Current IssueEditorial BoardOnline First ArticlesArchive
Current IssueEditorial BoardOnline First ArticlesArchive

Full Research Article

Effects of Seed Treatment and Bio-inoculants on Baikiaea plurijuga DC. and Pterocarpus angolensis Harms Seed Germination

K
Kamogelo Makgobota1
0000-0001-8520-2370
F
Flora Pule-Meulenberg2
0000-0001-9064-8078
A
Ayana Angassa1
0000-0002-6763-3959
M
Melusi Rampart1
0000-0002-4712-5308
W
Witness Mojeremane1,*
0000-0003-4015-4381
B
Barbara Reinhold-Hurek3
0000-0001-6570-9502
1Department of Range and Forest Resources, Botswana University of Agriculture and Natural Resources, Gaborone, Botswana.
2Department of Crop and Soil Sciences, Botswana University of Agriculture and Natural Resources, Gaborone, Botswana.
3CBIB Centre for Biomolecular Interactions Bremen, Department of Microbe-Plant Interactions, Faculty of Biology and Chemistry, University of Bremen, Bremen, Germany.

ABSTRACT

Background: This study examined the effect of pre-sowing seed treatments and microbial inoculation on the germination of Pterocarpus angolensis and Baikiaea plurijuga at the Botswana University of Agriculture and Natural Resources.

Methods: This tree nursery study employed a 2 × 4 × 2 factorial, randomised experiment to test four pre-sowing treatments: mechanical scarification, soaking in rainwater and warm water (40°C) and untreated seeds- alongside two levels of microbial inoculation: with and without B. ripae.

Result: Mechanical scarification slightly improved germination, leading to earlier and more uniform germination in both species, with P. angolensis exhibiting a mean germination time of 10.5 days and a germination velocity coefficient of 9.57. P. angolensis germination decreased from 80.83% to 66.7%, indicating an incompatibility with B. ripae. Conversely, B. plurijuga germination increased from 70.84% to 91.67%. The findings demonstrate that scarification enhances germination in both species and can be used to improve native seedling protocols for the regeneration of dryland forests. 

INTRODUCTION

Forests and woodlands provide essential ecosystem services, including climate change mitigation (Vargas, 2022), habitat for numerous plant and animal species (Pan et al., 2011; Watson et al., 2018) and the supply of timber, fuelwood and non-timber forest products (Sunderlin et al., 2007). They aid in carbon sequestration (Pramova et al., 2012), enhance landscape aesthetics (Mundher et al., 2022; O’Brien et al., 2024) and improve urban environments by filtering pollutants, reducing temperatures and promoting mental well-being (Tyrväinen et al., 2005; McDonald et al., 2007). Pterocarpus angolensis (African teak or mukwa) and Baikiaea plurijuga DC (Zambezi teak) are key deciduous hardwood species native to Miombo woodlands (Iipumbu et al., 2024). P. angolensis offers durable, attractive timber used in veneering, flooring, carpentry, furniture and construction (Palgrave, 2002). Its bark and roots are also employed in traditional medicine to treat various ailments (Samie et al., 2009; Sadiki and Tshikhwawe, 2018). Additionally, this species contributes to nitrogen fixation and provides pollen and nectar for bees (Orwa et al., 2009). B. plurijuga supplies durable timber suitable for construction, furniture and tools, with its bark used in medicine and tanning (Palgrave, 2002).
       
B. Plurijuga, P. angolensis Harms and other native woody species are threatened by overexploitation, forest fires, pests, diseases, drought, herbivory and climate change. Their natural recruitment has declined due to droughts, seed shortages, litter loss, fire incidents and herbivory (McLaren and McDonald, 2003; Vieira and Scariot, 2006; Gavinet et al., 2018; Zhang et al., 2017). Furthermore, the germination of native woody species seeds in their natural habitats is hindered by tough seed coats, unfavourable environmental conditions and human activities; however, factors such as temperature fluctuations, abrasion and animal digestion (Ooi et al., 2014; Traveset et al., 2001; Renison et al., 2010), along with heat from the soil (Mbalo and Witkowski, 1997; Lamont and Pausas, 2023), contribute to breaking seed dormancy.
       
