A periodical of the Faculty of Natural and Applied Sciences, UMYU, Katsina
ISSN: 2955 – 1145 (print); 2955 – 1153 (online)
ORIGINAL RESEARCH ARTICLE
Rahanatu Adamu Kakudi*1, Tijjani Sabiu Imam2, Hauwa Bah Ibrahim1, Safiyya Balarabe1, Abdulmutallib Isah Muhammad3, and Ismail Adamu Kakudi4
1Department of Integrated Science, Federal University of Education, Kano, Nigeria.
2Biological Science Department, Federal University of Education, Kano, P.M.B 3045, Kano State, Nigeria
3Centre for Dryland Agriculture, Bayero University, Kano, Nigeria.
4Department of Animal Health and Technology, Audu Bako College of Agriculture.
Corresponding Author: kakudirayhan@gmail.com
Prosopis africana, a valuable tree species in West Africa, is the only species of Prosopis that is indigenous to tropical Africa. Due to extensive overexploitation, this species has become extinct in many parts of the southern Sahel and the adjacent Sudan savannahs. In the first experiment, scarified seeds were surface sterilized with 10% and 20% NaOCl solution for 10 and 20 minutes. In the second experiment, seeds were soaked in 1% antifungal solution for 30 and 60 minutes before final sterilization with 10% and 20% NaOCl. Treating seeds with 10% and 20% NaOCl alone for 10 and 20 minutes resulted in very low contamination levels of 5.0 ± 0.04 and 2.5 ± 0.03, respectively, with no significant difference between the treatments at p < 0.05. Seed germination was influenced by treatments, with significant differences at p < 0.05, where the highest germination was obtained with 20% NaOCL for 10 minutes (70.0 ± 13.00). In the second experiment, the lowest contamination (20% ± 2.41) and the highest germination (50% ± 14.1) were recorded for seeds treated with 10% NaOCL + 1% antifungal for 30 min. The best sterilization treatment was 20% NaOCL for 10 minutes, which had the highest germination and one of the lowest contamination percentages. Cocopeat can be successfully used as a low-cost medium to generate sterile explants of P. africana, which can be used for micropropagation and genetic improvement of the species. This can be used for the conservation of the threatened tree in Nigeria.
Key words: Prosopis africana, Sterilization, Sodium Hypochlorite, Germination, micropropagation
Cocopeat can be used as a substitute for an expensive gelling agent to generate sterile explants for plant tissue culture.
P. africana explants can be generated in vitro from seeds.
Sterile explants of P. africana can be generated for micropropagation and genetic modification of the tree species.
Prosopis africana, a valuable tree species in West Africa, is the only species of Prosopis that is indigenous to tropical Africa (Asoiro and Ohagwu 2017). It is highly valued for its wood, which is sought after for firewood, charcoal, and other essential products, as well as for its environmental services. Prosopis africana enriches the soil by fixing nitrogen; its leaves are rich in protein, and sugar pods are used as foodstuffs for feeding ruminants in Nigeria (Ancha et al., 2020; Annongu et al., 2004). However, the species is facing degradation and regeneration problems due to over-exploitation (Bello et al., 2019). Ekpo et al. (2021) stated that P. africana is among the most endangered species due to commercial charcoal production. Despite its numerous benefits, the P. africana is listed as “Least Concern” on the IUCN Red List but locally it has been classified as critically threatened (Bello et al., 2019; Danjuma & Abubakar, 2017; Nodza et al., 2024). Due to extensive overexploitation, this species has disappeared from extensive parts of the southern Sahel and the adjacent Sudan savannahs (Agboola, 2004). There are several reports on the in vitro propagation of Prosopis species (Rivera et al., 2020; Haroon et al., 2018; Morales-Domínguez et al., 2019; Ortiz-Robles et al., 2022), but no work has been reported yet for P. africana. Excessive falling, in conjunction with a slow growth rate and difficulty in seed germination, prevents trees from multiplying in considerable numbers in the wild. Biotechnological techniques are widely used in conservation practices, with micropropagation emerging as a suitable tool for in vitro conservation purposes. However, plant tissue culture cannot be achieved without establishing a sterilization protocol, as the success of the process depends on it. Another factor to consider in tissue culture is cost; a cheap yet efficient alternative is crucial for tissue culture, especially in developing countries like Nigeria. Several media (Murashige and Skoog Media, Woody Plant Media, White’s Media, etc.) are used in conjunction with gelling agents as substrates for in vitro plant growth, which is costly. The use of cocopeat as an alternative medium to generate sterile explants will reduce cost. Having excellent air permeability, water-holding and nutrient-holding capabilities, low bulk density, high cation exchange capacity, and high expansion up to four or five times in volume when combined with water are some of the key attributes of coco peat, the last attribute contributing more to its economic value (Kim & Yoo, 2024). The first step in establishing plant tissue-cultured plants is to develop a sterilization protocol to produce aseptic explants. This research is the first to aim at developing a sterilization protocol and cost-effective media for the in vitro generation of sterile explants, which can be used in the micropropagation and conservation of P. africana.
