Comparison of Temporary Immersion Bioreactor (SETIS TM ) and Classical Solid Culture in Micropropagation of ‘Grand Naine’ ( Musa spp.) Banana Cultivar

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Introduction
According to a study by El Barnossi et al. (2021), global banana production data for the year 2018 reached approximately 115.74 million tons.Among the top banana-producing countries, India emerged as the leader with a production of 30.80 million tons, followed by China with 11.22 million tons, Indonesia with 7.26 million tons, and Brazil with 6.75 million tons, as reported by Napoleã et al. (2021).The production rankings remained consistent in 2019, with India maintaining its top position by producing 30.4 million tons, followed by China with 11.6 million tons, Indonesia with 7.2 million tons, Brazil with 6.8 million tons, and Ecuador with 6.5 million tons (Alzate Acevedo et al., 2021).Although Turkey's banana production is relatively lower compared to these leading countries, it has been observed to demonstrate a consistent growth trend.According to FAO (2022) data from 2020, India produced 31.5 million tons, whereas Turkey's production stood at only 0.72 million tons.

Biotechnological Banana Production Techniques and Grand Naine as a Banana Variety
The production of the banana plant is conducted using different techniques around the world.Banana plants produce suckers with a solid vascular connection to the parent plant.These can be removed from the parents, planted separately, and propagated again.In production using suckers, the characteristics of the primary source are preserved (Heslop-Harrison & Schwarzacher, 2007).An average of 8-10 saplings are obtained from one plant yearly, which is insufficient for commercial production.In addition, plant tissue culture techniques are used in the propagation of banana plants in many countries because of their negative features, such as the high risk of disease and pest transmission.With this technique, it is possible to produce disease-free, fast, and commercially potentially proven products.In addition, it is an essential technique to ensure the conservation of gene resources and to carry out biotechnological studies.On the other hand, it is noteworthy that this technique is costly, and especially the hardener has a large share in this cost.Because of the high costs, production in the laboratories of small and medium-sized enterprises is insufficient in quantity and quality (Jekayinoluwa et al., 2019;Uma et al., 2021).
In vitro tissue culture and micropropagation techniques continue to be employed in commercial settings, and ongoing advancements in these methodologies are closely monitored.As biotechnological developments increased, more effective suspension cultures and in vitro micropropagations were directed.The first notable advantages of using liquid media in micropropagation are the cost per plantlet and the reduction of personnel load by evaluating the automation option (Aitken-Christie, 1991).In addition, some advantages exist, such as more homogeneous nutrient elements in liquid culture, easy renewal of nutrient media, and larger culture vessels (Etienne et al., 2006).On the other hand, it has some disadvantages depending on the shaking systems frequently used in suspension cultures.The high amount of electricity used for shaking, hyperhydricity, damage to the cells by shaking, and low level of ventilation are some of its negative features (Gupta & Prasad, 2006).
Temporary immersion bioreactor (TIB) or Temporary immersion system (TIS) is one of the most important technologies in today's agricultural biotechnology as a promising method to prevent all these negativities.This method combines the advantages of solid and liquid culture (Jain & Ishii, 2012) and was first described by Steward et al. (1952) and reported as having been developed by Steward and Shantz (1956) (Preil, 2005).The RITA ® system, introduced by Alvard et al. (1993), represents the initial instance of a temporary immersion system (TIS) with a single jar featuring upper and lower compartments (Posada-Pérez, 2017).This pioneering system was specifically employed in the study conducted by Alvard et al. (1993) concerning banana plants (Persson, 2012).Apart from this, the Twin-Flask system (BIT ® ) (Escalona et al., 1999), Plantform, Ebb-and-Flow, Rocker systems, and many more systems are explained in detail by Georgiev et al. (2014).Besides these systems, SETIS™ has been reported to be more advantageous than others, as it is relatively larger and easier to use (Hwang et al., 2022).
Temporary immersion systems are considered more economically viable in commercial settings due to several factors.Firstly, these systems eliminate the need for solidifying agar, resulting in cost savings.Additionally, the labor requirements are reduced compared to traditional methods.Moreover, temporary immersion systems effectively address the issue of vitrification, also known as hyperhydricity, which can occur when plants are directly immersed in liquid media.Studies by Etienne and Berthouly (2002) and Uma et al. (2021) have highlighted the ability of temporary immersion systems to mitigate vitrification problems.In addition to banana plants, temporary immersion systems have successfully reproduced various other plant species.For example, Topoonyanont et al. (2012) utilized these systems in sugarcane studies, Jiménez et al. (1999) in potato research, and Firoozabady and Gutterson (2003) as well as Daungban et al. (2017) in pineapple-related investigations.Temporary immersion systems have also proven useful in the reproduction studies of other species such as Abies nordmanniana (Steven) Spach (fir), Eucalyptus grandis × E. urophylla (eucalyptus), Betula pubescens Ehrh (hairy birch), and Betula pendula var.carelica (curly birch), as reported by Businge et al. (2017).Overall, temporary immersion systems offer economic advantages, alleviate vitrification concerns, and have demonstrated successful applications in the reproduction of various plant species, expanding their utility beyond banana plants.Temporary Immersion Bioreactor Systems (TIS) have been under investigation for the past three decades, resulting in the development of various prototypes.Notably, the RITA ® (Recipient for Automated Temporary Immersion System) and BIT ® (Twin Flasks) systems are widely utilized for clonal propagation in tissue culture.The initial version of the RITA ® system was introduced by Alvard et al. (1993), while subsequent modifications were made by Pavlov andBley (2006), andZhu et al. (2015).On the other hand, the BIC (Bioreactor with Internal Cylinders) systems were developed by Escalona et al. (1999) and have been employed for diverse plant species, as demonstrated in studies by Escalona et al. (2003), andWelander et al. (2007).TIS bioreactors have proven effective in the mass propagation of strawberries (Takayama and Akita, 1998), ornamental plants (Dewir et al., 2006), Vaccinium angustifolium (Debnath, 2009), potatoes (Piao et al., 2003), and various other plant species.However, the previous bioreactor designs encountered challenges due to their small size or excessive weight, rendering them impractical (Welander et al., 2007).The Plantform bioreactor is another novel temporary immersion system developed to overcome the limitations of previous temporary immersion bioreactors.This innovative system offers advancements in plant tissue culture and clonal propagation techniques.The Plantform bioreactor is designed to provide efficient and controlled immersion of plant tissue or explants in a nutrient medium.It features a well-engineered design that ensures optimal gas exchange, nutrient supply, and growth conditions for plant cultures.The essential advantage of the Plantform bioreactor lies in its practicality and scalability.Unlike earlier designs that were too small or heavy, the Plantform bioreactor strikes a balance, making it suitable for large-scale production.It enables the mass propagation of various plant species, including ornamental plants, fruits, and vegetables (Aka Kaçar et al., 2020;Umarusman et al., 2020).
TISs offer a combination of benefits derived from conventional semi-solid and liquid media used in tissue culture.When compared to classical tissue culture systems, TIS provides several advantages.These include enhanced and uniform contact between the culture media, plant material, and nutrient media, reducing vitrification and asphyxiation.TIS also minimizes the presence of toxic compounds secreted by cultures, leading to decreased browning compared to liquid media.The periodic change of atmosphere within the culture plates in TIS prevents the accumulation of harmful gases such as CO 2 and ethylene, contributing to improved growth conditions.Additionally, using larger culture plates in TIS allows for longer sub-culture durations.TIS offers easier and more efficient culture performance than solid nutrient media, which requires greater attention, care, and labor.Another advantage of TIS is its ability to promote cell division with the assistance of bubbles generated through air circulation.These advantages collectively contribute to an increased propagation coefficient and improved shoot quality within the TIS.In summary, TIS brings together the positive attributes of both semi-solid and liquid media in tissue culture, resulting in a system that offers uniform contact, reduced vitrification and asphyxiation, decreased browning, improved gas exchange, longer sub-culture durations, easier handling, and enhanced cell division.These advantages ultimately contribute to higher propagation coefficients and improved shoot quality (Lambardi et al., 2015).
The objective of this study is to assess the efficacy of micropropagation and rooting of the commercially promising banana variety 'Grand Naine' using the innovative Temporary Immersion Bioreactor (TIB) system, SETIS™, in comparison to the conventional tissue culture method.The study also aims to investigate the impact of immersion frequency and duration in TIB systems on the reproduction process, with a view to comparing the outcomes.The primary focus is to determine the success rate of 'Grand Naine' banana variety in terms of micropropagation and rooting when utilizing the SETIS™ TIB system.This novel system offers an alternative approach to the traditional tissue culture method.The immersion frequency and duration, which play crucial roles in TIB systems, will be closely examined to understand their influence on reproductive efficiency.By conducting a comparative analysis between the SETIS TM TIB system and the classical tissue culture method, the study aims to provide insights into the effectiveness and potential advantages of employing the innovative TIB system for the micropropagation and rooting of 'Grand Naine' bananas.Understanding the impact of immersion frequency and duration on the reproduction process will contribute to further optimizing the TIB system for commercial banana propagation.

