Seed Germination Enhancement of Two Balanites Species (B. aegyptiaca (L.) Del. and B. rotundifolia (Tiegh.) Blatt.) Using Different Presowing Treatments in Ethiopia

B. aegyptiaca and B. rotundifolia are known to be multipurpose trees with various uses and values. Therefore, the aim of this study was to investigate the seed germination behaviours of B. aegyptiaca and B. rotundifolia under different presowing treatments. Hence, seeds were collected from the Central and Southern Ethiopian Rift Valley regions. Then, a total of 864 fruits (seeds) subjected to eight different presowing treatments and planted in pots arranged in a completely randomized design (CRD) were tested for each species. The mean germination percentage (GP), mean daily germination percentage (GD), mean germination time (GT), and mean germination index (GI) were computed. One-way ANOVA showed the presence of significant GP, GD, GT, and GI among treatment groups at p < 0.05 under both Balanites species. For B. aegyptiaca, Tukey's HSD test showed that seeds soaked with 98% H2SO4 for 10 minutes (98HSO10m) and 20 minutes (98HSO20m) have the highest GPs (87 ± 8.8 and 82 ± 10.2, respectively) that are significant at p < 0.05. The seeds soaked in 75°C hot water for 10 minutes and subsequently cooled for 12 hours (HW75d), 98HSO10m, and 98HSO20m have the highest GDs (2%) that are significant at p < 0.05. Moreover, 98HSO20m, 98HSO10m, and seeds soaked in cold water for 48 hours at room temperature of 25°C (CW48h) have the shortest GTs (24 ± 2.2, 25 ± 0.5, and 25 ± 1.3, respectively), and 98HSO10m and 98HSO20m have the highest GIs (1.04 ± 0.09 and 1.01 ± 0.08, respectively) that are significant at p < 0.05. For B. rotundifolia, the control recorded the highest cumulative germination (i.e., 71), followed by CW48h (i.e., 51). However, Tukey's HSD tests generally indicated that no treatment group resulted in significant differences in the means of GP, GD, GT, and GI at p < 0.05. So, no treatment group was observed to enhance the germination of B. rotundifolia compared to the control. However, this study generally indicated potential seed enhancement technologies for B. aegyptiaca with greater implications for propagation, conservation, and sustainable utilization of the species in the agricultural and pastoral communities of Ethiopia.


Introduction
B. aegyptiaca and B. rotundifolia are known to be multipurpose trees with various uses and values [1].Te species are widely distributed in arid and semiarid ecosystems in Ethiopia [1][2][3].Despite all these products, the species are considered one of the underutilized, neglected arid zone tree species that need to be domesticated [4].Tree seeds always exhibit some degree of dormancy, resulting in a delay and irregularity in the germination of seeds in the nursery and even on forest foors [5,6].Te seeds of Balanites are generally highly variable among sources in weight and morphology [7], as well as in germination capacity [8,9].Te seed storage behaviour of Balanites is orthodox in which seed viability can be maintained for 2 or even several years in air-dry storage at cool temperatures or hermetic storage at 3 °C with 6-10% moisture content [1].
Diferent seed treatments, such as cold and hot water, were reported to enhance the germination of B. aegyptiaca [10].Fresh seeds of B. aegyptiaca are believed to germinate readily without treatments, and the presence of mesocarp is thought to delay the seed germination of B. aegyptiaca [9].
Te seed storage behaviour of B. aegyptiaca is orthodox in which seed viability can be maintained for two or even several years in air-dry storage at cool temperatures or hermetic storage at 3 °C with 6-10% moisture contents [1].Te species can be propagated by seedlings (seeds) using direct sowing as well as by applying presowing treatments to maximize the germination percentage [1][2][3].Diferent seed treatments, such as cold and hot water, have been reported to enhance the germination of Balanites [10][11][12].B. rotundifolia is a very drought-resistant tree, even more so than B. aegyptiaca [13,14].Te fruits of B. rotundifolia are brittle and smooth on the outside, with a fbrous and oily endocarp with a high moisture content [14].B. rotundifolia is the less-studied Balanites in terms of its seed germination behaviour compared to B. aegyptiaca.However, direct sowing of the seed (or fruits) without applying treatments has previously been suggested [14].
Efective propagation and seedling establishment are the basic requirements for sustainable management for a species with notable seed dormancy profles.Indigenous fruit trees play an important role in the livelihood of rural people in sub-Saharan Africa and are sources of nutrition for the local population [15].Hence, understanding the seed physiology of plants can enhance future conservation through aforestation.Previously, Elfeel [9] studied the germination of Saudi Arabia's B. aegyptiaca using water soaking and different sowing orientations.However, extensive studies are yet to be conducted for populations of B. aegyptiaca and B. rotundifolia in other geographical areas, including Ethiopia.Moreover, previously, no information was documented on the seed germination behaviour of Balanites in Ethiopia using the diferent seed treatments.In this regard, documentation of the seed germination behaviours of species is very important for successful utilization or conservation plans [16].It was hypothesized that presowing seed treatments could enhance the germination of B. aegyptiaca and B. rotundifolia.Terefore, the present study was carried out to evaluate the potentiality of diferent seed presowing treatments for enhanced B. aegyptiaca and B. rotundifolia germination.