Various methods, including mechanical, acid and water treatments, are employed to break seed dormancy and enhance germination (Tiwari et al., 2023; Mojeremane et al., 2017, 2024; Taghizadeh and Sajadi, 2023; Mohamed et al., 2024; Alzandi et al., 2025). The advantages of seed treatments include higher germination rates, uniform seedling emergence and protection of seeds or seedlings from early-season diseases and insect pests, which promotes crop emergence and growth (Govindaraj et al., 2017). Besides pre-sowing treatments, bio-inoculants are increasingly recognised as sustainable and environmentally friendly methods to improve seed germination, root growth and seedling development (Khan et al., 2011, 2014; Ahemad and Kibret, 2014; Mridha et al., 2016). They are especially crucial in semi-arid and savanna areas characterised by water shortages and poor soil quality (Maitra et al., 2021).  Despite growing interest, their effects on the germination of native trees remain largely unexplored. This study examined the impacts of seed pre-treatments and Bradyrhizobium ripae WR4T inoculation on germination in a nursery setting.

MATERIALS AND METHODS

Study area
 
This experiment was conducted at the forestry nursery of Botswana University of Agriculture and Natural Resources (BUAN) located in Sebele Content Farm (latitude 23°34′S, longitude 25°57′E, elevation 994 m), approximately 10 km north of Gaborone near the A1 highway (Rasebeka et al., 2014). The soil composition is a well-drained sandy loam. The semi-arid climate of Sebele is characterised by hot summers and mild winters, receiving between 250 and 600 mm of rainfall each year, mainly from November to March. The study was carried out from March 2024 to March 2025.
 
Seed and potting soil collection
 
Fruits were harvested from two experimental trees collected from Kazuma, Kasane and Chobe Forest Reserves in the Chobe District of Northern Botswana. A GPS device was used to record the locations of the mother trees. Pods were gathered using a long-hooked stick and a looper, subsequently packed into paper bags and transported to BUAN for extraction and storage. For B. plurijuga, the pods were manually crushed at the BUAN Herbarium to remove the seeds, whereas for P. angolensis, secateurs were used. Any damaged or immature seeds were discarded, while the healthy seeds were stored at 5°C until they were required for experiments. Soil samples were collected from beneath the two study species at Pandamatenga Farms in the Chobe District. An electric fence protects the farm from elephants and other animals. The soil was taken to a depth of 20 cm using a spade. It was,  then transported to BUAN in 25-kg plastic bags and subsequently filled into 2-litre black polyethene bags.
 
Experimental design and layout
 
This study employed a 2 × 4 × 2 randomised factorial design, incorporating four pre-sowing treatments: (i) mechanical scarification, (ii) soaking in rainwater, (iii) exposure to warm water at 40°C and (iv)seeds that were not treated. The second factor pertained to inoculation with Bradyrhizobium ripae WR4T, which was either applied or omitted. Each treatment was replicated three times, with 10 seeds per species in each replication. A single seed was sown in a 2-litre black polythene bag containing 2 kg of soil. The bags were placed on raised metal benches under a 40% shade net in the nursery.
 
Seed germination treatments
 
Each presowing treatment used 60 seeds, for a total of 240 per species. The 240 seeds were divided into two batches of 120 seeds each, one for presowing and the other for inoculation. Each treatment had three replicates, with 10 seeds in each. A nail cutter was used to scarify the seeds of both species by nipping the convex end opposite the embryo, avoiding damage. For a warm-water (40°C) soaking treatment, a water bath was filled with tap water and kept at 40°C. After reaching equilibrium, the water bath was turned off and the seeds were soaked for 24 hours. Rainwater was collected in a bucket overnight and transferred to a 250 ml beaker containing seeds. Seeds were soaked for 24 hours. Untreated seeds served as a control. The inoculation experiment employed seed batches that were mechanically scarified, pretreated in a water bath at 40°C and soaked in rainwater. B. ripae from the University of Bremen, Germany, was used as an inoculant. Approximately 5 g of inoculum was mixed with 25 mL of distilled water to create a slurry in a sterile 250 mL glass beaker. Seeds were, then coated with bacterial slurry in peat, following the method described by Somasegaran and Hoben (1994) and immediately sown in black polyethene bags. To prevent cross-contamination, inoculated seeds were sown after the non-inoculated ones.
 
