Micropropagation of Gapi Banana (Musa sp.) using Aloe vera Gel Juice and Vigna radiata Sprout Extract as Natural Plant Growth Regulators Sources

2.1. Research Place and Time

The research was conducted at the Plant Tissue Culture Laboratory, Faculty of Agriculture, Universitas Negeri Gorontalo, Province of Gorontalo, Indonesia, from July to December 2024.

2.2. Plant and Treatment Materials

The primary materials used in this study included Gapi banana suckers, Aloe vera gel juice, Vigna radiata sprout extract, BAP, and 2,4-D plant growth regulators (PGRs). Due to the limited availability of Gapi banana suckers as plant material for explants, a total of 26 explants (banana suckers) were used in this study, with two replications. This number excluded any explants that failed during the initial establishment phase.

2.3. Research Experimental Design and Treatments Media

The experiment was conducted under laboratory conditions using a Completely Randomized Design (CRD) consisting of 13 treatment combinations. The treatments were formulated using Murashige and Skoog (MS) basal media supplemented with Aloe vera gel juice (MS-LB) and Vigna radiata sprout extract (MS-ET) as natural plant growth regulator sources. These treatments were compared to control media, including MS without additives (MS-0), MS supplemented with 1 mg L^-1 BAP (MS-BAP), and MS containing a combination of BAP (1 mg L^-1) and 2,4-D at concentrations of 0.1, 0.3, and 0.5 1 mg L^-1 (MS-BAP-2,4-D).

In the MS-LB treatments, the medium was supplemented with Aloe vera gel juice at concentrations of 10,15, 20, and 25% (v/v), while the MS-ET treatments contained Vigna radiata sprout extract at concentrations of 10, 15, 20, and 25% (v/v). Each treatment was replicated twice due to the limited number of available explants (banana suckers). Data collection was carried out through qualitative observations, including contamination occurrence, degree of browning, and regeneration responses such as shoot and callus formation.

2.4. Preparation of Control and Treatment Media

The control and treatment media were prepared using Murashige and Skoog (MS) basal medium. The composition of the MS basal medium per liter included MS mineral nutrients (from Himedia) 4.43 g, sucrose 3% (w/v), myo-inositol 10 mg L^-1, and distilled water up to 1000 mL, with the pH adjusted to 5.8.

Aloe vera gel juice was obtained through the following procedure: the gel was separated from the skin and soaked in water overnight. It was then rinsed up to five times, drained, and blended until smooth. It was then passed through a coarse sieve to remove fibrous residua. The resulting gel juice was considered a crude extract and was directly used as a supplement in the Murashige and Skoog (MS) medium without further purification. Vigna radiata sprouts purchased from a local supplier were boiled with distilled water in a 1:1 ratio (w/v) until boiling. The resulting boiling water extract was collected and used directly as a crude supplement in MS medium without further purification. The filtration of Vigna radiata sprout extract was performed solely to separate the liquid fraction from the sprout residues.

Aloe vera gel juice was added to the MS medium for the MS-LB treatment media at concentrations of 10, 15, 20, and 25% (v/v). Vigna radiata sprout extract was added to the MS medium for the MS-ET treatment media at concentrations of 10, 15, 20, and 25% (v/v).

As a comparison, MS-0 medium was used as the control, along with MS medium supplemented with BAP 1 mg L^-1 and a combination of BAP 1 mg L^-1 with 2,4-D at concentrations of 0.1 and 0.3. and 0.5 mg L^-1. Finally, 5 g L^-1 of micropropagation agar was added. After thoroughly mixing all the ingredients, the solution was heated to dissolve the agar. The prepared medium was then distributed into culture bottles and sterilized using an autoclave at 121°C under 115 kPa pressure for 20 minutes.

2.5. Initiation and Sterilization of Explants

The explants were obtained from Gapi banana suckers, cleaned of any adhering soil, and the corm (pseudostem) was removed. The explants were then washed with a detergent solution while being brushed and rinsed with water until completely clean. The rhizome section was trimmed to approximately an adult's thumb size.

The sterilization process was conducted inside a laminar airflow cabinet (LAF) using a 20% (v/v) disinfectant solution for 20 minutes, followed by a 10% (v/v) disinfectant solution for 15 minutes. After each sterilization treatment, the explants were rinsed three times with sterile distilled water. The sterilized explants were then trimmed to a size of approximately 1 cm², forming a small stupa-like structure. The explants were then divided into two equal parts.