Seeds Sampling.
Te seeds were mainly collected from the Central and Southern Ethiopian Rift Valley regions (Figure 1).Te seeds collection areas mainly included the arid and semiarid ecosystems that include the Acacia-Commiphora woodland and bushland proper, Acacia wooded grassland of the Rift Valley, and Combretum-Terminalia woodland and wooded grassland ecosystems of Ethiopia [17].
Seeds collections were done from February to November 2022, following the previous observations from specimen sampling for morphological study and phenetic analysis of the Balanites genus in the study area.Hence, the seeds of B. aegyptiaca were collected from Boset district- Te seeds were collected from conspicuous superior (mother) trees of B. aegyptiaca and B. rotundifolia.So, about 30-40 fruits (seeds) per mother tree (i.e., about 200 fruits (seeds) per population) were collected and mixed up to represent the population.In other words, the fruits (seeds) from diferent populations (provenances) were collected from diferent parts of Ethiopia and thoroughly mixed up and homogenized to represent the species.Te homogenized seeds were further processed, cleaned, and purifed from inert and unwanted materials.
Te mesocarps, i.e., the feshy and edible parts of the fruit, were also removed (Figures 2 and 3).Te seeds were dried, and the moisture content (MC) was adjusted to 6.0% (checked with a German-made KERN-DBS 60-3 moisture tester).

Preparations of Seed Treatments and Experimental
Layout.For this purpose of study, seed treatments were prepared based on literature and ISTA protocols [1,9,10,[18][19][20]. Accordingly, the seven seed treatments (excluding the control) were prepared (Table 1).Te International Seed Testing Association (ISTA) recommends four replicates of 100 seeds each for standard germination tests, but there should not be fewer than 100 seeds in replicates of 25 or 50 seeds [21][22][23].
Following the random table of Gomez and Gomez [24], the experiment was laid out in a completely randomized design (CRD) with eight treatments and four replications each.Hence, 27 seeds per replication (i.e., 9 pots per replication, 3 seeds per pot, 108 seeds per treatment, and 864 fruits (seeds) for 8 treatments) were tested.Te germinations were done in a standard nursery soil mixture (3 : 2 : 1 ratio of topsoil, manure, and sand) placed in polyethylene plastic pots with a 16 cm diameter (Figure 4).Generally, B. aegyptiaca has fruits with lengths ranging from 2 to 4 cm and widths of 1.5 to 2.5 cm, while B. rotundifolia has fruits with lengths ranging from 2 to 3 cm and widths of 2 to 2.5 cm.So, fruits (seeds) were sown in each pot with a horizontal orientation of the seed stalk while fully covering the fruits (seeds) with the substrate (∼2.5 cm depth).Te sown seeds (or fruits) were then covered with transparent polythene sheets to optimize and regulate the moisture and temperature.
Te germination counts were taken every other day, starting one week after sowing, for three months, after which no more germination was observed in two weeks' time.In this study, a seed was considered to have germinated when the radicle emerged conspicuously [25,26].All experimental activities were carried out in the nursery house of the Central Ethiopia Forestry Development Center (CEFDC)-Ethiopian 2 Scientifca Forestry Development (EFD), Addis Ababa.Over the course of the germination test, the morning average temperature was 20 °C, the midday average was 26.5 °C, and the evening average was 19.5 °C.