Germination parameters
 
Seed germination was monitored daily from the day after sowing until measurements ceased after 12 months. The collected germination data were then used to calculate the specified parameters.
 
Seed germination percentage (GP)
 
The GP was determined using the method described by Scott et al., (1984).


Mean germination time (MGT)
 
Mean germination time was calculated using the method outlined by Ellis and Roberts (1981).

 
Where,
n= The number of seeds germinated on t.
t= Days from the start of the experiment to when those seeds germinated.
Σ (t*n)=  The product of the number of seeds that have  germinated and the corresponding number of days.
Σn= The total number of seeds that germinate successfully.
 
Germination index (GI)
 
The germination index was evaluated following Esechie (1994).
 
             Germination index = Σ (Gt/Tt)                ...(3)   
 
Where,
Gt= The quantity of seeds that have germinated by Tt.
Tt= The number of days from sowing to germination.
 
Coefficient of velocity of germination (CVG)
 
The velocity coefficient of germination was determined as described by Maguire (1962).
 
                 Coefficient of velocity of germination = [Σ (N×T)/ΣN ) ×100                  ...(4)
 
 
Where,
N= The number of seeds germinated on t.
T= Days from the start of the experiment to when those seeds germinated.
Σ(T*N)= The product of the number of seeds germinated each day.
ΣN= The total quantity of seeds that germinated.
 
Data analysis
 
Data analysis was conducted using Statistix Software (Version 10) using descriptive statistics and three-way ANOVA. The germination percentage data were transformed to meet normality assumptions before performing the ANOVA. Tukey’s HSD was employed to test mean differences at p<0.05.

RESULTS AND DISCUSSION

Table 1 presents the effects of seed pre-treatments and inoculation on the germination of P. angolensis seeds. No significant differences were observed among pre-sowing treatments (P>0.05), with germination rates ranging from 71.7% to 75%. Mechanical scarification and warm-water treatment resulted in a slight increase in germination, though this was not statistically significant. Germination time ranged from 10.5 to 22.81 days, with mechanical scarification being the fastest at 10.5 days. Soaking in warm water and untreated seeds reduced germination time compared to rainwater, but these differences were not significant. The germination index varied slightly, from 12 to 13 across treatments. Mechanical scarification had the highest germination velocity coefficient of 9.57; although this was not statistically significant, it indicated a faster germination rate. Other treatments showed lower velocities with no significant differences. Germination parameters were similar overall, but mechanical scarification was most effective, as evidenced by shorter germination time and a higher velocity coefficient.  

Table 1: Effect of presowing treatments and inoculation on the germination parameters of P. angolensis.


       
The results showed that inoculating P. angolensis seeds with B. ripae significantly (P<0.05) decreased the germination percentage (Table 1). There were no significant differences in mean germination time or the coefficient of velocity of germination between inoculated and non-inoculated seeds (Table 1). A slight, non-significant reduction in germination time was observed for inoculated seeds (Table 1). Although non-inoculated seeds exhibited a higher germination index and velocity, these differences were not statistically significant. Inoculation may negatively affect germination, possibly due to microbial competition. Seed pre-treatment and inoculation effects appear to be independent, with no significant interaction.
       
The results presented in Table 2 showed no significant differences between pre-treatments. The results revealed that germination rates varied from 73.33% to 88.33%, with the highest value recorded in the mechanically scarified seeds (88.33%), followed by untreated seeds (85.00%), rainwater-treated seeds (78.33%) and warm water-soaked seeds (73.33%). The results showed no significant differences between the mean germination time (ranged from 26.37 to 28.15 days). The results displayed slightly shorter mean germination times due to mechanical scarification of seeds, with a germination index of 0.18 and a coefficient of velocity of germination of 3.82, indicating marginally faster germination, with no significant differences.

Table 2: Effect of different seed pre-treatments and inoculum on germination of Baikiaea plurijuga seeds.


       
B. plurijuga seeds inoculated with B. ripae showed significantly (P<0.05) higher germination rates and a higher germination index than non-inoculated seeds (Table 2). Microbial inoculation appears to enhance germination by improving seed health and suppressing pathogens. No interaction was observed between pre-treatment and inoculation in B. plurijuga.
       
