Revista Facultad Nacional de Agronomía Medellín

In vitro establishment of Musa textilis Nee

For the in vitro establishment of Musa textilis Nee, corms from the varieties Bungalanon, Tangongon, Maguindanao, and Bribri were utilized as the initial plant material. The outer leaf sheaths were removed to expose the apical region, and segments of approximately 3-5 cm containing the shoot apices were excised. These explants were first immersed in a commercial liquid detergent solution and subjected to agitation at 150 rpm for 10 minutes. Subsequently, the detergent solution was discarded, and the explants were treated with an agrochemical mixture composed of Agri-mycin 16.5 WP and Afungil 50 WP (5 g L^-1 each). The explants were maintained in this solution under constant agitation (150 rpm) for 15 minutes. Following treatment, the agrochemical solution was removed, and the explants were rinsed twice with sterile distilled water.

The explants were subsequently transferred to a 5.25% (a.i.) sodium hypochlorite (NaOCl) solution and taken into a laminar flow cabinet, where they were disinfected under constant agitation (150 rpm) for 20 minutes. Following this step, the disinfectant solution was discarded, and the explants were rinsed three times with sterile distilled water. The segments were then trimmed to lengths of 2-3 cm and incubated in a 0.525% (a.i.) NaOCl solution supplemented with 0.5% (v/v) Tween® 20 to enhance surface wetting. After disinfection, the solution was discarded, and the explants were again rinsed three times with sterile distilled water. The explants were further reduced in size by carefully removing the outer leaf sheaths, exposing shoot apices measuring approximately 0.5-0.6 cm from base to distal tip. Finally, the apices were immersed in an antioxidant solution containing ascorbic acid (100 mg L^-1) for 10 minutes to minimize oxidative stress.

Following the 10-minute incubation in the antioxidant solution, the explants were longitudinally bisected to obtain symmetrical vertical sections of the shoot apices. These segments were then cultured in the initiation medium, which consisted of Murashige and Skoog (MS) salts and vitamins (Murashige and Skoog 1962), supplemented with 3% (w/v) sucrose, 2 mg L^-1 6-benzylaminopurine (BAP), 1 mg L^-1 indole-3-butyric acid (IBA), and 3.3 g L^-1 Gellan Gum®. Prior to autoclaving, the pH of the medium was adjusted to 5.7 using 1 N NaOH. All reagents were obtained from PhytoTech Labs® (Lenexa, KS, USA). Cultures were maintained at 25 °C in complete darkness for the first 7 days. Subsequently, explants were transferred to a photoperiod of 16 hours light / 8 hours dark, under an irradiance of 72 μmol m^-2 s^-1 and incubated for 3 to 4 weeks, depending on their morphogenic response.

Micropropagation and acclimatization of Musa textilis Nee vitro plants

Explants exhibiting sprouting after approximately four weeks of culture were sectioned at their base to stimulate the formation of multiple shoots. Each explant was bisected through the meristematic region prior to subculturing. The new culture medium consisted of Murashige and Skoog (MS) salts and vitamins (Murashige and Skoog 1962), supplemented with 3% (w/v) sucrose, 5 mg L^-1 BAP, 2 mg L^-1 α-naphthaleneacetic acid (NAA), and 3.3 g L^-1 Gellan Gum®. Prior to autoclaving, the pH was adjusted to 5.7 using 1 N NaOH. All reagents were obtained from PhytoTech Labs® (Lenexa, KS, USA). Cultures were maintained at 25 °C under a 16-hour photoperiod with an irradiance of 72 μmol m^-2 s^-1 for 3 to 4 weeks, until the next subculture. During subsequent subcultures, newly formed shoots were separated and transferred to fresh medium of the same composition and maintained under the previously described environmental conditions.