Statistical Data Analysis.
Te data analysis was based on untransformed seed germination percentages.Te dataset was checked with a histogram and has a normal distribution.Te nineteen germination measurements (or parameters) were generated (see Supplementary Tables 1 and 2), of which four parameters, namely, the mean germination percentage (GP), mean daily germination percentage (GD), mean germination time (GT), and mean germination index (GI), were analysed and presented for this study report.Te GP, which is an estimate of the germinability of the population of seeds, was calculated as follows: where n i is the number of seeds germinated in the i th time and N is the total number of seeds used.Te GD, which is the mean number of seeds germinated per day (i.e., the number of seeds germinated daily relative to the maximum number of germinated seeds), was also calculated using the following Adams and Farrish [27]:   4 Scientifca where CP is the fnal cumulative germination percentage and T n T is the total number of intervals required for fnal germination.Following Ellis and Roberts [28], the GT, a measure of the rate and time spread of the germination, of each treatment was calculated using the formula: where "n" is the number of seeds germinated on each day, "d" is the number of days from the beginning of the test, and "N" is the total number of seeds germinated at the termination of the experiment.Furthermore, the GI, which is a measure of the percentage and speed of germination, was calculated using the following formula [29]: Te higher values for this measure indicate a greater rate of germination [30].
Moreover, a one-way ANOVA was computed to see the level of signifcant diference among the treatment means for the germination parameters at p ≤ 0.05.Moreover, Tukey's Honestly Signifcant Diference (HSD) was computed to assess the signifcance of the diference between pairs of treatments at p ≤ 0.05 [31], allowing all possible pairwise comparisons while keeping the family-wise error rate low.5 illustrates the newly emerging germinate and fully grown seedlings of B. aegyptiaca.After the six weeks of the germination period, no new germinates were observed in most of the treatments (Figure 6).

Germination of B. aegyptiaca. Figure
Te highest cumulative germination percentage (94%) was recorded by seeds soaked in 98% H 2 SO 4 for 10 minutes and subsequent rinsed with water (98HSO10m), followed by seeds soaked in 98% H 2 SO 4 for 20 minutes and subsequent rinsed with water (98HSO20m) with 89% cumulative germination percentage, and seeds soaked in hot water at 75 °C for 10 minutes, and left to cool for 12 hours at room temperature of 25 °C (HW75d) with 78% cumulative germination percentage.Te lowest cumulative germination percentage was recorded by seeds soaked in cold water for 48 hours at room temperature of 25 °C (CW48h) with a 58% cumulative germination percentage (Figure 6).
Te one-way ANOVA also indicated that there exist signifcant mean variations among treatment groups for the diferent germination parameters (Table 2).Tukey's HSD test also showed that 98HSO10m (87 ± 8.8) and 98HSO20m (82 ± 10.2) have the highest GPs which are signifcant at p < 0.05 compared to the other treatment groups.Moreover, Tukey's HSD indicated that HW75d, 98HSO10m, and 98HSO20m have the highest GDs (2%) that are signifcant at p < 0.05 compared to the other treatment groups (Table 3).Tukey's HSD for GT also showed that 98HSO20m (24 ± 2.2), 98HSO10m (25 ± 0.5) and CW48h (25 ± 1.3) have the shortest GTs that are signifcant at p < 0.05 compared to the other treatment groups (Table 3).Most importantly, Tukey's HSD resulted that 98HSO10m (1.04 ± 0.09) and 98HSO20m (1.01 ± 0.08) have the highest GIs that are signifcant p < 0.05 compared to the other treatment groups (Table 3).

Germination of B. rotundifolia.
Tis study generally demonstrated that B. rotundifolia germinates rapidly in a couple of weeks.Figure 7 illustrates the newly emerging germinate and fully grown seedlings of B. rotundifolia.In this regard, it was observed that the seeds of B. rotundifolia have shorter and faster germination time trends compared to those of B. aegyptiaca (Figure 8).
Hence, the highest cumulative germination was recorded by the Ctrl (i.e., 71), followed by CW48h (i.e., 51) (Figure 8).Te one-way ANOVA also indicated that there exist signifcant mean variations among treatment groups for the diferent germination parameters (Table 4).However, Tukey's HSD test showed that the control has the highest GP (66 ± 10.2) that is signifcant at p < 0.05 compared to the other treatment groups (Table 5), while the lowest signifcant GP at p < 0.05 was observed by HW75d (34% ± 7.0).
Overall, except for HW75d and 98HSO20m, the GDs of the diferent treatments were found to be 2 germination parentages per day that are signifcant at p < 0.05.Te species tend to have a mean germination time (GT) of less than 10 days regardless of the treatment type applied.Tukey's HSD test for GT indicated that there exist no signifcant mean diferences among treatment groups, except HW75d, at p < 0.05.Tukey's HSD test for GI also showed that the control has not signifcant diferent GI compared to the remaining treatment groups, except HW75d and 98HSO20m, at p < 0.05.

