Optimization of Surface Sterilization Protocol for in Vitro Initiation of African Star Grass (Hypoxis Schimperi)

The micropropagation of African star grass (Hypoxis schimperi) is often hindered by microbial contamination, necessitating an optimized surface sterilization protocol for effective tissue culture initiation. This study investigated the effects of different concentrations and exposure times to sodium hypochlorite (NaOCl) on the initiation of Hypoxis schimperi corm explants. Experiments were conducted at the Egerton University Tissue Culture Laboratory. The study used a completely randomized design (CRD) with factorial treatment combinations of concentration and exposure time replicated thrice. Data were analyzed using SAS (version 9.4) and Microsoft Excel. Corm explants were collected from Njombe-Tanzania, incubated in a growth chamber at 28°C ± 2°C, and exposed to artificial illumination of 2000–2500 lx for 16 h daily over 15 days. Sodium hypochlorite (1%, 2%, 3%, and 4%) was tested at varying exposure times 10 and 15 min each. The results indicated that the highest contamination rate (93.3%) was observed when explants treated with 1% NaOCl for 10 min, whereas the lowest contamination rate (46.7%) occurred in explants treated with 4% NaOCl for 15 min. The optimal treatment was determined to be 4% NaOCl for 15 min, which yielded a survival rate of 53.3%. This optimized protocol offers a reliable foundation for initiating micropropagation of Hypoxis. schimperi, supporting its conservation and potential for large-scale propagation in medicinal plant biotechnological programs.

Introduction

Hypoxis schimperi Baker belongs to the Hypoxidaceae family. This species is commonly known as African star grass, which is a slender, herbaceous perennial plant that produces a cluster of grass-like leaves from a corm-like tuber [1]. Hypoxis species are associated with medicinal properties and have been used for many generations by indigenous people and traditional healers to treat various ailments, as well as food [2]. The dried, powdered corm is mixed with hot water or tea and drunk as a treatment for coughs and convulsions in children, and as an aphrodisiac in males [3]. Traditionally, Hypoxis species are regenerated using seeds that take a long time to germinate because they seem to have strong seed coats and deep dormancy [4]. An efficient in-vitro propagation protocol is the most essential and pragmatic alternative method for the production of large-scale Hypoxis schimperi plantlets throughout the year, aiming for their conservation and sustainable utilization [5]. A successful in-vitro regeneration protocol begins with effective explant sterilization. Maintaining aseptic conditions is essential for successful tissue culture procedures [6]. Unfortunately, contamination is a serious universal problem in tissue culture studies, and is experienced in all tissue culture laboratories [7]. Some contaminants are visible, including bacteria, yeast, and fungi; others are hidden or cryptic and are often difficult to detect, causing serious problems [8]. The aim of every researcher in tissue culture studies is to eliminate or prevent contamination. Unfortunately, contamination cannot be eliminated completely but can only be managed to reduce both the frequency of occurrence and the seriousness of its consequences, and this can be achieved through chemical sterilization [9]. Hence, it is important to determine the appropriate concentration of the sterilant and exposure time to standardize the sequence of sterilant use to minimize explant injury. Unfortunately, to date, limited information exists on surface sterilization protocols for H. schimperi. This is crucial because materials obtained from ex vitro environments contain high levels of microorganism contamination [10]. This study established the most viable and effective protocol for surface sterilization of Hypoxis schimperi Baker using corm explants to facilitate in vitro propagation.

Materials and Methods

Study Area

This study was conducted at the Egerton University Tissue Culture Laboratory, Njoro-Kenya, located at 0°22^′11.0″ S, 35°55′ 58.0″ E (latitude: −0.369734; longitude: 35.932779). The laboratory recorded maximum and minimum temperatures of 22°C and 17°C. The experimental design was Completely Randomized (CRD) with three replicates per treatment.

Plant Materials

Young corms of Hypoxis schimperi were collected from Njombe-Tanzania. Corms were used as the source of explants collected from the wild and transported to the Biotechnology and Molecular Laboratory at Egerton University Njoro-Kenya for explant preparation for tissue culture experiments.