Samples were collected from Yobe state at Dazigaw village in Nangere Local Government Area of Yobe State, North-eastern Nigeria at latitudes of 11̊42’44” N and Longitudes of 11̊ 0’ 33” E. The experiment was conducted at the Plant Tissue Culture Laboratory of the Centre for Dryland Agriculture, Bayero University, Kano, Nigeria.
Seeds were scarified in 98% Sulfuric acid (H2SO4, Sigma Aldrich) for 20 minutes, then washed under running tap water until no traces of acid were left (Dera et al., 2019) with modification. Seeds were then soaked for 48 hours for water imbibition.
Cocopeat was soaked in water and drained, then fifteen grams (15g) was weighed into test tubes (50ml) and sterilized by autoclaving at 121 °C and 103.4 kPa pressure for 20 minutes.
In the first sterilization method, soaked seeds were surface sterilized in the laminar flow hood by stirring in 10 and 20% (v/v) NaOCl solution (3.5% Jik, Uniliver) with 2 drops of liquid soap (Morning fresh Uniliver) for 10 and 20 minutes, then rinsed several times with sterile distilled water to remove all traces of NaOCl.
In the second setup, seeds were pretreated with 1% w/v antifungal (SAAF Carbendazim 12% + Mancozeb 63%) for 30 and 60 mins then then soaked for 48 hours. After soaking, they were sterilized in a laminar flow hood by shaking in 20% NaOCl with one drop of liquid soap for 20 minutes, followed by several rinses with sterile distilled water. The sterilized seeds were inoculated on sterilized coconut coir (cocopeat) and kept in a dark room for germination. Seeds that germinated were removed from the dark room and incubated at 28 ± 2 °C and in the light (16 h light /8 h dark) for seedling growth.
Germination, defined as the emergence of the radicle from the seed coat, was recorded every week after the first radicle emerged. Final germination was evaluated after 60 days for all germination experiments.
Final germination percentage (FG) was calculated with the following equations (NASR et al., 2013):
FG = n/N × 100 (Where n is the number of germinated seeds, N is the total number of seeds).
The experiments were laid out in a completely randomized design (CRD) with 20 explants per treatment, making a total of 160 seeds. Each treatment consisted of an average of 2 replicates, with 10 seeds in each replicate. The number of contaminated cultures after sterilization was recorded 2 days after culturing and continued weekly for 8 weeks.
One-way Analysis of Variance (ANOVA) was used to test the statistical significance, and the significance differences among means were carried out using the Least Significant Difference (LSD) at a 5% probability. All results were analyzed using Microsoft Excel software version 2016.