In vitro-derived plantlets (vitroplants) cultured for 12 weeks and reaching heights of 10-15 cm were then transferred to greenhouse conditions for a two-week acclimatization period. Prior to transplantation, all residual culture medium was carefully removed from the root systems. The plantlets were then transplanted into plastic trays containing a sterile substrate composed of a 1:1 ratio of soil and peat moss. Irrigation was performed every 3 days. Additionally, during the first four weeks post-transplantation, plants received weekly applications of Fertiplant NPK® fertilizer (Seracsa, Tres Ríos, Costa Rica).

Characterization of abaca varieties through SSR markers

Genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega Corporation©, Fitchburg, WI, USA), following the manufacturer's protocol with specific modifications to accommodate the high fiber content of Musa textilis Nee leaves. Leaf tissue from greenhouse-grown plants of each variety was stored in a desiccator to maintain a dry and contaminant-free environment. For each variety, genomic DNA was extracted from three biological replicates, with each replicate corresponding to an independent greenhouse-grown plant. The extraction protocol was modified as follows: (1) 30 mg of dried leaf tissue were used per sample; (2) all steps, including centrifugations, were carried out under cold conditions (4 °C), using chilled isopropanol and ethanol; (3) in step 6 of the protocol, 250 μL of protein precipitation solution were added, followed by centrifugation at 8,000 rpm for 5 minutes; and (4) in steps 9 and 10, centrifugation was performed at 5,000 rpm for 3 minutes.

The protocol for Simple Sequence Repeat (SSR) analysis via PCR was based on that proposed by Yllano et al. (2020). Table 1 details the SSR markers used. Thirty-microliter reactions contained 17 μL of 2× Master Mix, 2 μL of each primer (forward/reverse), 6 μL of nuclease-free water, and 3 μL of genomic DNA. The thermocycling program was: 95 °C for 3 min; 35 cycles of 95 °C for 30 s, Ta for 30 s, and 72 °C for 30 s; and the final extension at 72 °C for 5 min. Annealing temperatures actually used: R07=62 °C; R39=56 °C; R40=58 °C; R45=56 °C; R150=53 °C; R231=58 °C; and R264=57 °C. T_m values were obtained by a temperature-gradient PCR; gradient ranges and summary are shown in Table 2.

Table 1: Primers used for the amplification of Single Sequence Repeat (SSR) markers to identify abaca (M. textilis Nee) varieties.

Table 2: Annealing temperatures (Tm) recommended for the amplification of different Simple Sequence Repeat (SSR) markers in abaca (M. textilis Nee).

The temperature gradient was used to determine the ideal annealing temperature (T_m) for each molecular marker, between the temperatures recommended by the manufacturer and those proposed by Yllano et al. (2020) (Table 2). The tested annealing gradients were as follows: 53-62 °C for R07, 52-57 °C for R39, 54-60 °C for R40, 52-57 °C for R45, 51-57 °C for R150, 55-60 °C for R231, and 55-62 °C for R264. These temperature intervals were selected to evaluate amplification efficiency and specificity under variable stringency conditions.

To obtain higher quality and resolution in the gel (agarose 1.5% w/v), both the loading buffer and the molecular weight marker were prepared in advance. The first was prepared by adding 1 μL of Gel Red (Gold Biotechnology, St. Louis, MO, USA) and 100 μL of 6X loading buffer; for this, Gel Red was not added during gel preparation, to ensure better band resolution. Meanwhile, the molecular weight marker was prepared by adding 2 μL of nuclease-free water, 1 μL of 6X loading buffer, and 1 μL of molecular weight marker solution. For the analysis of PCR samples, the QIAxcel Advance system and QIAxcel ScreenGel v1.6 software were used. This analysis was carried out using the AL420 method, a 15-1,500 bp molecular weight marker, and a 15-3,000 bp annealing marker.

Genetic diversity analysis

Alleles were called from QIAxcel electropherograms (AL420) as discrete fragment sizes per accession and locus after binning to integer base pairs with a global tolerance of ±1-2 bp to account for sizing jitter, and filtering stutter/low-intensity peaks (retaining peaks with S/N≥3 and height ≥10% of the highest peak in that trace, discarding ladder-like stutter within ±1-4 bp of a main allele if height <15% of the main peak). Assuming diploidy, genotypes were limited to ≤2 alleles per accession.

