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Article

Temporary Immersion Bioreactor (TIB) System for Large-Scale Micropropagation of Musa sp. cv Kluai Numwa Pakchong 50

by
Sudarat Thanonkeo
1,
Haruthairat Kitwetcharoen
2,
Pornthap Thanonkeo
2,3 and
Preekamol Klanrit
2,3,*
1
Walai Rukhavej Botanical Research Institute (WRBRI), Mahasarakham University, Maha Sarakham 44150, Thailand
2
Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen 40002, Thailand
3
Research Center for Value Added Agricultural Products (FerVAAPs), Khon Kaen University, Khon Kaen 40002, Thailand
*
Author to whom correspondence should be addressed.
Horticulturae 2024, 10(10), 1030; https://doi.org/10.3390/horticulturae10101030
Submission received: 9 August 2024 / Revised: 25 September 2024 / Accepted: 26 September 2024 / Published: 27 September 2024
(This article belongs to the Section Propagation and Seeds)
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Versions Notes

Abstract

:
Conventional in vitro propagation using semisolid Murashige and Skoog (MS) culture systems is costly, labor-intensive, and requires substantial space for large-scale plant production. This study investigated the application of a temporary immersion bioreactor (TIB) system for the micropropagation of the banana cultivar Kluai Numwa Pakchong 50, as a promising platform for economical commercial production. The cultivation parameters affecting plantlet multiplication, including plant growth regulator (PGR) use, explant density, and immersion frequency, were examined. Additionally, the ex vitro acclimatization of well-developed in vitro plantlets was also evaluated. Using liquid MS medium supplemented with 7.5 mg/L 6-benzylaminopurine (BAP) in the TIB system yielded significantly better results than the conventional semisolid MS control system, producing more shoots (5.60 shoots/explant) and leaves (2.80 leaves/explant) with longer shoot length (2.19 cm). Optimal conditions in the TIB system included an inoculum density of five explants per culture vessel and an immersion frequency of once every 6 or 8 h for 2 min. For root induction, 0.5 mg/L indole-3-butyric acid (IBA) proved more effective than 1-naphthaleneacetic acid (NAA). After 30 days of ex vitro acclimatization, plantlets regenerated from the TIB system demonstrated high survival rates, vegetative growth performance, and root formation efficiency comparable to those from the semisolid culture system. These findings establish the TIB system as a promising platform for the mass propagation of the Kluai Numwa Pakchong 50 banana. The protocol developed in this study could potentially be adapted for large-scale production of other banana varieties.

1. Introduction

Banana (Musa spp.) is one of the most popular tropical fruits globally, with production reaching approximately 120 million tons in 2020 and an estimated value exceeding 30 billion USD [1]. As global demand continues to rise, production is projected to reach 133 million tons by 2029 [1]. While India leads in banana production, Thailand ranks among the top twenty producing countries, with an output of approximately 1.3 million tons [1,2].
In Thailand, three banana varieties are cultivated commercially: Kluai Hom (AAA group), Kluai Khai (AA group), and Kluai Numwa (ABB group). Among these, Kluai Numwa is the most widely consumed due to its adaptability to various growing conditions. However, this variety is highly susceptible to Fusarium wilt disease, caused by Fusarium oxysporum f. sp. cubense, which can lead to severe growth problems and total yield loss. Currently, there are no effective fungicides or eradication methods for this pathogen [3]. To address this challenge, the production of disease-free, high-quality plantlets has become a crucial strategy. Modern plant biotechnology, particularly plant tissue culture, offers a promising approach for large-scale production of pathogen-free and genetically uniform plantlets. However, conventional tissue culture methods, both semisolid and liquid systems, have some limitations. For instance, explants exhibit a slow growth rate, solidifying agents are required, automated processes require supervision, and there are labor and large area requirements for a semisolid culture system. For a liquid cultivation system, several disadvantages include a high energy input for shaking, increased hyperhydricity of the plantlets due to long periods of direct immersion in the culture medium, damage to the plant cells caused by shearing stress during shaking, and a low level of ventilation during cultivation [2,4].
Temporary immersion bioreactors (TIBs) have emerged as a novel system to overcome these challenges. TIBs combine the advantages of semisolid and liquid cultures while offering economic benefits for large-scale micropropagation [2,5]. These systems provide uniform medium contact, reduce vitrification and browning, improve gas exchange, and enhance propagation efficiency [2]. Various types of TIB systems have been developed during the past decade, such as the RITA®, BIT® (twin flask bioreactor), Plantform, Ebb-and-Flow, Rocker, Balloon Type Bubble Column Bioreactor (BTBCB), and SETISTM systems [6,7,8,9,10]. However, RITA® and BIT® are the most widely used systems for the clonal propagation of plant materials.
While TIB systems have been successfully employed for various plant species, including Thapsia garganica [11], goji [12], chrysanthemum, strawberry, Cnidium officinale [10], cannabis [13], pineapple [14], and some banana cultivars [2,15,16], there is limited information on their application to the Kluai Numwa Pakchong 50 banana cultivar. The efficiency of TIB systems depends on several factors, including system configuration, explant density, culture volume, immersion frequency, and plant growth regulators (PGRs). Therefore, these cultivation parameters must be optimized and standardized for each plant species.
This study investigates the factors influencing the in vitro growth of Kluai Numwa Pakchong 50 using a Temporary Immersion Bioreactor (TIB) system. The research objectives encompass optimizing cultivation parameters for the TIB system, evaluating the effects of plant growth regulators (PGRs) on shoot and root induction, and comparing the ex vitro acclimatization performance of TIB-cultivated plantlets with those from conventional semisolid cultivation systems. The study aims to develop an efficient protocol for the large-scale production of disease-free Kluai Numwa Pakchong 50 plantlets, contributing to sustainable banana cultivation in Thailand and potentially other regions facing similar challenges.