Poor germination, often caused by seed coat hardness in woody species, is common in arid regions and hampers nursery propagation (Mojeremane et al., 2021). This study investigated the impact of seed pre-treatments and inoculation on P. angolensis and B. plurijuga, two ecologically and economically significant species in Botswana. In nature, seed coats are typically broken by fire (Walters et al., 2004). To counter dormancy caused by seed coat hardness and promote faster, more uniform germination, several methods have been suggested (Fredrick et al., 2017; Cañizares et al., 2025; Motbaynor et al., 2025). The study found that seed responses to pre-treatment vary by species and method. All treatments increased germination, with mechanically scarified seeds showing higher rates; however, this difference was not statistically significant. Results for P. angolensis align with studies reporting higher germination in scarified seeds (Chisha-Kasumu et al., 2007; Botsheleng et al., 2014; Tselakgosi, 2021; Latiwa et al., 2023). Similarly, Botsheleng et al. (2014) observed higher but insignificant germination in scarified B. plurijuga seeds. The findings suggest that seed treatment may not be essential for B. plurijuga and P. angolensis; however, the germination rate (speed) is crucial, as slower germination can lead to uneven seedling growth (Chisha-Kasumu et al., 2007). Pre-treatments did not significantly influence mean germination time, germination index, or coefficient of velocity of germination. Scarified seeds showed marginally faster germination, indicating that this process could be expedited.
       
The benefits of mechanical scarification have been observed in various woody species in Botswana (Koobonye et al., 2018; Botumile et al., 2020; Mojeremane et al., 2021; 2024) and in other regions (Fredrick et al., 2017; Salazar and Ramírez, 2018; Alzandi et al., 2025). However, this method has its limitations. It is physically demanding and labour-intensive, especially when only a small number of seedlings are needed (Todd-Bockarie et al., 1993) and it is not suitable for large-scale seed treatment (Danthu et al., 1992).
       
Soil inoculants can affect seed germination rates depending on the type of inoculant and the plant species (Balshor et al., 2017; Mawarda et al., 2020). This study shows the different responses of B. plurijuga and P. angolensis seeds to B. ripae. The effects of inoculants on woody seed germination vary depending on the strain used (Lindström and Mousavi, 2020; Mendoza-Suárez et al., 2021). In this case, inoculated P. angolensis seeds had lower germination rates than non-inoculated seeds, although germination metrics remained unchanged. Since P. angolensis naturally forms an effective symbiosis, introducing a non-native strain might have disrupted essential early plant-microbe communication (Bünger et al., 2021). Incompatibility between host and inoculant strains can reduce seed germination and seedling vigour in legumes, especially if the non-native strain fails to form a symbiosis (Allito et al., 2021; Etesami, 2025). Inoculating B. plurijuga significantly improved germination and the germination index, indicating higher rates and more vigorous, uniform sprouting. These results agree with other studies, which demonstrate that inoculants, either alone or in combination, can enhance seed germination in woody plants (Khan et al., 2011, 2014; Singh et al., 2011; Sreedhar and Mohan, 2014; Mridha et al., 2016).

CONCLUSION

Seeds of P. angolensis and B. plurijuga can germinate without pre-treatment, but mechanical scarification proved to be the most effective method for both species. It produces the highest, though statistically insignificant, germination rates, with faster germination and better uniformity. This approach is suitable for small seed quantities due to its labour intensity. Inoculation reduced germination in P. angolensis but enhanced it for B. plurijuga. These findings highlight the importance of tailoring propagation strategies to meet the unique biological needs of each species.

ACKNOWLEDGEMENT

The authors thank the German Federal Ministry of Education and Research (BMBF) for funding this study. We also appreciate the Southern African Science Service Centre for Climate Change and Adaptive Land Management (SASSCAL) and the Botswana University of Agriculture and Natural Resources as the implementing agencies. Additionally, the authors thank the University of Bremen, Germany, for providing the inoculant.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for the experiments were approved by the Committee of Experimental Animal and handling techniques were approved by the University of Animal Care Committee. 