Generating aseptic explants is the most important step in tissue culture studies. The use of explants from mature plants growing in the field is often difficult or even impossible, mainly due to the high level of contamination (Bonga et al., 2010; Kirika et al., 2015). Therefore, sterile explants need to be produced in vitro using the most suitable sterilization protocol. Contamination control is mostly determined by the disinfectant used and the duration of exposure. Only microbial contamination should be removed during sterilisation; living material shouldn't lose its biological activity. Consequently, the plant material needs to be surface sterilized using a disinfectant for a predetermined amount of time and at an appropriate concentration (Anđelić et al., 2024). Contamination can be external, resulting from laboratories and used materials (media, glassware, culture vessels, tools, and explants), whereas it can also be internal, related to the endophytic microbes in mother plants (Abdalla et al., 2020). It is easier to control external contamination, but very difficult when contamination is endophytic and can lead to an increase in the cost of producing explants. The high costs of equipment, water, chemicals (such as plant growth hormones or regulators, surface sterilants, disinfectants, etc.), and culture losses due to in vitro culture contamination, among other factors, can cripple several in vitro plant culture techniques in many developing countries, especially in Africa. Possibly, this is the main reason we have limited or no interest in investing in tissue culture laboratories in Africa, except for academic institutions, which use them solely for learning the technique (Mng'omba et al., 2012). Several chemicals have been used to generate aseptic explants, including mercuric chloride, calcium hypochlorite, sodium hypochlorite, ethanol, antibiotics, and fungicides (Chornobrov & Tkachova, 2021; Payamnour et al., 2014), with high success rates.
Even though the concentration of sterilant is very important when establishing aseptic cultures. Table 1 shows the contamination and germination percentages of P. africana seeds following different treatments with NaOCl. The lowest contamination (2.5% ± 0.03) was recorded when seeds were immersed in 10% and 20% NaOCl for 20 minutes. Similarly, the highest contamination (5.0% ± 0.04) was observed with 10 % and 20% NaOCL immersion for 10 minutes. The concentration of sterilant and exposure time of seeds were not significantly different at (p<0.05). In a Procedure to establish aseptic seedlings of Zehneria capillacea, sterile seedlings were obtained using 20-100% NaOCl, with no difference in contamination percentages (Agogbua et al., 2022). Sometimes higher concentrations require optimized exposure time (Connell et al., 2016). If the treatment was too brief or too prolonged, microbial cells could survive, especially in micropores or crevices that strong NaOCl cannot fully reach (Rozman & Doull, 2000). However, germination was influenced by concentration and exposure time, with significant differences even though not across all treatments. Treatment concentration of 20% NaOCL resulted in higher germination percentages (70.0% ± 0.07 and 67.5% ± 0.07) compared to 10%. This record indicated a high germination occurrence compared to 40.0 ± 2.46 and 52.5 ± 6.50 recorded for treatments with 10% NaOCl for 10 and 20 minutes, respectively (Table 1 .0). The difference is statistically significant at (p<0.05). A high concentration of sterilant, with lower contamination and higher germination percentages, has been reported in other works. A similar result was reported by Maina et al. (2010), recording (40%) survival using the lower concentration of NaOCl (0.525%) and a higher survival percentage of 66% corresponding to the higher concentration of 1.050% NaOCl in ICGV-12991 groundnut variety.
This result is in conformity with other seeds of various plants which have been successfully sterilized with 10% or 20% NAOCl: Parkia biglobosa (Abbas et al., 2018), Ficus religiosa (Hesami et al., 2017), Prosopis laevigata (Morales et al., 2019), Khaya senegalensis (Hung and Trueman, 2011), and Boscia senegalensis (Khalafalla et al., 2011). Treating the seeds with 20% NAOCl for 10 minutes and 20 minutes did not show a statistically significant difference in the germination percentage of P. africana seeds. Therefore, a 20% NAOCl solution was used for 20 minutes throughout the research. Healthy seedlings were produced, which could be used as explants for micropropagation of P. africana (Plate 1).
Table 1.0: Effect of NAOHCL Treatment on Sterilization and Germination of P. africana Seeds
| Treatment | Contamination% | Germination % |
|---|---|---|
| 10% NaOHCL 10min | 5.0 ± 0.04 a | 40.0 ± 5.10 a |
| 10% NaOHCL 20min | 2.5 ± 0.03 a | 52.5 ± 7.08 ab |
| 20% NaOHCL 10min | 5.0 ± 0.04 a | 70.0 ± 13.00 b |
| 20% NaOHCL 20min | 2.5 ± 0.03 a | 67.5 ± 11.13 b |
Values represent means ±SD. Means followed by the same letter within columns are not significantly different (P < 0.05) using LSD.