2. Materials and Methods

2.1. Chemicals

Murashige and Skoog (MS) basal medium and GelzanTM agar were purchased from PhytoTech Labs (Lenexa, KS, USA). PGRs, including 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA), were obtained from Sigma Aldrich Corporation (Burlington, MA, USA). Other chemicals, such as hydrochloric acid (HCl), sodium hydroxide (NaOH), and sucrose, were acquired from a local supplier in Khon Kaen Province, Thailand.
Thirty-day-old sterile plantlets of the banana cultivar Kluai Numwa Pakchong 50, kindly provided by Mrs. Nuntipa Khumkarjorn, Hana Garden Plant Lab, Co., Ltd., Udonthani, Thailand, were used in the present study. The plants were cultured on semisolid MS medium with 0.2% (w/v) agar and supplemented with 30 g/L sucrose and 7.5 mg/L BAP and maintained in a standard cultivation room with a light intensity of 3000 lux, a photoperiod of 16 h, a relative humidity of 80%, and a temperature of 25 ± 2 °C. The plantlets were subcultured into fresh MS medium under the abovementioned conditions every 30 days.

2.2. The TIB System Setup

A TIB system with two main compartments (twin culture vessel system) was used (Figure 1). The upper compartment (600 mL), where the explants were placed, was used as a growth chamber, while the lower portion (600 mL) was used as a nutrient reservoir for the culture medium. These two compartments are connected by a small stainless-steel tube through an autoclavable rubber support placed on both compartments. The culture medium was supplied periodically to the upper compartment by an air pump controlled by an electronic timer switch. The airflow was sterilized through a 0.2 μm syringe filter before entry into and exit from the bioreactor (Minisart® NML, Sartorius Stedim Biotech Gmbh, August-Spindler-Strasse 11, Goettingen, Germany).

2.3. Effect of BAP on Shoot Proliferation in the TIB System

Thirty-day-old sterile explants with shoot lengths of approximately 1.5 cm were transferred into the growth chamber of the TIB units containing 250 mL of MS medium supplemented with 30 g/L sucrose and different concentrations of BAP (0.0, 2.5, 5.0, 7.5, and 10.0 mg/L). One explant was placed into each culture vessel of the TIB system, and all explants were cultivated in a standard culture room at 25 ± 2 °C with a light intensity of 3000 lux under a 16/8 h (light/dark) photoperiod. The immersion frequency of the explants in the culture medium was set at 2 min every 4 h. Each treatment was performed twice, with five replications. After 30 days of cultivation, the number of shoots and leaves and shoot length were determined and compared with those in the control treatment, which was performed by culturing the explants in a 240 mL (8 oz) culture vessel containing 30 mL semisolid MS medium with 0.2% agar and supplemented with 30 g/L sucrose and 7.5 mg/L BAP. One explant per culture vessel was also used for the control treatment with ten replications.

2.4. Effect of Explant Number on Shoot Proliferation in the TIB System

Thirty-day-old sterile explants with shoot lengths of approximately 1.5 cm were transferred into the growth chamber of the TIB units containing 250 mL of MS medium supplemented with 30 g/L sucrose and 7.5 mg/L BAP. Different numbers of explants, i.e., 1, 5, and 10 explants/culture vessel, were tested. The explants were cultivated in a standard culture room at 25 ± 2 °C with a light intensity of 3000 lux under a 16/8 h (light/dark) photoperiod. The culture medium was supplied to the explants every 4 h for 2 min. All treatments were tested twice, with five replications. After 30 days of cultivation, the number of shoots and leaves and shoot length were determined and compared with those of the control treatment, which was performed as mentioned earlier using one explant per culture vessel.

2.5. Effect of Immersion Frequency on Shoot Proliferation in the TIB System

The effect of the explant immersion frequency in culture medium on the shoot proliferation of the banana cultivar Kluai Numwa Pakchong 50 was investigated. Thirty-day-old sterile explants with shoot lengths of approximately 1.5 cm were transferred into the growth chamber of the TIB units containing 250 mL of MS medium supplemented with 30 g/L sucrose and 7.5 mg/L BAP. Five explants were placed in the growth chamber of the TIB units, and all treatments were performed twice, with five replications. The explants were exposed to the culture medium for 2 min every 4, 6, or 8 h in a standard cultivation room with a light intensity of 3000 lux and a 16 h light photoperiod. The number of shoots and leaves and shoot length were determined after 30 days of cultivation, and the data were compared with those of the control treatment, which was performed as mentioned earlier using one explant per culture vessel.

2.6. Effect of Auxins on Root Formation

Thirty-day-old sterile explants with 2–3 leaves were transferred into the growth chamber of the TIB units with one explant per culture vessel. MS liquid media supplemented with 30 g/L sucrose and different concentrations of NAA and IBA (0.0, 0.1, 0.5, and 1.0 mg/L) were used in this experiment. The explants were immersed in culture medium for 2 min every 6 or 8 h. The control treatment with one explant per culture vessel was performed using a semisolid MS medium (0.2% agar) containing 30 g/L sucrose, and supplemented with 0.5 mg/L NAA or 0.5 mg/L IBA. All experiments were conducted twice, with each treatment having ten replications. After 30 days of cultivation in a standard cultivation room, as previously mentioned, the number of roots and the length of the roots were monitored.

2.7. Ex Vitro Acclimatization of the Plantlets

For the TIB system, thirty-day-old rooted shoots were selected, transplanted in 2 × 5 in2 nursery bags filled with 1:1 peat moss and vermiculite, and placed in a growth chamber at 25 ± 2 °C with a light intensity of 3000 lux and relative humidity of 45–55%. For the semisolid culture system, thirty-day-old rooted shoots were also selected, and their roots were thoroughly washed with running tap water to remove all trace elements from the culture medium. All of the selected plantlets were transplanted in the same manner as those of the TIB system. The experiment was performed three times, each with ten replications. After 30 days of acclimatization, the survival rate, plant height, number of roots, and root length were recorded.