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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    Full Research Article

    Effects of Seed Treatment and Bio-inoculants on Baikiaea plurijuga DC. and Pterocarpus angolensis Harms Seed Germination

    K
    Kamogelo Makgobota1
    0000-0001-8520-2370
    F
    Flora Pule-Meulenberg2
    0000-0001-9064-8078
    A
    Ayana Angassa1
    0000-0002-6763-3959
    M
    Melusi Rampart1
    0000-0002-4712-5308
    W
    Witness Mojeremane1,*
    0000-0003-4015-4381
    B
    Barbara Reinhold-Hurek3
    0000-0001-6570-9502
    1Department of Range and Forest Resources, Botswana University of Agriculture and Natural Resources, Gaborone, Botswana.
    2Department of Crop and Soil Sciences, Botswana University of Agriculture and Natural Resources, Gaborone, Botswana.
    3CBIB Centre for Biomolecular Interactions Bremen, Department of Microbe-Plant Interactions, Faculty of Biology and Chemistry, University of Bremen, Bremen, Germany.

    ABSTRACT

    Background: This study examined the effect of pre-sowing seed treatments and microbial inoculation on the germination of Pterocarpus angolensis and Baikiaea plurijuga at the Botswana University of Agriculture and Natural Resources.

    Methods: This tree nursery study employed a 2 × 4 × 2 factorial, randomised experiment to test four pre-sowing treatments: mechanical scarification, soaking in rainwater and warm water (40°C) and untreated seeds- alongside two levels of microbial inoculation: with and without B. ripae.

    Result: Mechanical scarification slightly improved germination, leading to earlier and more uniform germination in both species, with P. angolensis exhibiting a mean germination time of 10.5 days and a germination velocity coefficient of 9.57. P. angolensis germination decreased from 80.83% to 66.7%, indicating an incompatibility with B. ripae. Conversely, B. plurijuga germination increased from 70.84% to 91.67%. The findings demonstrate that scarification enhances germination in both species and can be used to improve native seedling protocols for the regeneration of dryland forests. 

    INTRODUCTION

    Forests and woodlands provide essential ecosystem services, including climate change mitigation (Vargas, 2022), habitat for numerous plant and animal species (Pan et al., 2011; Watson et al., 2018) and the supply of timber, fuelwood and non-timber forest products (Sunderlin et al., 2007). They aid in carbon sequestration (Pramova et al., 2012), enhance landscape aesthetics (Mundher et al., 2022; O’Brien et al., 2024) and improve urban environments by filtering pollutants, reducing temperatures and promoting mental well-being (Tyrväinen et al., 2005; McDonald et al., 2007). Pterocarpus angolensis (African teak or mukwa) and Baikiaea plurijuga DC (Zambezi teak) are key deciduous hardwood species native to Miombo woodlands (Iipumbu et al., 2024). P. angolensis offers durable, attractive timber used in veneering, flooring, carpentry, furniture and construction (Palgrave, 2002). Its bark and roots are also employed in traditional medicine to treat various ailments (Samie et al., 2009; Sadiki and Tshikhwawe, 2018). Additionally, this species contributes to nitrogen fixation and provides pollen and nectar for bees (Orwa et al., 2009). B. plurijuga supplies durable timber suitable for construction, furniture and tools, with its bark used in medicine and tanning (Palgrave, 2002).
           
    B. Plurijuga    , P. angolensis Harms and other native woody species are threatened by overexploitation, forest fires, pests, diseases, drought, herbivory and climate change. Their natural recruitment has declined due to droughts, seed shortages, litter loss, fire incidents and herbivory (McLaren and McDonald, 2003; Vieira and Scariot, 2006; Gavinet et al., 2018; Zhang et al., 2017). Furthermore, the germination of native woody species seeds in their natural habitats is hindered by tough seed coats, unfavourable environmental conditions and human activities; however, factors such as temperature fluctuations, abrasion and animal digestion (Ooi et al., 2014; Traveset et al., 2001; Renison et al., 2010), along with heat from the soil (Mbalo and Witkowski, 1997; Lamont and Pausas, 2023), contribute to breaking seed dormancy.
           