For each SSR, the following parameters were computed: Na (number of distinct alleles observed), Ho (fraction of accessions that are heterozygous at the locus), and He (Nei's gene diversity, He=1−∑i pi², with pi representing the allele frequency based on counts across 2N gene copies). Given the small sample size, PIC was approximated as PIC ≈ He (a standard practice for multiallelic codominant SSRs under limited N). The exact PIC value can be recalculated when a larger panel becomes available.

In vitro establishment and micropropagation

Micropropagation of species within the Musaceae family represents a fundamental strategy for the large-scale production of pest- and disease-free planting material (Shah et al. 2020). Moreover, this biotechnological approach enables the rapid multiplication of genetically uniform and stable plantlets, which is essential for commercial propagation systems (Abdalla et al. 2022; Cardoso et al. 2018). However, the establishment and maintenance of in vitro culture systems can be cost-prohibitive due to the need for specialized infrastructure, consumables, and skilled labor (Abdalla et al. 2022). Therefore, the development and implementation of optimized and cost-effective micropropagation protocols is crucial to increase efficiency and reduce production costs, as emphasized by Abdalla et al. (2022).

In this context, the results obtained in the present study demonstrate that the micropropagation protocol originally developed for banana can be successfully applied to abaca, achieving a high multiplication rate and a significant increase in the number of shoots per explant. This finding highlights the adaptability and efficiency of the protocol across related species, offering an alternative that is both technically feasible and cost-effective for the large-scale propagation of abaca. The ability to obtain a greater amount of plant material under similar culture conditions suggests that this approach can contribute to reducing production time and optimizing resource use in commercial and research settings for abaca.

According to Abdelnour-Esquivel et al. (2020), prior to in vitro establishment, superficial disinfection of the explant material is essential due to the ubiquitous presence of microbial contaminants. The application of fungicidal, bactericidal, and antibiotic agents has been shown to significantly reduce both fungal and bacterial contamination in donor tissues, as reported by Ray and Ali (2017) and Orlikowska et al. (2017). Among the various disinfectants available, sodium hypochlorite (NaOCl) is one of the most commonly used biocides, owing to its broad-spectrum antimicrobial activity, ease of availability, and low cost (Abdelnour-Esquivel et al. 2020; Orlikowska et al. 2017).

The disinfection, establishment, and in vitro multiplication protocols implemented for Musa textilis Nee explants resulted in successful shoot induction and plant development (Figure 1). Figures 1A, 1B, and 1C depict the preparation of apical explants from processed corms, including size reduction and transverse sectioning prior to culture initiation. Figure 1D shows an apical segment one week after culture initiation, exhibiting signs of greening and metabolic activation. Subsequent stages of shoot induction and plantlet formation are illustrated in Figures 1E through 1H. The described protocols proved effective, producing between 5 and 10 shoots per explant within 3-4 weeks. Under the established conditions of temperature, light, and culture medium composition, complete in vitro plantlet production was achieved within 8 to 12 weeks.

Figure 1: Stages of the micropropagation process of abaca (Musa textilis Nee). A. Explant prior to disinfection. B. Size-reduced explant prepared for culture initiation. C. Explant recently inoculated onto semisolid culture medium. D. Explant 1-week post-inoculation, showing signs of greening and metabolic activation. E and F. Explants at 4 weeks post-inoculation, exhibiting the emergence of initial shoots. G and H. Plantlets after eight weeks of culture in multiplication medium, and I. Acclimatized plants grown under greenhouse conditions.

Rooting occurred successfully without the need for supplementary additives in the multiplication medium. Furthermore, from the twelfth week onward, the in vitro plantlets exhibited morphological and physiological characteristics appropriate for acclimatization and subsequent cultivation under greenhouse conditions (Figure 1I).