2.8. Experimental Design and Data Analysis

The experiments were carried out using a completely randomized design (CRD). All experiments were performed at least twice, and the experimental data were processed statistically using the SPSS program for Windows (IBM SPSS Statistics 28, IBM Corporation, Armonk, NY, USA). The mean difference between each treatment was determined based on Duncan’s multiple range test (DMRT) at a probability level of 5%. The experimental data are presented as the means ± standard deviations (SDs).

3. Results and Discussion

3.1. Effect of BAP on Shoot Proliferation in the TIB System

Several cytokinins have been used for plant proliferation, and BAP is the most effective PGR for shoot induction in many plant species, including bananas [16,17]. Based on this information, the effect of BAP on the shoot proliferation of the banana cultivar Kluai Numwa Pakchong 50 was investigated using the TIB system. As shown in Table 1, MS medium supplemented with 7.5 mg/L BAP yielded the greatest number of shoots (5.60 shoots/explant), followed by medium supplemented with 5.0 mg/L BAP (5.20 shoots/explant) and 2.5 mg/L BAP (4.60 shoots/explant). Based on the present results, the shoot formation efficiency of the TIB system using MS medium supplemented with 7.5 mg/L BAP was approximately 1.3 times greater than that of the control treatment using a semisolid culture system (4.40 shoots/explant). While these results suggest a potential advantage of the TIB system over the semisolid culture system for this banana cultivar, it is important to note that the difference, though statistically significant (p < 0.05), is relatively modest. Further research is needed to fully understand the mechanisms underlying this difference and to determine whether it translates to meaningful improvements in large-scale propagation efforts. Previous studies have proposed that TIB systems may offer advantages in nutrient absorption and gas exchange [18,19,20], but these factors were not directly measured in our study. Future investigations could focus on quantifying these parameters to provide a more comprehensive understanding of the TIB system’s effects on banana micropropagation.
Considering the shoot length and the number of leaves, the TIB system using MS medium supplemented with 2.5 mg/L BAP yielded the greatest shoot length (2.68 cm) and leaf number (3.20 leaves/explant), followed by medium supplemented with 5.0 mg/L BAP (2.36 cm and 2.90 leaves/explant) and 7.5 mg/L BAP (2.19 cm and 2.80 leaves/explant). The shoot length and leaf number of the control treatment using a semisolid cultivation system were 1.85 cm and 2.30 leaves/explant, respectively, which were similar to the values observed in the TIB system using MS medium without BAP supplementation. Notably, a high concentration of BAP (10 mg/L) in the medium yielded the lowest shoot length and leaf number, possibly due to hormonal imbalance. It has been previously reported that an excess of one phytohormone may alter the action of another, leading to the suppression of shoot growth and leaf formation [21,22]. The results obtained in this study are similar to those of a study by Klanrit et al. [23], who reported that a high concentration of BAP reduced the formation of shoots and leaves of Philodendron plants grown on semisolid MS medium. Another study by Ruta et al. [12] also indicated that a high concentration of BAP reduced the formation of shoots and shoot length of Lycium barbarum cultured using the TIB system.
The MS medium supplemented with 7.5 mg/L BAP yielded the greatest number of shoots (5.60 shoots/explant) while maintaining a relatively high shoot length (2.19 cm) and leaf number (2.80 leaves/explant), comparable to those observed in media supplemented with 2.5 and 5.0 mg/L BAP. In selecting the optimal BAP concentration for subsequent experiments, multiple factors were considered, including shoot proliferation efficiency, shoot quality (assessed by shoot length and leaf number), and potential for further development. The primary goal was to maximize the number of viable shoots produced, which is crucial for efficient micropropagation while ensuring that the shoots maintained quality parameters within an acceptable range for successful further growth. Balancing these factors, it was determined that the marginal increase in shoot length and leaf number at lower BAP concentrations did not outweigh the significant gain in shoot number at 7.5 mg/L BAP. The shoots produced at this concentration still maintained quality parameters suitable for successful micropropagation, and the higher shoot numbers could lead to more efficient use of culture media and space. Therefore, MS medium supplemented with 7.5 mg/L BAP was selected for subsequent experiments to optimize overall propagation efficiency while maintaining adequate plantlet quality.