Discussion
In the current study, some presowing treatment techniques enhanced the germination capability of the seeds of B. aegyptiaca compared to seeds receiving no prior presowing treatment (i.e., the control).Generally, soaking the Scientifca fruits (seeds) in 98% H 2 SO 4 and water (hot or cold) enhances the germination percentages of B. aegyptiaca.In particular, seeds soaked with 98% H 2 SO 4 for 10 and 20 minutes effectively broken the seeds dormancy and signifcantly improved the mean germination percentages (GP).Te soaking of fruits (seeds) with hot water for up to 20 minutes has also a promising efect on the germination of B. aegyptiaca.Te   6 Scientifca fbrous endocarp of fruits needs to be digested to increase the inlet of moisture and oxygen to the embryo for germination to start.A study by Ahmed et al. [32] reported that the highest germination percentage (>80%) was observed for seeds of B. aegyptiaca from Saudi Arabia soaked with cold distilled water for 24 hours.Similarly, a study by Elfeel [9] in Saudi Arabia also reported that seeds soaked with cold water for 18 and 24 hours resulted in a higher germination percentage compared to seeds soaked for 48 hours.Similarly, soaking the seed in cold water for 24 hours is recommended by Bekele-Tesemma [2,3], and a germination rate of 50-70% can be attained.Te observation of such variations in seed germination is likely due to the variability of the physical, physiological, and genetic quality of the seeds of the diferent populations of B. aegyptiaca distributed in wider ranges of ecological conditions.Terefore, the physical, physiological, and genetic diferences among populations greatly afect their rate of water and chemical uptake by the seeds [33,34].Seed soaking duration afects embryonic physiological activities toward germination and ultimately determines the seed germination percentage, both negatively and positively [35,36].Increasing the soaking duration in both cold and hot water is very likely to decrease the germination percentage of B. aegyptiaca [9,32], which was also observed in our present study.Similarly, prolonged soaking of seeds in highly concentrated H 2 SO 4 could cause damage in both the external and internal parts of the seeds, which ultimately damages the embryonic tissue, resulting in lower germination percentages [29,37].Overall, the optimal seed soaking duration required for attaining a better germination percentage varies with the species and the ecological conditions under which it evolved.Furthermore, apart from presowing treatments, the sowing orientations of seeds in the soil substrate also afect the germination percentages.For instance, for B. aegyptiaca, the horizontal orientation of the seed stalk was recommended to attain higher germination [9], while Hall and Walker [10] recommend the seed be sown vertically with the stalk end downwards.
In this study, seeds treated with 98% H 2 SO 4 were observed to reduce the germination time required for the seed   Scientifca populations to complete their germination.Te shorter the germination time, the shorter the nursery period, and the faster the species reach for plantation and forestry work.In the contrary, a study by Elfeel [9] reported that seeds soaked with water for 18 and 24 hours, as well as untreated seeds, resulted in the shortest germination time compared to seeds  Te germination of B. rotundifolia, however, was not enhanced by any treatment applied.Tis might be due to the morphology of the fruit and germplasm of B. rotundifolia, which has softer fruits and seed germplasm that are very likely injured by the treatments used, either mechanical or biochemical ones.However, for B. rotundifolia, having a good germination percentage (>70%) without treatments is very advantageous in terms of propagation and conservation strategies.Seed propagation without treatment application is by far the most cost-efective approach.Te shorter germination time and faster germination rate observed by B. rotundifolia are the two additional advantages of the species for easy propagation and cultivation.Terefore, the application of treatments to the fruit (seed) of B. rotundifolia is not necessary [14].Te fruits of B. rotundifolia are brittle and smooth on the outside, with a fbrous and oily endocarp and high moisture content.Tis probably helped the species maintain the sufcient moisture required for germinating in dryland.Tat is why the species is a very drought-resistant tree, even more so than B. aegyptiaca [13,14].