    Various methods, including mechanical, acid and water treatments, are employed to break seed dormancy and enhance germination (Tiwari et al., 2023; Mojeremane et al., 2017, 2024; Taghizadeh and Sajadi, 2023; Mohamed et al., 2024; Alzandi et al., 2025). The advantages of seed treatments include higher germination rates, uniform seedling emergence and protection of seeds or seedlings from early-season diseases and insect pests, which promotes crop emergence and growth (Govindaraj et al., 2017). Besides pre-sowing treatments, bio-inoculants are increasingly recognised as sustainable and environmentally friendly methods to improve seed germination, root growth and seedling development (Khan et al., 2011, 2014; Ahemad and Kibret, 2014; Mridha et al., 2016). They are especially crucial in semi-arid and savanna areas characterised by water shortages and poor soil quality (Maitra et al., 2021).  Despite growing interest, their effects on the germination of native trees remain largely unexplored. This study examined the impacts of seed pre-treatments and Bradyrhizobium ripae WR4T inoculation on germination in a nursery setting.

    MATERIALS AND METHODS

    Study area
     
    This experiment was conducted at the forestry nursery of Botswana University of Agriculture and Natural Resources (BUAN) located in Sebele Content Farm (latitude 23°34′S, longitude 25°57′E, elevation 994 m), approximately 10 km north of Gaborone near the A1 highway (Rasebeka et al., 2014). The soil composition is a well-drained sandy loam. The semi-arid climate of Sebele is characterised by hot summers and mild winters, receiving between 250 and 600 mm of rainfall each year, mainly from November to March. The study was carried out from March 2024 to March 2025.
     
    Seed and potting soil collection
     
    Fruits were harvested from two experimental trees collected from Kazuma, Kasane and Chobe Forest Reserves in the Chobe District of Northern Botswana. A GPS device was used to record the locations of the mother trees. Pods were gathered using a long-hooked stick and a looper, subsequently packed into paper bags and transported to BUAN for extraction and storage. For B. plurijuga, the pods were manually crushed at the BUAN Herbarium to remove the seeds, whereas for P. angolensis, secateurs were used. Any damaged or immature seeds were discarded, while the healthy seeds were stored at 5°C until they were required for experiments. Soil samples were collected from beneath the two study species at Pandamatenga Farms in the Chobe District. An electric fence protects the farm from elephants and other animals. The soil was taken to a depth of 20 cm using a spade. It was,  then transported to BUAN in 25-kg plastic bags and subsequently filled into 2-litre black polyethene bags.
     
    Experimental design and layout
     
    This study employed a 2 × 4 × 2 randomised factorial design, incorporating four pre-sowing treatments: (i) mechanical scarification, (ii) soaking in rainwater, (iii) exposure to warm water at 40°C and (iv)seeds that were not treated. The second factor pertained to inoculation with Bradyrhizobium ripae WR4T, which was either applied or omitted. Each treatment was replicated three times, with 10 seeds per species in each replication. A single seed was sown in a 2-litre black polythene bag containing 2 kg of soil. The bags were placed on raised metal benches under a 40% shade net in the nursery.
     
    Seed germination treatments
     
    Each presowing treatment used 60 seeds, for a total of 240 per species. The 240 seeds were divided into two batches of 120 seeds each, one for presowing and the other for inoculation. Each treatment had three replicates, with 10 seeds in each. A nail cutter was used to scarify the seeds of both species by nipping the convex end opposite the embryo, avoiding damage. For a warm-water (40°C) soaking treatment, a water bath was filled with tap water and kept at 40°C. After reaching equilibrium, the water bath was turned off and the seeds were soaked for 24 hours. Rainwater was collected in a bucket overnight and transferred to a 250 ml beaker containing seeds. Seeds were soaked for 24 hours. Untreated seeds served as a control. The inoculation experiment employed seed batches that were mechanically scarified, pretreated in a water bath at 40°C and soaked in rainwater. B. ripae from the University of Bremen, Germany, was used as an inoculant. Approximately 5 g of inoculum was mixed with 25 mL of distilled water to create a slurry in a sterile 250 mL glass beaker. Seeds were, then coated with bacterial slurry in peat, following the method described by Somasegaran and Hoben (1994) and immediately sown in black polyethene bags. To prevent cross-contamination, inoculated seeds were sown after the non-inoculated ones.
     