3.2. Effect of Explant Number on Shoot Proliferation in the TIB System

The number of explants per culture vessel or explant density also plays a significant role in shoot multiplication under the TIB system. The optimum number of explants for high shoot proliferation efficiency depends on several factors, specifically the plant species, culture volume, and type and configuration of the TIB system utilized [5,19]. Therefore, the number of explants per culture vessel should be standardized to maximize shoot number. In this study, the effects of different explant densities, including 1, 5, and 10 explants/vessel, on the shoot proliferation of the banana cultivar Kluai Numwa Pakchong 50 were determined. As summarized in Table 2, five explants/vessel yielded the greatest number of shoots (5.80 shoots/explant), followed by one explant/vessel (5.20 shoots/explant). The control treatment using a semisolid cultivation system yielded 4.50 shoots/explant, which was significantly lower than the number obtained using one or five explants/vessel in the TIB system.
Notably, a high explant density (10 explants/vessel) resulted in a relatively low number of shoots (3.90 shoots/explant), approximately 1.5 and 1.2 times lower than those obtained using five explants/vessel and a semisolid culture system, respectively. This observation aligns with previous studies on other plant species cultured in TIB systems. For instance, Uma et al. [5] demonstrated that a high density of explants (12 explants/vessel) caused a dramatic decrease in shoot formation in the banana cultivar Rasthali when cultured in the TIB system with an immersion frequency of 2 min every 6 h. Similarly, Rico et al. [13] noted that a high explant density of cannabis (16 explants/vessel) led to a decrease in shoot proliferation and quality when cultured in the TIB system with an immersion frequency of 1 min every 8 h.
The consistent observation of reduced proliferation at high explant densities across different plant species suggests a common underlying mechanism. While we did not directly measure nutrient availability and gas exchange in our study, these factors have been implicated in previous research. For instance, Escalona et al. [7] found that increasing the number of pineapple shoots per vessel in a TIB system led to a decrease in the concentration of nutrients in the medium and a reduction in CO2 levels inside the vessel. This information provides a rationale for our observations, suggesting that the reduction in shoot proliferation efficiency at high explant densities may be due to competition for resources. However, further research specifically measuring nutrient uptake and gas exchange in banana TIB cultures at different explant densities would be valuable to confirm these hypotheses and optimize culture conditions.
The shoot length was highest (2.28 cm) when five explants per culture vessel were applied in the TIB system, followed by one explant/vessel (1.94 cm), whereas the shortest shoot length (1.39 cm) was detected in the treatment using ten explants/vessel. The average shoot length of the control treatment cultivated in a semisolid culture system was 1.83 cm, which was shorter than that of the TIB system cultivated with one or five explants/vessel. Considering the number of leaves, treatment using five explants/vessel yielded the highest value (2.40 leaves/explant), followed by the treatments using one explant/vessel (2.30 leaves/explant). The lowest value was detected in the treatment using ten explants/vessel (1.20 leaves/explant). The control treatment using a semisolid cultivation system yielded 2.10 leaves/explant, which was slightly lower than that of the TIB system using one or five explants/vessel. As previously discussed, the lowest shoot and leaf numbers and the shortest shoot length under a high explant density may be attributed to insufficient nutrients, gases, and light necessary for the growth and development of individual plantlets [8]. Thus, a low number of explants per culture vessel is preferable for cultivating the banana cultivar Kluai Numwa Pakchong 50 using the TIB system. Based on the results obtained in this study, the TIB system with five explants/vessel was selected for the next experiment since it provided the greatest number of shoots, shoot length, and number of leaves.

3.3. Effect of Immersion Frequency on Shoot Proliferation in the TIB System

The shoot proliferation efficiency in the TIB system is also dependent on the immersion frequency. Previous studies have demonstrated that low immersion frequencies reduce multiplication rates due to a lower nutrient supply, while high immersion frequencies not only enhance microshoot formation but also increase hyperhydric shoot growth with low survival rates after ex vitro acclimatization [13,24,25,26]. Different types of explants or different plant species displayed different multiplication efficiencies in terms of immersion frequency. Therefore, the immersion frequency of explants in the culture medium should be optimized for each plant species. As shown in Table 3, compared with other treatments, a low immersion frequency (every 8 h for 2 min or three times per day) seemed to be the best condition for promoting microshoot proliferation. The frequency of immersion once every 8 h for 2 min yielded the greatest number of shoots, with a value of 8.20 shoots/explant, which was approximately 1.9 times greater than that of the control treatment using a semisolid cultivation system (4.40 shoots/explant). Increasing the frequency of immersion from once every 8 h to once every 6 h (or four times per day) and 4 h (six times per day) resulted in a lower number of shoots per explant. Furthermore, the occurrence of hyperhydric shoots also increased, specifically at an immersion frequency of every 4 h. This finding is consistent with a study by Rico et al. [13], who reported that a high frequency of immersion once every 4 h for 1 min caused a reduction in the multiplication coefficient. On the other hand, the percentage of hyperhydric shoots in the plantlets also increased by approximately 63%. Previous studies by Uma et al. [5] and Erol et al. [15] also noted the same phenomenon, i.e., a high immersion frequency (every 4 h for 2 min) caused a reduction in shoot proliferation of the banana cultivars Rasthali and Grande Naine, respectively. It should be noted from the study of Uma et al. [5] that the frequency of immersion once every 6 h for 2 min resulted in the highest number of shoots (24.2 shoots/explant), which is different from the results obtained in the current study, where an immersion frequency of every 8 h yielded the highest value. The difference between our results and those of Uma et al. [5] might be due to differences in the plant variety and the types and configuration of the TIB system used in the experiment. This study used the banana cultivar Kluai Numwa Pakchong 50, while Uma et al. [5] used Rasthali. Regarding the TIB system, this study utilized a custom TIB system with a cylindrical bottom container of 600 mL capacity filled with 300 mL of culture medium. In contrast, Uma et al. [5] employed the RITA® system, which has a circular bottom with a 1000 mL lower part capacity, filled with 250 mL of medium, and a 2000 mL capacity upper portion. The differences in system design and medium volume may influence shoot proliferation efficiency in several ways. The ratio of air space to medium volume, the distribution of medium during immersion, and the gas exchange dynamics could all be affected. For instance, the TIB system used in this study has a higher medium-to-air ratio and might provide more nutrients per immersion but could potentially reduce gas exchange. The RITA® system has a larger upper portion, which might allow for better aeration but could result in different humidity levels. These factors could contribute to the observed differences in optimal immersion frequencies and shoot proliferation rates between the two studies.
Considering the average shoot length, an immersion frequency of 2 min every 6 h had the greatest effect (2.24 cm), followed by an immersion frequency of once every 4 h (1.98 cm) or 8 h (1.84 cm). Notably, the control treatment using a semisolid cultivation system yielded an average shoot length of 1.87 cm, which was similar to that of the TIB system in which the frequency of immersion was once every 8 or 4 h but significantly lower than that in which the frequency of immersion was once every 6 h. These findings are similar to those of Uma et al. [5], Rico et al. [13], and Uma et al. [16]. However, a greater average shoot length using a semisolid cultivation system than the TIB system has also been reported. For instance, Erol et al. [15] demonstrated that cultivation of the banana cultivars Grande Naine and Azman using a semisolid culture system yielded greater shoot lengths than those obtained using Plantform and SETISTM TIB systems with a frequency of immersion once every 10 and 4 h, respectively.
The highest number of leaves (2.40 leaves/explant) was obtained using the frequency of immersion once every 4 h, followed by once every 6 h (2.20 leaves/explant) and 8 h (2.13 leaves/explant). Although the frequency of immersion once every 4 h yielded the greatest number of leaves, the generated microshoots displayed more hyperhydric symptoms than those in the other treatments, similar to the findings of a study by Rico et al. [13]. This observation highlights the need to balance proliferation rates with plant quality in practical applications of the TIB system.
Notably, the control treatment using a semisolid cultivation system yielded approximately 2.13 leaves/explant, a similar value to that obtained using the TIB system with the frequency of immersion once every 8 h. Based on the present results, the TIB system employing different immersion frequencies seemed to be more suitable for the multiplication of the banana cultivar Kluai Numwa Pakchong 50 than a semisolid culture system, which was different from a study reported by Erol et al. [15], who noted that a semisolid culture system provided a greater number of leaves of the banana cultivar Grande Naine than that obtained using the TIB system.
Notably, the increased hyperhydricity observed at higher immersion frequencies presents a challenge for large-scale banana propagation using the TIB system. Several strategies could be employed to address this issue and maintain high proliferation rates. These strategies include (1) optimization of immersion frequency and duration: further studies could explore intermediate frequencies (e.g., every 5 h) or shorter immersion periods to find an optimal balance between proliferation and plant quality; (2) modification of culture medium: adjusting cytokinin concentrations or incorporating anti-hyperhydricity agents like silicon or phloroglucinol may help reduce hyperhydric symptoms while maintaining high multiplication rates; (3) improved bioreactor design: enhancing ventilation and gas exchange within the bioreactor could help reduce humidity and prevent water accumulation in plant tissues, potentially mitigating hyperhydricity; and (4) use of porous support materials: incorporating materials such as vermiculite or perlite into the culture system could improve aeration and reduce waterlogging, thus decreasing the risk of hyperhydricity. These strategies, along with careful monitoring of plant quality throughout the multiplication process, could help optimize the TIB system for large-scale banana propagation. Future research should focus on implementing and evaluating these approaches to develop a robust and efficient protocol for commercial-scale production of high-quality banana plantlets.
The results obtained in this study demonstrated that the TIB system using a frequency of immersion once every 6 and 8 h for 2 min yielded a greater multiplication coefficient than the control using a semisolid application system. Therefore, these immersion regimes were selected for further investigation. As previously reported, the frequency of immersion once every 6 and 8 h not only provided a high multiplication efficiency but also lowered the risk of contamination, as observed in other plants, such as sugarcane [27] and cannabis [13].