Conclusions
Tis study identifed the potentiality of presowing seed treatment as a tool for enhancing the seeds germination of B. aegyptiaca.In general, soaking of seeds in 98% concentrated H 2 SO 4 , hot water as well as in normal cold water has proven to be efective methods to break the seed dormancy, improving the germination percentages, and reducing the mean germination time required to complete the germination.Yet, the untreated seeds of B. aegyptiaca also exhibited satisfactory germination (>50%), with implications of the high degree of regeneration and subsequent ecological succession of the species under the natural conditions of recurrent climate change and habitat fragmentation.However, no presowing treatments applied enhanced the seeds germination of B. rotundifolia, where more than 70% of seed germination was attained without the application of treatments.Tis has an implication of the high degree of regeneration and subsequent ecological succession of B. rotundifolia under the natural conditions of recurrent climate change and habitat fragmentation.Terefore, both species can be generally considered as alternatives for dryland rehabilitation and forestry development.Overall, these fndings will contribute to the conservation and sustainable utilization of the species in the agricultural and pastoral communities of Ethiopia.

Table 1 :Figure 4 :
Figure 4: Te nursery beds and the arrangement of pots, sown seeds, and sown seeds covered with transparent polythene sheet.

Figure 5 :Figure 6 :
Figure 5: Illustration of newly emerging germinate and fully grown seedlings of B. aegyptiaca.
between pairs of treatments at p ≤ 0.05 is calculated using Tukey's HSD test.Te values denoted with the same alphabet(s) along the vertical column of each germination parameter are not signifcantly diferent at p ≤ 0.05, and vice versa.

Figure 7 :
Figure 7: A newly emerging germinate (a) and fully grown seedlings of B. rotundifolia (b).

Table 2 :
One-way ANOVA of the mean signifcance diference among treatment groups for their efect on mean germination percentage (GP), daily germination percentage (GD), germination time (GT), and germination index (GI) of B. aegyptiaca.

Table 3 :
Summary of GP, GD, GT, GI, standard deviation (SD), and standard error (SE) of the germination parameters of B. aegyptiaca.

Table 4 :
One-way ANOVA of the mean signifcance diference among treatment groups for their efect on mean germination percentage (GP), daily germination percentage (GD), germination time (GT), and germination index (GI) of B. rotundifolia.** * highly signifcant if p ≤ 0.001, * * very signifcant if p ≤ 0.01, and * signifcant if p ≤ 0.05. Figure 8: Cumulative germination of B. rotundifolia under diferent treatments over days; Ctrl: control, RuSP: rubbed with sandpaper,CW24h: soaked in cold water for 24 hours at room temperature of 25 °C, CW48h: soaked in cold water for 48 hours at room temperature of 25 °C, HW65d: soaked in hot water at 65 °C for 10 minutes and left to cool for 12 hours at room temperature of 25 °C, HW75d: soaked in hot water at 75 °C for 10 minutes and left to cool for 12 hours at room temperature of 25 °C, 98HSO10m: soaked in 98% H 2 SO 4 for 10 minutes and subsequent rinsed with water, and 98HSO20m: soaked in 98% H 2 SO 4 for 20 minutes and subsequent rinsed with water.

Table 5 :
[3]]ary of GP, GD, GT, GI, standard deviation (SD), and standard error (SE) of the germination parameters of B. rotundifolia.Scientifca soaked with water for 48 hours.According to Hall and Walker[10]and Bekele-Tesemma[3], the germination of B. aegyptiaca generally occurs in 1-4 weeks, which also agrees with our present study.Overall, the current study indicated the potential of seed presowing treatment technologies to enhance seed germination in B. aegyptiaca, which has greater implications for the conservation and sustainable utilization of the species in the semiarid ecosystems of Ethiopia and beyond.
Te signifcance diference between pairs of treatments at p ≤ 0.05 is calculated using Tukey's HSD test.Te values denoted with the same alphabet(s) along the vertical column of each germination parameter are not signifcantly diferent at p ≤ 0.05, and vice versa.8