In this context, Shah et al. (2020) and Sugiyono et al. (2021) report that BAP is the most suitable cytokinin for in vitro culture due to its high chemical stability. Furthermore, BAP plays a critical role in shoot induction and proliferation (Priyanka 2020). Consistent with the present study, previous research has demonstrated that BAP concentrations ranging from 4.0 to 6.0 mg L^-1 in the culture medium effectively promote shoot emergence in Musaceae species (Khatun et al. 2017; Ngomuo et al. 2013; Priyanka 2020; Uzaribara et al. 2015). Nevertheless, the balanced and combined application of cytokinins and auxins enhances shoot development, delays tissue senescence, and facilitates root formation (Priyanka 2020; Sugiyono et al. 2021). Kalimuthu et al. (2007) and Priyanka (2020) further emphasize that the concomitant use of BAP and NAA in in vitro cultures of Musaceae, as implemented in this study, results in improved shoot proliferation and rooting.

Characterization of abaca varieties through SSR analysis

Comparing annealing temperatures is essential for determining the optimal conditions to amplify each molecular marker (Dewangan et al. 2020). Furthermore, it is essential, as small variations can significantly influence the efficiency of amplification and the specificity of PCR (Ishii and Fukui 2001). To determine the optimal annealing temperatures for the amplification of SSR markers in Musa textilis Nee, a gradient PCR protocol was implemented, applying temperature ranges adjusted to the physicochemical characteristics and melting temperatures of each primer pair.

In most cases present in this study, the ideal temperatures obtained during the PCR gradient repetitions were close to those recommended by the manufacturer. However, some discrepancies were identified, such as in the case of the R39 marker, where the ideal temperature is slightly lower than that recommended by the manufacturer. These discrepancies could be due to specific factors related to the abaca samples or to variations in the laboratory conditions (Porta and Enners 2012).

Following electrophoretic analysis, the optimal annealing temperatures were determined as 62 °C for R07, 56 °C for R39, 58 °C for R40, 56 °C for R45, 53 °C for R150, 58 °C for R231, and 57 °C for R264. These annealing values were subsequently employed in all downstream amplifications to ensure consistent PCR performance, high resolution of allelic variants, and robust marker reproducibility across the different abaca varieties analyzed.

In the analysis of the R40 marker, a significant variation in the length of the obtained amplicons was observed, compared to the expected 540 bp (Figure 2A). For the Tangongon variety, a 207 bp fragment was obtained; meanwhile, for the Maguindanao variety, two amplicons of 214 and 231 bp were detected, indicating variability in the length of the PCR products. For the Bungalanon material, a 215 bp fragment was obtained; meanwhile, the Bribri material showed three amplicons with lengths of 178, 212, and 228 bp.

Figure 2: Results of capillary electrophoresis coupled to fluorescence for SSR markers: A. R40, B. R07, C. R231, D. R45, E. R39, F. R264, and G. R150.

For the R07 marker (165 bp), divergent results were observed in the analyzed abaca samples (Figure 2B). The Tangongon material presented two amplicons of 133 and 177 bp. In the Maguindanao variety, 120 and 133 bp fragments were detected. For the Bungalanon material, 133 and 176-bp fragments were observed. Lastly, the Bribri material showed two amplicons of 106 and 135 bp.

Regarding the R231 marker, a 286 bp fragment was expected; however, the results revealed shorter amplicons (Figure 2C). The Tangongon material showed an amplification of 223 bp, while the Maguindanao material had an amplification of 224 bp. The Bungalanon and Bribri varieties each presented an amplicon of 226 bp.

In the case of the R45 marker, a 275 bp amplicon was expected, but slightly larger fragments were observed (Figure 2D). The Tangongon material presented an amplicon of 277 bp, and in the Maguindanao variety, a fragment of 276 bp was detected. In the Bungalanon material, a 280 bp fragment was detected, while in the Bribri material, a 278-bp amplicon was obtained.