3.4. Effect of Auxins on Root Formation in the TIB System

Several auxins have been used for in vitro root induction, and among them, NAA and IBA exhibit good potential for enhancing root formation in several plant species [11,14,23]. Therefore, the effects of NAA and IBA on the formation of the roots of the banana cultivar Kluai Numwa Pakchong 50 were evaluated using the TIB system. As shown in Table 4, the TIB system using MS medium supplemented with NAA or IBA, specifically at 0.5 and 1.0 mg/L, yielded a greater number of roots and longer root lengths than did the control treatment using semisolid MS medium supplemented with either NAA or IBA at 0.5 mg/L. The TIB system using MS medium supplemented with a low concentration of auxins (0.1 mg/L of either NAA or IBA) yielded a relatively lower number of roots and shorter root lengths than did the control system using a semisolid cultivation system. In contrast, no root formation was detected in the TIB system using MS medium without auxin, which might be attributed to insufficient endogenous auxin levels for root multiplication. These findings aligned with those reported by López et al. [11], Ruta et al. [12], and Klanrit et al. [23]. Notably, the explants obtained from the treatment with immersion once every 6 h yielded a markedly greater number of roots and longer root lengths than those obtained from the treatment with immersion once every 8 h. This can be attributed to the fact that the high immersion frequency (once every 6 h) allowed the explants to absorb more nutrients than the low immersion frequency, which may favor the root formation of the banana cultivar Kluai Numwa Pakchong 50. The present results are in line with those reported by Bozkurt et al. [2], who demonstrated that a high immersion frequency yielded greater root lengths and root numbers of the banana cultivar Grand Naine than did a low immersion frequency.
As previously reported, IBA is the most effective rooting hormone for several plant species, such as L. barbarum [12], Philodendron erubescens [23], and Ananas comosus [14]. However, some plant species, such as T. garganica, may respond well to NAA compared to IBA [11]. Thus, the root formation efficiency depends on the plant species and the type and concentration of auxins being evaluated. As shown in the present study, the application of IBA resulted in a greater number of roots and longer root lengths than did the application of NAA, similar to the findings in L. barbarum, P. erubescens, and A. comosus. Among the concentrations tested, 0.5 mg/L IBA yielded the greatest number of roots and longest root lengths in both TIB systems, with immersion once every 6 h (2.90 roots/explant and 1.48 cm) or 8 h (2.70 roots/explant and 1.37 cm), which are approximately 1.4 and 1.3 times greater, respectively, than those of the control using a semisolid cultivation system. Similarly, 0.5 mg/L NAA also provided the greatest number of roots and longest root lengths in both TIB systems compared to the other concentrations. It should be noted from this study that increasing the concentration of IBA or NAA from 0.5 mg/L to 1.0 mg/L tended to reduce the number of roots and root length. Therefore, it can be concluded from these findings that cultivation using the TIB system and IBA rooting hormone, specifically at 0.5 mg/L, is the most effective method for inducing root formation in the banana cultivar Kluai Numwa Pakchong 50.