    Germination parameters
     
    Seed germination was monitored daily from the day after sowing until measurements ceased after 12 months. The collected germination data were then used to calculate the specified parameters.
     
    Seed germination percentage (GP)
     
    The GP was determined using the method described by Scott et al., (1984).


    Mean germination time (MGT)
     
    Mean germination time was calculated using the method outlined by Ellis and Roberts (1981).

     
    Where,
    n= The number of seeds germinated on t.
    t= Days from the start of the experiment to when those seeds germinated.
    Σ (t*n)=  The product of the number of seeds that have  germinated and the corresponding number of days.
    Σn= The total number of seeds that germinate successfully.
     
    Germination index (GI)
     
    The germination index was evaluated following Esechie (1994).
     
                 Germination index = Σ (Gt/Tt)                ...(3)   
     
    Where,
    Gt= The quantity of seeds that have germinated by Tt.
    Tt= The number of days from sowing to germination.
     
    Coefficient of velocity of germination (CVG)
     
    The velocity coefficient of germination was determined as described by Maguire (1962).
     
                     Coefficient of velocity of germination = [Σ (N×T)/ΣN ) ×100                  ...(4)
     
     
    Where,
    N= The number of seeds germinated on t.
    T= Days from the start of the experiment to when those seeds germinated.
    Σ(T*N)= The product of the number of seeds germinated each day.
    ΣN= The total quantity of seeds that germinated.
     
    Data analysis
     
    Data analysis was conducted using Statistix Software (Version 10) using descriptive statistics and three-way ANOVA. The germination percentage data were transformed to meet normality assumptions before performing the ANOVA. Tukey’s HSD was employed to test mean differences at p<0.05.

    RESULTS AND DISCUSSION

    Table 1 presents the effects of seed pre-treatments and inoculation on the germination of P. angolensis seeds. No significant differences were observed among pre-sowing treatments (P>0.05), with germination rates ranging from 71.7% to 75%. Mechanical scarification and warm-water treatment resulted in a slight increase in germination, though this was not statistically significant. Germination time ranged from 10.5 to 22.81 days, with mechanical scarification being the fastest at 10.5 days. Soaking in warm water and untreated seeds reduced germination time compared to rainwater, but these differences were not significant. The germination index varied slightly, from 12 to 13 across treatments. Mechanical scarification had the highest germination velocity coefficient of 9.57; although this was not statistically significant, it indicated a faster germination rate. Other treatments showed lower velocities with no significant differences. Germination parameters were similar overall, but mechanical scarification was most effective, as evidenced by shorter germination time and a higher velocity coefficient.  

    Table 1: Effect of presowing treatments and inoculation on the germination parameters of P. angolensis.


           
    The results showed that inoculating P. angolensis seeds with B. ripae significantly (P<0.05) decreased the germination percentage (Table 1). There were no significant differences in mean germination time or the coefficient of velocity of germination between inoculated and non-inoculated seeds (Table 1). A slight, non-significant reduction in germination time was observed for inoculated seeds (Table 1). Although non-inoculated seeds exhibited a higher germination index and velocity, these differences were not statistically significant. Inoculation may negatively affect germination, possibly due to microbial competition. Seed pre-treatment and inoculation effects appear to be independent, with no significant interaction.
           
    The results presented in Table 2 showed no significant differences between pre-treatments. The results revealed that germination rates varied from 73.33% to 88.33%, with the highest value recorded in the mechanically scarified seeds (88.33%), followed by untreated seeds (85.00%), rainwater-treated seeds (78.33%) and warm water-soaked seeds (73.33%). The results showed no significant differences between the mean germination time (ranged from 26.37 to 28.15 days). The results displayed slightly shorter mean germination times due to mechanical scarification of seeds, with a germination index of 0.18 and a coefficient of velocity of germination of 3.82, indicating marginally faster germination, with no significant differences.

    Table 2: Effect of different seed pre-treatments and inoculum on germination of Baikiaea plurijuga seeds.


           
    B. plurijuga     seeds inoculated with B. ripae showed significantly (P<0.05) higher germination rates and a higher germination index than non-inoculated seeds (Table 2). Microbial inoculation appears to enhance germination by improving seed health and suppressing pathogens. No interaction was observed between pre-treatment and inoculation in B. plurijuga.
           