Media Preparation

Murashige and Skoog [11] powder was used for medium preparation according to [12]. To prepare a Liter of MS media, 4.4 g of MS powder were measured using a fine electronic balance and added to approximately 800 ml of distilled water in a beaker, followed by the addition of 30 g of sucrose. The Beaker was then placed in a magnetic stirrer and stirred until the solution was dissolved. The solution was poured into a measuring cylinder, topped to 1000 ml using distilled water, and then transferred to a beaker. The pH of the solution was measured using a magnetic stirrer and adjusted to pH 5.8, using 0.1N NaOH and 0.1N HCl solutions. Additionally, using an electronic balance, 8 g of gerlite powder was added to a beaker containing the solution to complete the media. The medium was poured into sterilized Manson jars under aseptic conditions. The Manson jars containing media were autoclaved at 121°C and 15 psi for 20 min. Finally, the media was cooled and maintained properly with a temperature of 25°C ± 2°C and photoperiod of 16 h light and 8 h dark with a light intensity of 2000 lx and relative humidity between 50%–60%.

Preparation and Sterilization of Explants

Explants were taken from the corms, peeled using a sterilized knife, and sliced to a size between (1 cm and 2 cm). Surface sterilization of explants was initiated by washing the corms explants with running tap water for 30 min to reduce the load of contaminants. The explants were soaked in the liquid detergent Tween 20 for 20 min with vigorous shaking using a rotary shaker. The explants were washed with distilled water five times to remove traces of detergent. Followed by explants treated with 0.2% Carbendazim solution (Antifungal) for 30 min, then explants were soaked in 70% ethanol for 1 min, followed by various concentrations of sodium hypochlorite solutions (1%, 2%, 3% and 4%) (Table I) and exposure durations of 10 and 15 min each. The explants were then trimmed under a laminar flow hood and rinsed four times in sterile distilled water after sterilization to remove any lingering sterilant [13]. Finally, the explants were inoculated into Manson jars containing media, with each jar containing only one explant. Manson jars containing explants were kept in a growth chamber at a light intensity of 1500 lx from white fluorescent light and 16-hour photoperiod. The room temperature was set at 25°C ± 14°C. The cultures were continuously examined for 10–15 days after inoculation to determine the aseptic and survival percentages. After the examination, cultures with no signs of microbial infection were considered for culture establishment.

Table I. Different Sterilization Treatments on Corms Explants of Hypoxis schimperi

Treatments	Concentration	Exposure time
Control	Distilled water	15 min
T1	1% NaOCl	10 min
T2	1% NaOCl	15 min
T3	2% NaOCl	10 min
T4	2% NaOCl	15 min
T5	3% NaOCl	10 min
T6	3% NaOCl	15 min
T7	4% NaOCl	10 min
T8	4% NaOCl	15 min

Data Analysis

Observation was made on the number of contaminated explants, number of clean and surviving explants, and type of contaminating microorganisms. Data obtained were analyzed statistically by Descriptive Statistics method using Microsoft Excel computer applications and SAS software (version 9.4), and least significant difference (LSD) test was used to separate the means where required at a probability level of 5%. Results are expressed as mean ± standard error (S.E). In addition, qualitative data, such as the visual performance of explants, were captured and presented in the form of an image to support quantitative data:

$$\begin{array}{rl}& PercentageofSurvivalExplants\\ & =\frac{NumberofSurvivalexplants}{TotalnumberofExplantsCultured}\times 100\mathrm{\%}\end{array}$$

$$\begin{array}{rl}& PercentageofContaminatedExplant\\ & =\frac{NumberofContaminatedExplant}{TotalnumberofExplants}\times 100\mathrm{\%}\end{array}$$

Results

These results highlight the impact of different treatments (T1 to T8) on aseptic conditions. The control group (T0) exhibited 100% contamination, clearly demonstrating the critical importance of surface sterilization in plant tissue cultures. As treatment levels increased from T1 to T8, a progressive decline in contamination rate was observed from 93.3% to 46.7%, with a corresponding increase in the survival rate from 6.7% to 53.3% as shown in (Fig. 1). The treatments demonstrated a dose-response effect, where higher treatment levels (T5–T8) showed superior performance compared to lower treatment levels (T1–T4). These findings suggest that the treatments effectively controlled microbial contamination, either by reducing their viability or by promoting competitive exclusion by the desired culture.