3.5. Ex Vitro Acclimatization of the Plantlets

Ex vitro acclimatization is an important stage during in vitro plant propagation because it involves the gradual transition from artificial culture conditions to the natural living environment. In the acclimatization stage, optimal culture conditions are necessary to obtain high survival rates and good growth properties. Various planting materials have been used for ex vitro acclimatization of in vitro plantlets, and their ability to promote plant survival and plant growth and development depends on the plant species and the acclimatization process. Among the plant materials being reported, peat moss and vermiculite are not only the most widely used but also exhibit good properties in supporting plant growth and development due to their high water-holding capacity and high levels of nutrients, specifically peat moss [28,29,30]. The application of peat moss and vermiculite as a single planting material or in combination has been previously reported [14,23,31]; however, a mixture of both materials seemed to be the best for vegetative growth and root formation. Therefore, a mixture of peat moss and vermiculite at a ratio of 1:1 was used as the planting material for ex vitro acclimatization of in vitro plantlets of the banana cultivar Kluai Numwa Pakchong 50. After 30 days of acclimatization, the survival rate, plant height, number of roots, and root length were recorded, and the results are summarized in Figure 2 and Figure 3. The in vitro plantlets from the control treatment grown in a semisolid culture system exhibited approximately 93.3% survival, which is slightly greater than that of the plantlets from the TIB system with immersion once every 6 h (TIB1, 83.3% survival) and 8 h (TIB2, 86.7% survival) (Figure 2A). These findings suggested that most of the established in vitro plantlets in this study successfully acclimatized to the ex vitro conditions with relatively high survival rates (more than 80%), similar to those reported by Uma et al. [16], Erol et al. [15], and Bozkurt et al. [2]. The slightly lower survival rate obtained in this study than those previously reported might be attributed to differences in plant varieties, types and configurations of the TIB system, and cultivation conditions.
Considering the plant growth properties, there were no significant differences in plant height between the control treatment and either of the TIB systems. The acclimatized plantlets from the control treatment were approximately 29.1 cm in length, while those from the TIB system with a frequency of immersion once every 6 h (TIB 1) and 8 h (TIB 2) were 27.6 and 28.0 cm, respectively (Figure 2B), suggesting that the TIB system had no effect on the vegetative growth of the well-established plantlets in this study. The development of the root system is also crucial during the acclimatization stage. As shown in Figure 2C, all of the plantlets exhibited similar root formation efficiencies, with values ranging from 4.3 to 4.8 roots/explant. The plantlets from the TIB system with immersion once every 6 h (4.8 roots/explant) and 8 h (4.7 roots/explant) exhibited a greater number of roots than did those from the control treatment (4.3 roots/explant). A similar result was also reported by Bozkurt et al. [2], who found that the established plantlets of the banana cultivar Grand Naine from the TIB system exhibited a greater rooting rate than those from the solid culture system. The observed enhancement of root formation in the TIB system can be attributed to several potential mechanisms. For instance, the liquid medium in the TIB system allows for better nutrient uptake and gas exchange, with Georgieva et al. [32] demonstrating that improved aeration in liquid cultures led to increased expression of genes involved in root initiation and development. Periodic immersion may induce mild mechanical stress, potentially triggering ethylene production and auxin redistribution, as Ramos-Castellá et al. [33] found that temporary immersion systems enhanced auxin signaling and root primordia formation in agave plantlets. The liquid medium also allows for a more uniform distribution of plant growth regulators. Additionally, roots formed in the TIB system appeared to have more branching and root hairs, potentially due to the intermittent exposure to air and nutrients. To further elucidate these mechanisms, future studies will include histological analysis of root primordia formation at different culture stages, gene expression profiling focusing on key genes involved in root initiation and development, hormonal analysis to quantify changes in auxin and cytokinin levels during the culture period, and root morphological analysis using scanning electron microscopy. These investigations will provide a more comprehensive understanding of the physiological and molecular basis for enhanced root formation in the TIB system, potentially leading to further optimization of this propagation method for bananas and other important crop species.
The root length of plantlets from the semisolid culture system (7.91 cm) was comparable to those from the TIB system with immersion frequencies of once every 6 h (7.90 cm) and 8 h (7.96 cm), as shown in Figure 2D. While no significant difference in root length was observed, the TIB system showed slightly higher root formation efficiency. This subtle enhancement can be attributed to several factors, such as better nutrient accessibility and a more uniform distribution of growth regulators of the TIB system, which potentially stimulates root primordia formation, as previously discussed. Additionally, the improved gas exchange in the TIB system could enhance oxygen availability to developing roots, supporting their growth and function. The similar root lengths across systems suggest that once initiated, root elongation proceeds comparably in both environments. However, the marginally higher root count in TIB cultures indicates a potential advantage in root initiation. These findings suggest that the TIB cultivation system not only avoids negative impacts on root formation and development in the banana cultivar Kluai Numwa Pakchong 50 but may offer slight benefits. The comparable morphology observed across all plantlets (Figure 3) further supports the viability of the TIB system for banana micropropagation.
In a semisolid cultivation system, plantlets must be carefully removed from the culture vessel, and their roots must be cleaned to remove the solidifying agents. This process often damages roots and increases the risk of infection, resulting in a reduction in the survival rate and an increase in the abnormal development of acclimatized plantlets [34]. One of the advantages of the TIB system is that no cleaning and removal of gelling agents is needed, simplifying the acclimatization process and making it applicable for the large-scale production of plantlets with relatively low operating costs and low labor requirements.
The morphological and physiological parameters of the plantlets produced through the TIB system are also important determining factors. Several parameters, such as photosynthetic pigments, stomatal density, and epidermal cell size, have been analyzed, and the results demonstrated that the plantlets regenerated through the TIB system exhibited greater values of all these parameters than those regenerated using a semisolid cultivation system [5,10,15,16]. The greater growth performance of the plantlets regenerated through the TIB system may be associated with these parameters. Thus, the morphological and physiological parameters of the banana cultivar Kluai Numwa Pakchong 50 obtained from the TIB system should be evaluated and compared with those obtained using a semisolid cultivation system.
The genetic stability of plantlets obtained through in vitro propagation is also an important factor in determining the sustainability of large-scale plant production. It has previously been reported that high concentrations of PGRs or an increased number of subcultures of explants may cause genetic variation, such as chromosome rearrangements, DNA methylation, or point mutations [35,36]. Therefore, the genetic uniformity of the established plantlets derived through the TIB system should be assessed using different genetic markers, such as simple sequence repeats (SSRs) and inter-simple sequence repeats (ISSRs) [5,15,16].