2.6. Inoculation and Incubation of Explants

After preparation, the Gapi banana explants were inoculated into the respective treatment media inside culture bottles. Due to the limited availability of explant sources, only two culture bottles were prepared for each treatment. Each bottle contains one explant. The culture bottles were then placed in an incubation room at a temperature of 25 ± 2°C, under LED light (9 watts), with a photoperiod of 16 hours of light and 8 hours of dark. Each month, subculturing was performed into fresh medium treatments.

2.7. Observations and Data Analysis

All data collected from culture observations were analyzed both qualitatively and statistically. Observations were recorded at one-month intervals, focused primarily on the percentage of contamination, the percentage of browning, and explant growth responses, specifically whether shoots/roots, calluses were formed in the 1st, 2nd, and 3rd months.

2.7.1. Contamination

The percentage of contamination was calculated to evaluate the frequency of microbial infection during culture initiation. Contamination (%) was determined using the following formula:

The calculation clarifies how values such as 0% contamination were obtained in the experimental results.

2.7.2. Browning

Browning was recorded as the progressive darkening of Gapi banana explants, beginning at the cut surfaces, and caused by phenolic oxidation. In this study, browning was defined qualitatively based on the intensity and spread of discoloration: mild browning (localized at the cut edge) indicated viability, whereas extensive browning across most tissue indicated severe damage. Observations were compared across Aloe vera, Vigna radiata, and synthetic PGR treatments to assess their effects on reducing oxidative stress and maintaining explant viability. Browning (%) was determined using the following formula:

(1)

3.1. Results

3.1.2. Culture Condition in The Micropropagation of Gapi Banana (1st, 2nd, and 3rd Month)

The culture responses of Gapi banana explants differed among treatments during the three months of observation (Table 3). In the control (MS-0) medium, explants remained dormant with no visible growth throughout the culture period (Fig. 1).

In the MS-LB treatment (10 to 25% (v/v)) (Fig. 2), the explant initially blackened during the first month but began to exhibit growth in the second and third months in some treatments. MS-LB 10% and 15% (v/v) started to show either shoot or root growth in the second month, which later developed into shoots in the third month. The MS-LB 20% (v/v) treatment exhibited the best growth, with shoot formation in the second month and the development of a complete plantlet in the third month. Conversely, MS-LB 25% (v/v) failed to develop throughout the three months, indicating that a higher concentration (25% v/v) of Aloe vera gel juice did not optimally support the growth of Gapi banana explants.

Table 3.

S.No.	Culture Media Treatment	Culture Condition in the 1st Month	Culture Condition in the 2nd Month	Culture Condition in the 3rd Month
1.	MS-0 (Control)	Not growing	Not growing	Not growing
2.	MS-LB 10%	Blackened	Shoots grow	Potential shoots
3.	MS-LB 15%	Blackened	Roots grow and appear green at one end of the inoculant	shoots
4.	MS-LB 20%	Blackened	Shoots grow	Plantlets
5.	MS-LB 25%	Blackened	Not growing	Not growing
6.	MS-ET 10%	Shoots grow	Shoots grow	Shoots grow bigger
7.	MS-ET 15%	Blackened	Not growing Stay black	Not growing Stay black
8.	MS-ET 20%	Blackened	Looks like going to sprout	Shoots grow
9.	MS-ET 25%	Blackened	Stay black	Stay black
10.	MS-BAP 1 mg L-1	The inoculant enlarges	Shoots and callus grow	Several shoots are formed
11.	MS-BAP 1 mg L-1 - 2,4 D 0.1 mg L-1	The inoculant enlarges	Forms a compact callus	compact callus
12.	MS-BAP 1 mg L-1 - 2,4 D 0.3 mg L-1	The inoculant enlarges	Forms a compact callus	compact callus
13.	MS-BAP 1 mg L - 2,4 D 0.5 mg L-1	Blackened	Stay black	Stay black

Note: MS-LB= MS+Aloe vera gel juice, MS-ET= MS+Vigna radiata sprout extract, BAP = 6-Benzylaminopurine, 2,4-D = 2,4-Dichlorophenoxy acid.