For the R39 marker, fragments of nearly 400 bp were expected (Figure 2E). The Tangongon material presented two amplicons with lengths of 324 and 355 bp. In the Maguindanao variety, a 327 bp fragment was detected, while the Bungalanon material showed two amplicons of 324 and 356 bp. Two amplicons were obtained from the Bribri material, with lengths of 318 and 352 bp.

For the R264 marker, a 274 bp amplicon was expected, but shorter fragments were detected in all abaca samples (Figure 2F). The Tangongon material presented three different amplicons (180, 270, and 255 bp), while the Maguindanao variety showed four different fragments with lengths of 186, 210, 230, and 248 bp. In the Bungalanon and Bribri varieties, three amplicons (189, 210, and 228 bp) and two amplicons (211 and 230 bp) were detected, respectively.

Finally, in the case of the R150 marker, a 270 bp fragment was expected, but longer fragments were detected in all abaca samples (Figure 2G). The Tangongon and Maguindanao materials presented a single fragment of 355 bp. In the Bungalanon and Bribri varieties, a single amplicon was also detected, with lengths of 354 and 356 bp, respectively.

The results of this study reveal notable differences in the length of the amplicons obtained for each variety using the same molecular markers. This variability suggests the presence of significant genetic differences among the analyzed materials and supports the hypothesis that it is possible to differentiate abaca varieties through molecular marker analysis. In addition, the detection of multiple amplicons in some varieties is significant, as it suggests greater genetic complexity in these genotypes (Chen et al. 2020). For example, the R40 marker revealed multiple amplifications in the Bribri variety, with lengths of 178, 212, and 228 bp. These differences in the length and number of amplicons within the same variety indicate significant genetic variability. Beyond qualitative size shifts, the SSR panel exhibited clear multi-allelic polymorphism across accessions (Table 3). Marker informativeness was highest for R40, R39, and R264 (He≈PIC≥0.80), while R231 and R150 showed moderate informativeness (PIC≈0.63). Observed heterozygosity (Ho) peaked at R40 and R264 (Ho=1.00), and was null for R231, R45, and R150, consistent with their predominantly single-band profiles. These indices quantitatively support the genetic differentiation already apparent from the amplicon-size patterns.

Table 3: Marker-level diversity indices estimated from QIAxcel runs.

Furthermore, the greater the differences in these amplicons, the stronger the evidence of genetic differentiation among varieties (Patil et al. 2021). However, it is important to acknowledge the limitations of using SSR markers alone for genetic differentiation, as they may not capture the full extent of genomic diversity (Adamek et al. 2023). Additional analyses, such as whole-genome sequencing, could provide a more comprehensive understanding of genetic variation among abaca varieties (Jagadeesan et al. 2019). Within the analyzed panel, R40 emerged as one of the most informative loci (Table 3) and represents a valuable tool for differentiating abaca varieties, given its ability to detect significant genetic differences (Marimuthu Somasundaram et al. 2019). In contrast, markers that generate more homogeneous profiles are less useful for varietal discrimination because they offer limited resolution and produce simpler amplicon patterns (Gaikwad et al. 2023).

Although the SSR panel differentiated the accessions evaluated in this study, the inference space is constrained by the limited varietal breadth (four varieties). With such a small number of varieties, resolution among closely related genotypes and the stability of allele-frequency metrics (e.g., PIC/He in Table 3) remain limited; therefore, these values should be interpreted as panel-level indicators rather than population parameters. Replicability was ensured by analyzing three biological replicates per variety (independent greenhouse-grown plants) and by repeating PCR and capillary electrophoresis. Under the adopted binning and stutter-filter criteria, allele bins were consistent across replicates, supporting the technical repeatability of genotype calls. Regarding applicability to in vitro explants, the same SSR primers (including R40) can be used to assess genetic diversity and clonal fidelity during micropropagation. Nevertheless, potential sources of error-such as somaclonal variation, occasional chimerism, and allele dropout related to DNA quality or quantity-should be anticipated and minimized through biological replication and standardized allele-calling thresholds.