4. Conclusions

The present study demonstrated that the TIB is an effective system for mass propagation of the banana cultivar Kluai Numwa Pakchong 50 compared to conventional in vitro propagation using a semisolid cultivation system. Cultivating the banana explants in the TIB system using MS medium supplemented with 7.5 mg/L BAP and applying five explants per vessel with an immersion frequency of once every 6 or 8 h for 2 min yielded promising results. This TIB-based approach produced a higher number of shoots, longer shoot length, and greater leaf formation compared to the control treatment using a semisolid culture system. Furthermore, all the in vitro plantlets regenerated from the TIB system exhibited high survival rates, high vegetative growth performance, and efficient root formation, comparable with those regenerated from the semisolid cultivation system. These findings indicate that the TIB system is a promising technique for the mass production of the Kluai Numwa Pakchong 50 banana cultivar. Moreover, this approach could potentially be extended to other banana cultivars for large-scale commercial propagation.

Author Contributions

Conceptualization, S.T., P.T. and P.K.; methodology, S.T., P.T. and P.K.; software, P.T.; validation, S.T., P.T. and P.K.; formal analysis, S.T., H.K. and P.K.; investigation, S.T., H.K. and P.K.; resources, S.T. and P.K.; data curation, S.T., P.T. and P.K.; writing—original draft preparation, S.T., P.T. and P.K.; writing—review and editing, S.T., P.T. and P.K.; visualization, P.T. and P.K.; supervision, S.T. and P.K.; project administration, S.T. and P.K.; funding acquisition, P.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Research and Graduate Studies, Khon Kaen University, through the Research Program of the Fermentation Research Center for Value Added Agricultural Products, Khon Kaen University (RP-FerVAAP-67).

Data Availability Statement

The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.