The MS medium supplemented with Vigna radiata sprout extract (MS-ET) exhibited variable results. The MS-ET 10% (v/v) treatment proved to be the most effective in stimulating stable shoot growth, with shoots continuing to enlarge until the third month. The MS-ET 20% (v/v) treatment also showed positive explant growth, as the explant began turning green in the second month and developed shoots in the third month (Fig. 3). In contrast, the explants in the MS-ET 15 and 25% (v/v) treatments remained blackened and showed no growth or development. This indicates that the formulation of these media concentrations was unsuitable for explant regeneration.

In the first month, the explants exhibited size enlargement in the MS-BAP 1 mg L^-1 treatment and in the combination treatments of MS-BAP 1 mg L^-1 + 2,4-D at 0.1 and 0.3 mg L^-1. In the second month, explants in the MS-BAP 1 mg L^-1 treatment and the MS-BAP 1 mg L^-1 + 2,4-D (0.1 and 0.3 mg L^-1) combinations began to form shoots and compact callus. Meanwhile, the explants in the MS-BAP 1 mg L^-1 + 2,4-D 0.5 mg L^-1 treatment showed no growth and remained blackened. This indicates an inhibitory effect at this concentration (Fig. 4).

In the third month, the explant in the MS-BAP 1 mg L^-1 treatment successfully developed multiple shoots, while the combinations of MS-BAP 1 mg L^-1 + 2,4-D at 0.1 and 0.3 mg L^-1 remained as compact callus. This indicates that these treatment media have a high potential for inducing callus formation, which may later develop into shoots. However, in the MS-BAP 1 mg L^-1 + 2,4-D 0.5 mg L^-1 treatment, the explant remained blackened up to the third month, suggesting that an increased concentration of 2,4-D in this medium may have hurt explant viability (Fig 4).

Overall, differential growth responses were recorded, with explants showing active development mainly in MS-LB 20%, MS-ET 10%, and MS-BAP-based media (Table 3). These findings formed the basis for the subsequent statistical evaluation using Chi-square analysis.

4.1. The Effect of Aloe vera Gel Juice on the Micropropagation Development of Gapi Banana

The research results on the MS-LB treatment medium, which consists of MS medium supplemented with Aloe vera gel juice, showed that the explant initially blackened, resembling cell death symptoms. However, over time, it recovered and began to develop shoots. This phenomenon can be explained by the bioactive compounds in Aloe vera gel, which play a crucial role in supporting cell regeneration and the growth of plant tissue.

Aloe vera gel contains polysaccharides, enzymes, vitamins, minerals [26], and natural plant growth hormones such as auxins and gibberellins. These growth hormones are known to stimulate cell division and tissue regeneration. Additionally, polysaccharides in Aloe vera exhibit antioxidant properties, which help reduce oxidative stress caused by the accumulation of phenolic compounds [27]. Phenolic compounds are the primary cause of browning and tissue death in plant tissue culture, making the antioxidant function of Aloe vera particularly beneficial for enhancing the survival and development of explants.

Additionally, the amino acids and phenolic compounds present in Aloe vera gel can act as cell protectors against toxic compounds while accelerating the recovery of damaged cells [21]. Therefore, although the explant initially blackened due to tissue culture stress, the presence of Aloe vera gel juice in the medium helped in tissue recovery [28] and stimulated shoot growth, demonstrating its potential as a natural supplement to enhance the success of Gapi banana micropropagation. This finding also highlights Aloe vera gel as a possible solution to the challenge of obtaining Gapi banana explants for in vitro culture, providing an alternative approach to improve explant viability and regeneration [26]. A previous study [29] showed that Aloe vera gel coating can extend the shelf life of lime by up to 4 days by slowing down colour changes without affecting the physicochemical quality of the fruit.

4.2. The Effect of Vigna Radiata Sprout Extract on the Culture Development of Gapi Banana

The research results on the MS-ET treatment medium, which consists of MS medium supplemented with Vigna radiata sprout extract, showed varied growth responses among the explants. Some explants turned green and developed shoots, while others remained blackened without further growth. Vigna radiata sprout extract is rich in natural plant growth hormones, particularly cytokinin. Germinated Vigna radiata sprouts accumulate bioactive metabolites and exhibit cytokinin-related biological activity, supporting their role as natural PGR sources [30]. Cytokinin is crucial in stimulating cell division and organogenesis in tissue culture. They also function to delay tissue culture senescence (aging) and accelerate shoot formation by inducing the expression of genes associated with cell division and differentiation [31]. Additionally, Vigna radiata sprout extract contains proteins, essential amino acids, and antioxidant enzymes [32-34], which can enhance explant viability and reduce the impact of stress caused by in vitro culture. However, this study observed that not all explants in the MS-ET medium successfully developed shoots.