    Poor germination, often caused by seed coat hardness in woody species, is common in arid regions and hampers nursery propagation (Mojeremane et al., 2021). This study investigated the impact of seed pre-treatments and inoculation on P. angolensis and B. plurijuga, two ecologically and economically significant species in Botswana. In nature, seed coats are typically broken by fire (Walters et al., 2004). To counter dormancy caused by seed coat hardness and promote faster, more uniform germination, several methods have been suggested (Fredrick et al., 2017; Cañizares et al., 2025; Motbaynor et al., 2025). The study found that seed responses to pre-treatment vary by species and method. All treatments increased germination, with mechanically scarified seeds showing higher rates; however, this difference was not statistically significant. Results for P. angolensis align with studies reporting higher germination in scarified seeds (Chisha-Kasumu et al., 2007; Botsheleng et al., 2014; Tselakgosi, 2021; Latiwa et al., 2023). Similarly, Botsheleng et al. (2014) observed higher but insignificant germination in scarified B. plurijuga seeds. The findings suggest that seed treatment may not be essential for B. plurijuga and P. angolensis; however, the germination rate (speed) is crucial, as slower germination can lead to uneven seedling growth (Chisha-Kasumu et al., 2007). Pre-treatments did not significantly influence mean germination time, germination index, or coefficient of velocity of germination. Scarified seeds showed marginally faster germination, indicating that this process could be expedited.
           
    The benefits of mechanical scarification have been observed in various woody species in Botswana (Koobonye et al., 2018; Botumile et al., 2020; Mojeremane et al., 2021; 2024) and in other regions (Fredrick et al., 2017; Salazar and Ramírez, 2018; Alzandi et al., 2025). However, this method has its limitations. It is physically demanding and labour-intensive, especially when only a small number of seedlings are needed (Todd-Bockarie et al., 1993) and it is not suitable for large-scale seed treatment (Danthu et al., 1992).
           
    Soil inoculants can affect seed germination rates depending on the type of inoculant and the plant species (Balshor et al., 2017; Mawarda et al., 2020). This study shows the different responses of B. plurijuga and P. angolensis seeds to B. ripae. The effects of inoculants on woody seed germination vary depending on the strain used (Lindström and Mousavi, 2020; Mendoza-Suárez et al., 2021). In this case, inoculated P. angolensis seeds had lower germination rates than non-inoculated seeds, although germination metrics remained unchanged. Since P. angolensis naturally forms an effective symbiosis, introducing a non-native strain might have disrupted essential early plant-microbe communication (Bünger et al., 2021). Incompatibility between host and inoculant strains can reduce seed germination and seedling vigour in legumes, especially if the non-native strain fails to form a symbiosis (Allito et al., 2021; Etesami, 2025). Inoculating B. plurijuga significantly improved germination and the germination index, indicating higher rates and more vigorous, uniform sprouting. These results agree with other studies, which demonstrate that inoculants, either alone or in combination, can enhance seed germination in woody plants (Khan et al., 2011, 2014; Singh et al., 2011; Sreedhar and Mohan, 2014; Mridha et al., 2016).

    CONCLUSION

    Seeds of P. angolensis and B. plurijuga can germinate without pre-treatment, but mechanical scarification proved to be the most effective method for both species. It produces the highest, though statistically insignificant, germination rates, with faster germination and better uniformity. This approach is suitable for small seed quantities due to its labour intensity. Inoculation reduced germination in P. angolensis but enhanced it for B. plurijuga. These findings highlight the importance of tailoring propagation strategies to meet the unique biological needs of each species.

    ACKNOWLEDGEMENT

    The authors thank the German Federal Ministry of Education and Research (BMBF) for funding this study. We also appreciate the Southern African Science Service Centre for Climate Change and Adaptive Land Management (SASSCAL) and the Botswana University of Agriculture and Natural Resources as the implementing agencies. Additionally, the authors thank the University of Bremen, Germany, for providing the inoculant.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    All animal procedures for the experiments were approved by the Committee of Experimental Animal and handling techniques were approved by the University of Animal Care Committee. 

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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