed Quality Parameters.
Te correlation analysis showed that seed germination percentage, speed of germination, seed viability, seedling length, seedling dry weight, and seed vigor indices I and II had highly signifcant with positive correlations with each other. In contrast, hard seeds had highly signifcant with negative correlations with germination percentage, speed of germination, seed viability, seedling length, seedling dry weight, and seed vigor indices I and II (Table 7). High germination percentages, viability, and rapid seedling growth are characteristics of vigorous seeds. Te ability of a seed to germinate, together with its germination percentage, germination speed, and seedling growth, are all combined to form seed vigor. Terefore, the potential performance of viable seeds is Note. Mean value within columns followed by the same letter (s) of trait (s) were not signifcantly diferent at 5% probability level; LSD, least signifcant diference; CV (%), is coefcient of variation in percent; GA 3, gibberellic acid; KH 2 PO 4 , potassium dihydrogen phosphate. Note. * * , highly signifcant at 0.01 levels; GS, germination speed; HS, hard seed; SV, seed viability; SL, seedling length; SVI, seed vigor index I; SDW, seedling dry weight; SVII, seed vigor index II. described by seed germination and vigor. Germination uniformity, germination speed, and seedling growth are the three basic aspects of seed vigor [39,40]. Seed vigor index is directly refected in seed germination percentage and seedling growth. Te other most important factor afecting the seed vigor index is speed of germination, which is also the seed germination parameter and most strongly linked to seed germination and vigor [41,42]. Hard seed, on the other hand, hinders uniform germination, slows germination speed, and reduces the germination of the seed because it restricts water absorption in the seeds, which has an impact on the seed germination and establishment of seedlings. As a result, there is a negative correlation between hard seed and the abovementioned seed quality parameters.

Conclusion
In general, the study results showed that seeds of diferent okra genotypes showed diferent responses to diferent seed priming treatments for seed quality. Te seeds of okra genotypes treated by diferent seed priming treatments on seed germination, seed vigor, and reduction of seed hardiness revealed that the GA 3 seed priming treatment was better than any other seed priming treatment. Tap water, cow urine, and KH 2 PO 4 seed priming treatments can be used as an alternative to GA 3 seed priming treatment. Seed priming treatment is therefore a useful technique for improving the germination percentage, speed of germination, seedling growth, seed vigor, and reducing seed hardiness of okra seeds. Yet, more research is needed to know the efects of diferent seed priming treatments on the morphological traits and yield of okra genotypes.

Data Availability
Te datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
Te author declares that there are no conficts of interest.