Fig. 1. A graph showing the effects of Treatments on contamination and survival rate of African star grass (Hypoxis schimperi) corm explants.

In this study, treatments T1 and T2 showed slight reduction in contamination rate to 93.3% and 86.7%, respectively, whereas survival rates modestly increased from 6.7% to 13.3%. Although these rates were significantly better than those of the control group, they remained suboptimal. A more substantial improvement was observed in T3 (80% contamination rate, 20% survival rate) and T4 (73.3% contamination rate, 26.7% survival rate). With contamination dropping to 66.7% (T5) and 60% (T6) and survival increasing to 33.3% and 40%, respectively, these treatments marked a significant turning point. This trend indicates that a more optimized sterilization environment is sufficient to eliminate most pathogens while maintaining explant viability. T7 and T8 represent the most successful protocols tested, showing a contamination rates of 53.3% and 46.7%, and the highest survival rates of 46.7% (T7) and 53.3% (T8).

The differences in the rate of explant contamination owing to the combined effects of both concentrations of the sterilizing agent and exposure time to the sterilant chemicals were highly significant (P < 0.001) (Table II). The highest loss of cultured explants (93.3% and 86.7%) due to microbial contamination was recorded when explants were treated for 10 min and 15 min, respectively, with the sodium hypochlorite solution containing 1% active chlorine (Table I).

Table II. Mean Square Values for the Effect of Different Concentrations of Sodium Hypochlorite and Exposure Time on Contamination Rate of African Star Grass Explants ^** = P < 0.05

Source of variation	Mean square
	Degree of freedom	Contamination
Chemical Concentration	3	1705.56**
Exposure time	1	150.00**
Concentration * Exposure time	3	16.67**

The relationship between exposure time to sterilizing agents and its effects on both the contamination and survival rates of Hypoxis schimperi explants is shown in (Fig. 2). The graphs reveal a clear inverse relationship between the contamination rate and exposure time; as the exposure time increased, the contamination rate decreased significantly. The results revealed a significant increase in both the survival and contamination rates with an increase in the treatment level from 10 to 15. At treatment level 10 min, the survival rate was relatively low at 26.675%, whereas the contamination rate stood at 33.325%. Upon increasing the treatment to level 15 min, the survival rate increased markedly to 73.325%, indicating enhanced efficacy or improved conditions for survival with increasing of exposure time.

Fig. 2. A graph showing the effect of exposure time on the contamination and survival rate of Hypoxis schimperi corms explants.

A general reduction trend in contamination rate of African star grass explants (from 93.33% to 46.67%) was observed with increasing concentrations of the sodium hypochlorite solution from 1% to 4% active chlorine, together with a concomitant increase in exposure time from 10 min to 15 min (Table III). In this experiment the highest the highest contamination free (53.3%) of explants were observed from a treatment that involved a 15 min sterilization of corms explants with 4% sodium hypochlorite solution, followed by a 10 min treatment of explants with a similar level of active chlorine concentration. In most cases, sodium hypochlorite did not exert any adverse effects on the explants; thus, the surviving explants from the respective treatments were actively growing on the initiation media.

Table III. Effect of Sterilant Chemical Concentrations and Durations of Treatment Exposure Time on the Contamination Rates of Africa Star Grass Explants After 2 Weeks of in vitro Culture

Treatment	Sodium hypochlorite (%)	Exposure time (min)	Contamination rate (Mean ± S.E)
Control	Distilled water	15	100.00 ± 0.02a
T1	1%	10	93.33 ± 0.07a
T2	1%	15	86.67 ± 0.09b
T3	2%	10	80.00 ± 0.11bc
T4	2%	15	73.33 ± 0.12c
T5	3%	10	66.67 ± 0.13d
T6	3%	15	60.00 ± 0.13de
T7	4%	10	53.33 ± 0.13e
T8	4%	15	46.67 ± 0.13f

Fungi and bacterial contaminants were readily identifiable by the naked eye because of the visible symptoms produced in the culture medium. Fungal contamination was particularly noticeable and characterized by a fuzzy texture and a variety of colors resulting from mycelial growth.