This could be due to the suboptimal concentration of sprout extract or the presence of certain compounds that may cause an imbalance in explant growth. Therefore, optimizing the Vigna radiata sprout extract concentration in the culture medium is necessary to maximize the positive effects of cytokinin and other bioactive components, thereby improving shoot regeneration in the micropropagation of Gapi banana plants. According to [35], mung beans contain flavonoids, polysaccharides, and peptides, which act as antioxidants [36], neutralizing free radicals, reducing inflammation, and preventing chronic diseases such as diabetes and cancer [37].

4.3. The Effect of Benzylaminopurine (BAP) and 2,4-Dichlorophenoxy acid (2,4-D) on the Culture Development of Gapi Banana

The research results showed that the addition of BAP and 2,4-D in the culture medium had a significant influence on the development of Gapi banana explants. In the treatment with MS medium + BAP 1 mg L^-1, the explants initially enlarged, then began to form shoots and callus, eventually producing several small shoots. This indicates that BAP, as a cytokinin, plays a role in stimulating cell division and shoot differentiation [38, 39]. Cytokinins are known to regulate plant growth by promoting shoot formation and accelerating cell division in the meristematic region.

However, when BAP was combined with 2,4-D at different concentrations, the explant responses varied significantly. In the MS medium containing BAP 1 mg L^-1 + 2,4-D 0.1 mg L^-1, the explants enlarged and formed compact callus, but no shoot formation was observed. A similar phenomenon occurred in the treatment with BAP 1 mg L^-1 + 2,4-D 0.3 mg L^-1, where the explants continued to form compact callus without any shoot differentiation. This can be explained by the dominant effect of auxin (2,4-D) in inducing callus formation [40], while cytokinin at the same concentration was not strong enough to stimulate shoot formation. Auxin plays a key role in callus induction and tissue regeneration initiation, but when its concentration is higher than that of cytokinin, shoot formation may be inhibited [39].

In the treatment with BAP 1 mg L^-1 + 2,4-D 0.5 mg L^-1, the explants turned black and remained black until the end of the observation period, indicating necrosis or tissue death. In this study, necrosis was defined as intense blackening in more than 75% of explant tissue that persisted until the end of the observation period. The high concentration of 2,4-D likely caused physiological stress on the explants, disrupting cellular metabolism and inhibiting growth. Additionally, an increase in auxin levels in the medium is known to trigger phenolic accumulation, leading to browning and tissue death in in vitro cultures [38].

Thus, the combination of BAP and 2,4-D must be optimized to promote shoot proliferation without causing excessive callus formation or necrosis [40]. Based on these findings, BAP without 2,4-D was more effective in inducing shoot growth, whereas low concentrations of 2,4-D were more suitable for callus induction. Meanwhile, a high concentration of 2,4-D is not recommended, as it can damage plant tissues [39]. According to the previous studies [38, 41], the presence of cytokinin, along with a small amount of auxin, is required for haploid tissue to form callus and somatic embryos. Understanding the interaction between these two hormones is essential for developing a more optimal tissue culture technique for Gapi banana, whether for shoot multiplication or callus production in further studies.

Overall, the findings suggest that Aloe vera gel juice and Vigna radiata sprout extract have the potential for the micropropagation of Gapi banana. These natural supplements demonstrated comparable to, or even superior to, conventional synthetic PGRs, thereby emphasizing their practical value in plant tissue culture. While the current study was limited to controlled laboratory conditions, the consistency of the results provides a solid basis for considering broader applications. Building upon these findings, it is essential to reflect on the potential prospects and future directions that may further enhance the applicability of plant-based PGRs in banana micropropagation and beyond.

The findings of this study highlight the practical potential of Aloe vera gel juice and Vigna radiata sprout extract as natural plant growth regulators (PGR) sources in banana micropropagation. Beyond the current laboratory-based observations, these results open prospects for applying locally available plant extracts as low-cost and eco-friendly supplements in tissue culture media. Further studies are recommended to validate these findings under larger sample sizes, explore concentration optimization, and assess reproducibility in field-scale trials. Such efforts will strengthen the potential for large-scale banana propagation and contribute to the development and commercialization of bio-based PGRs.