The need to incorporate an antifungal often arises when dealing with contamination from various sources (Kakade and Nutan 2017). In Table 2.0, records show a contamination percentage of 20 ± 2.41% and 25 ± 4.20% for 10% NaOCL + Antifungal after 30 minutes. Using 10% NaOCl + Antifungal for 60 minutes and 20% NaOCl + Antifungal for 30 minutes had the same effect (25% ± 4.20), with no statistical difference between the three treatments. Similarly, sterilizing seeds of Parkia biglobosa (Fabaceae) with 30% NaOCl and 60% NaOCl all resulted in 50% contamination percentages and a survival percentage of 25% and 37%, respectively (Abbas et al., 2018). Kirika et al. (2015) also found 10% NaOCl to be the most effective sterilizing agent, yielding 55% clean seeds compared to 50% clean seeds with a 30% NaOCl treatment. Contrary to the efficiency of the sterilant used at high concentration and longer exposure time, 20% NaOCl + 1% antifungal for 60 minutes resulted in the highest contamination and a significant difference at p < 0.05. This could be explained by extended exposure to antifungal drugs, which may weaken seed defenses or reduce beneficial microbial populations, thereby facilitating the growth of opportunistic pathogens after treatment. High amounts of NaOCl may be phytotoxic. This might damage the outer seed coat or compromise the integrity of plant tissue. During sterilisation, damaged seeds may have been injected undetected, leaving them more susceptible to microbial invasion and subsequent contamination (Carmello & Cardoso, 2018). Both during culture initiation and from outside sources, contamination may be introduced. Germination, although higher at lower treatment levels, was not statistically different. A similar result was obtained by Silveira et al. (2022). Reports have shown that high levels of sterilization, which correspond to low contamination rates, usually result in low germination due to the inhibitory action of the chemicals in the sterilants (Akin et al., 2014; Hesami et al., 2017). Similar results obtained in this research also confirm that different concentrations and treatment durations of sodium hypochlorite did not affect in vitro germination of Zehneria capillacea seeds (Agogbua and Okoli 2022).
Table 2.0: Effect of NaOHCL and Antifungal on Sterilization and Germination of P. africana Seeds
| Treatment | Contamination (%) | Germination (%) |
|---|---|---|
| 10% NaOCL + 1% FUNGICIDE 30MINS | 20 ± 2.41a | 50 ± 14.1 a |
| 10% NaOCL + 1% FUNGICIDE 60MINS | 25 ± 4.20 a | 30 ± 9.11a |
| 20% NaOCL + 1% FUNGICIDE 30 MIN | 25 ± 4.20a | 35 ± 8.11a |
| 20% NaOCL + 1% FUNGICIDE 60 MIN | 60 ± 12.33b | 25 ± 5.10a |
Values represent means ±SD. Means followed by the same letter within columns are not significantly different (P < 0.05) using LSD.
Plate 1: Sterile P. africana seedlings generated on cocopeat
For the first time, an effective sterilization protocol was developed for P. africana. A 10% NaOCl treatment for 20 minutes resulted in a low contamination percentage of 5% and a high germination percentage of 70%, making it the most suitable treatment. Exposure time and concentration of NaOCl did not affect the contamination percentage but had a significant effect on germination percentage. Adding an antifungal to NaOCl resulted in a higher contamination percentage compared to NaOCl alone in this experiment. Sterile explants of P. africana can be successfully generated on low-cost cocopeat as a new medium to produce sterile explants. This research introduces a technique that can be utilized for the conservation of P. Africana. Future research should focus on multiplication using cocopeat-grown explants and rooting to establish a micropropagation protocol for the species, including the assessment of genetic stability in vitro seedlings and genetic improvement of the plant. Additionally, the isolation of fungi can help identify the best antifungal treatment.
We appreciate the financial support given by the Nigerian Tertiary Education Trust Fund (TETFUND). We also thank the Centre for Dryland Agriculture at Bayero University, Kano, Nigeria, for providing the necessary support to accomplish this research.
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