Overall, fungi were the predominant source of contamination, accounting for approximately 73.93% of the cases (Fig 3), whereas bacterial contamination was relatively rare, accounting for only approximately 26.02% of total contamination. The fungal colonies appeared white in color (Fig. 4). In all instances, the microbial growth was more aggressive and extensive than that of the explants, especially when lower concentrations of active chlorine and/or shorter sterilant exposure times were used.

Fig. 3. The mean percentage of Fungi and Bacteria contamination rate.

Fig. 4. Contamination types observed on in vitro culture initiation of African star grass corms explants: (a) Bacteria contaminants on cultured corms explants, (b) and (c) Different colors of fungi contaminants.

Discussion

The presented results evaluated the effectiveness of sodium hypochlorite treatments by analyzing contamination and survival rates across eight treatments (T1 to T8) compared to a control group because the creation of aseptic cultures is a crucial stage in the in vitro propagation process, as highlighted by Lal et al. [13]. The trends observed in the results clearly demonstrate that increasing both the concentration of sodium hypochlorite and the exposure time significantly reduces contamination rates while influencing survival rates in a nonlinear pattern. The control group showed the highest contamination and no survival, confirming the importance of disinfection to remove microbial contaminants, as supported by Babu et al. [14], and providing a baseline for assessing other treatments. Contamination rates decreased from T1 to T8, with lower concentration treatments (T1–T3) proving less effective due to lower disinfectant concentration and shorter exposure time compared to the treatments with lower concentrations and longer exposure times. The reduced survival rates at T1 (6.7%) and T2 (13.3%) were most likely due to inadequate disinfection, leading to ineffective microbial control." Improved outcomes from T4 to T6 suggest optimized sterilization that preserves tissue viability, which is in agreement with Gammoudi et al. [15], who showed the importance of optimizing sterilization protocols to reduce contamination without damaging regenerative capacity. T7 and T8, with 4% NaOCl for 10 min and 15 min, showed the lowest contamination rates, confirming the effectiveness of higher concentrations and adequate exposure time in minimizing microbes without damaging the explants [16]. The results showed that increased concentrations of sodium hypochlorite and exposure time reduced the contamination rate of explants, which is in agreement with Hashim et al. [17], who reported that microbial contaminants were reduced in Clinacanthus nutans, with a slight increase in concentration and exposure time. Approximately 73.98% of the total contamination was attributed to fungal species, potentially as a result of environmental factors conducive to their growth [18]. A high level of fungal contamination suggests favorable environmental conditions and insufficient antifungal sanitation [19]. Fungi are particularly problematic in in vitro culture environments, where their spores can rapidly colonize media surfaces and outcompete explants for nutrients. Previous studies have demonstrated that high humidity and organic matter create ideal conditions for fungal growth [20]. Bacterial contaminants accounted for 26.02% of total contamination, significantly lower than fungal contamination and was still notable. Unlike fungi, bacterial contamination likely originated from within plant tissues, as the explants were sourced from field-grown plants [21]. This indicates that although surface sterilization can eliminate external microbes, it may not be sufficient to remove internalized bacteria, which can persist and multiply during culture. Therefore, according to Tewelde et al. [22], addressing bacterial contamination requires additional strategies, including the careful use of antibiotics such as streptomycin or gentamicin in the culture medium, considering the potential risk of phytotoxic effects.

Conclusion

This study successfully optimized a surface sterilization protocol for the micropropagation of Hypoxis schimperi, addressing the challenge of microbial contamination during tissue culture initiation. The results demonstrated that a 4% sodium hypochlorite solution for 15 min effectively minimized contamination (46.7%), while maintaining a relatively high explant survival rate (53.3%). This optimized sterilization protocol is crucial for the successful initiation of tissue culture in H. schimperi and lays a solid foundation for its conservation and large-scale propagation in medicinal plant biotechnology programs. These findings contribute to the enhancement of the viability of H. schimperi for future biotechnological applications.

Recommendation

Based on the results of the optimized surface sterilization protocol, further research should aim to improve this protocol, particularly the use of sodium hypochlorite. Refinement of the 4% sodium hypochlorite treatment in terms of concentration, exposure time, and supplementary sterilizing agents could lead to lower contamination rates. Additionally, alternative cost-effective sterilants should be explored.