Acknowledgments

The authors would like to thank Surakiat Kreeb-choho, Jennarong Sirata, Theppon Leuangsuk, and Sadanan Thongsaman for their technical support.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The temporary immersion bioreactor (TIB) system used for multiplying banana cultivar Kluai Numwa Pakchong 50. The upper and lower compartments are the growth chamber and nutrient reservoir, respectively.
Figure 1. The temporary immersion bioreactor (TIB) system used for multiplying banana cultivar Kluai Numwa Pakchong 50. The upper and lower compartments are the growth chamber and nutrient reservoir, respectively.
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Figure 2. Ex vitro acclimatization of banana cultivar Kluai Numwa Pakchong 50 using peat moss and vermiculite at a ratio of 1:1 as planting material. The survival rate (A), plant height (B), number of roots (C), and root length (D) were recorded after 30 days of ex vitro acclimatization. TIB1 and TIB2 correspond to an immersion frequency of 2 min every 6 and 8 h, respectively.
Figure 2. Ex vitro acclimatization of banana cultivar Kluai Numwa Pakchong 50 using peat moss and vermiculite at a ratio of 1:1 as planting material. The survival rate (A), plant height (B), number of roots (C), and root length (D) were recorded after 30 days of ex vitro acclimatization. TIB1 and TIB2 correspond to an immersion frequency of 2 min every 6 and 8 h, respectively.
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Figure 3. Plantlets of banana cultivar Kluai Numwa Pakchong 50 after 30 days of acclimatization. The acclimatized plantlets of the control treatment using a semisolid MS system (A) and the TIB system with the frequency of immersion once every 6 h (B) and 8 h (C).
Figure 3. Plantlets of banana cultivar Kluai Numwa Pakchong 50 after 30 days of acclimatization. The acclimatized plantlets of the control treatment using a semisolid MS system (A) and the TIB system with the frequency of immersion once every 6 h (B) and 8 h (C).
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Table 1. Effect of BAP on shoot proliferation of the banana cultivar Kluai Numwa Pakchong 50 cultured in the TIB system and semisolid cultivation system for 30 days.
Table 1. Effect of BAP on shoot proliferation of the banana cultivar Kluai Numwa Pakchong 50 cultured in the TIB system and semisolid cultivation system for 30 days.
BAP Concentration (mg/L)Number of Shoots (Shoots/Explant)Average Shoot Length (cm)Number of Leaves (Leaves/Explant)
Semisolid MS4.40 ± 1.26 abc1.85 ± 0.41 b2.30 ± 0.67 b
0.03.50 ± 1.08 c1.73 ± 0.65 b2.30 ± 0.95 b
2.54.60 ± 1.58 abc2.68 ± 0.78 a3.20 ± 1.03 a
5.05.20 ± 1.14 ab2.36 ± 0.94 ab2.90 ± 0.88 ab
7.55.60 ± 1.26 a2.19 ± 0.78 ab2.80 ± 0.79 ab
10.04.00 ± 1.15 bc0.66 ± 0.30 c1.00 ± 0.47 c
The concentration of BAP in the semisolid MS medium was 7.5 mg/L. For the TIB system, the immersion frequency was set at 2 min every 4 h. The data are presented as the means ± SDs. Different letters within the same column indicate significant differences based on DMRT analysis (p ≤ 0.05).
Table 2. Effect of explant density on shoot proliferation of the banana cultivar Kluai Numwa Pakchong 50 cultured in the TIB system and semisolid cultivation system for 30 days.
Table 2. Effect of explant density on shoot proliferation of the banana cultivar Kluai Numwa Pakchong 50 cultured in the TIB system and semisolid cultivation system for 30 days.
Number of Explants/VesselNumber of Shoots (Shoots/Explant)Average Shoot Length (cm)Number of Leaves (Leaves/Explant)
Semisolid MS4.50 ± 0.85 bc1.83 ± 0.31 a2.10 ± 0.74 a
15.20 ± 1.03 ab1.94 ± 0.41 a2.30 ± 0.95 a
55.80 ± 1.23 a2.28 ± 0.49 a2.40 ± 0.97 a
103.90 ± 0.99 c1.39 ± 0.65 b1.20 ± 0.42 b
The MS medium supplemented with 7.5 mg/L BAP was used. For the TIB system, the immersion frequency was set at 2 min every 4 h. The data are presented as the means ± SDs. Different letters within the same column indicate significant differences based on DMRT analysis (p ≤ 0.05).
Table 3. Effect of immersion frequency of the TIB system on shoot proliferation of the banana cultivar Kluai Numwa Pakchong 50 after cultivation for 30 days.
Table 3. Effect of immersion frequency of the TIB system on shoot proliferation of the banana cultivar Kluai Numwa Pakchong 50 after cultivation for 30 days.
Immersion FrequencyNumber of Shoots(Shoots/Explant)Average Shoot Length (cm)Number of Leaves (Leaves/Explant)
Semisolid MS4.40 ± 0.97 c1.87 ± 0.40 a2.13 ± 1.06 a
Every 4 h5.20 ± 1.03 c1.98 ± 0.63 a2.40 ± 0.91 a
Every 6 h6.40 ± 1.26 b2.24 ± 0.56 a2.20 ± 0.94 a
Every 8 h8.20 ± 1.14 a1.84 ± 0.56 a2.13 ± 0.74 a
The MS medium supplemented with 7.5 mg/L BAP was used. Five explants per vessel were tested in the TIB system, while one explant per vessel was tested in the semisolid cultivation system. The data are presented as the means ± SDs. Different letters within the same column indicate significant differences based on DMRT analysis (p ≤ 0.05).
Table 4. Effect of auxins on the root formation of the banana cultivar Kluai Numwa Pakchong 50 cultured in the TIB and semisolid cultivation systems for 30 days.
Table 4. Effect of auxins on the root formation of the banana cultivar Kluai Numwa Pakchong 50 cultured in the TIB and semisolid cultivation systems for 30 days.
TreatmentConcentration
(mg/L)		Immersion Frequency of Every 6 h
for 2 min		Immersion Frequency of Every 8 h
for 2 min
NR
(Roots/Explant)		RL
(cm)		NR
(Roots/Explant)		RL
(cm)
Semisolid + NAA0.52.10 ± 0.57 ab1.00 ± 0.20 b2.00 ± 0.67 ab0.93 ± 0.20 b
TIB + NAA0.00.00 ± 0.00 c0.00 ± 0.00 c0.00 ± 0.00 c0.00 ± 0.00 c
0.11.80 ± 0.63 b0.96 ± 0.18 b1.60 ± 0.84 b0.90 ± 0.16 b
0.52.50 ± 0.53 a1.29 ± 0.19 a2.40 ± 0.84 a1.25 ± 0.22 a
1.02.50 ± 0.85 a1.23 ± 0.13 a2.20 ± 0.63 ab1.10 ± 0.18 a
Semisolid + IBA0.52.10 ± 0.99 b1.02 ± 0.17 c2.10 ± 0.57 b1.08 ± 0.15 b
TIB + IBA0.00.00 ± 0.00 c0.00 ± 0.00 d0.00 ± 0.00 c0.00 ± 0.00 c
0.12.20 ± 0.92 ab1.21 ± 0.17 b2.00 ± 0.67 b1.04 ± 0.21 b
0.52.90 ± 0.88 a1.48 ± 0.22 a2.70 ± 0.67 a1.37 ± 0.31 a
1.02.80 ± 0.63 ab1.43 ± 0.22 a2.40 ± 0.52 ab1.35 ± 0.26 a
One explant per culture vessel was tested in this study. The experiments were performed three times, each with ten replications, and the data are presented as the means ± SDs. Different letters within the same column and the same group (NAA or IBA) indicate significant differences based on DMRT analysis (p ≤ 0.05). NR is the number of roots, and RL is the root length.
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Thanonkeo, S.; Kitwetcharoen, H.; Thanonkeo, P.; Klanrit, P. Temporary Immersion Bioreactor (TIB) System for Large-Scale Micropropagation of Musa sp. cv Kluai Numwa Pakchong 50. Horticulturae 2024, 10, 1030. https://doi.org/10.3390/horticulturae10101030

AMA Style

Thanonkeo S, Kitwetcharoen H, Thanonkeo P, Klanrit P. Temporary Immersion Bioreactor (TIB) System for Large-Scale Micropropagation of Musa sp. cv Kluai Numwa Pakchong 50. Horticulturae. 2024; 10(10):1030. https://doi.org/10.3390/horticulturae10101030

Chicago/Turabian Style

Thanonkeo, Sudarat, Haruthairat Kitwetcharoen, Pornthap Thanonkeo, and Preekamol Klanrit. 2024. "Temporary Immersion Bioreactor (TIB) System for Large-Scale Micropropagation of Musa sp. cv Kluai Numwa Pakchong 50" Horticulturae 10, no. 10: 1030. https://doi.org/10.3390/horticulturae10101030

APA Style

Thanonkeo, S., Kitwetcharoen, H., Thanonkeo, P., & Klanrit, P. (2024). Temporary Immersion Bioreactor (TIB) System for Large-Scale Micropropagation of Musa sp. cv Kluai Numwa Pakchong 50. Horticulturae, 10(10), 1030. https://doi.org/10.3390/horticulturae10101030

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