Method
Rhizomes of 'Grand Naine' banana cultivars were used as plant material from a greenhouse belonging to farmers in Alanya/Mersin/Türkiye.By cleaning the coarse dirt of the rhizomes, the root, leaf, and upper stem parts were removed and the meristematic shoot tips in the inner part of the rhizomes were used as an explant source.The rhizomes, which were selected and collected from the greenhouses, were brought to the laboratory and shrunk to a size that the tips of the shoots could be accessed with the help of a knife.The shrunken explants were subjected to surface sterilization.The shoot tips, which were cut and reduced, were first washed under running tap water for 20 min.

Surface Sterilization
The shoot tips, which were cleaned of coarse dirt by washing were taken into a laminar flow cabinet and kept in a mixture containing Tween 20 (Sigma-Aldrich) and 0.35% (v/v) sodium hypochlorite for 5 min then washed 3 times with sterile water.

Media and Culture Conditions
Murashige and Skoog (MS, 1962) medium supplemented with 30 g⋅l -1 sucrose and 2 mg⋅l -1 BAP was used as the culture medium.The pH of the culture medium was adjusted to 5.7-5.8 using 0.1N HCl and NaOH, and 2.4-2.6 g⋅l -1 Gelrite (Duchefa Biochemie, NL) was used as the gelling agent.The prepared culture medium was sterilized by autoclaving at 121C and 1.05 kg⋅cm -2 for 30 minutes.Afterward, the media was poured into culture containers in a sterile cabinet and left to solidify.
The explants, whose surface sterilization was completed, were shrunk to appropriate sizes in a sterile cabinet, transferred to the nutrient medium, and cultured under a 16/8-h (light/dark) photoperiod at 25±2 °C.Enough plants were reproduced by subculturing in the same environment and culture conditions every 4 weeks.The desired amount was reached in the 4th subculture and TIB trials were established after this stage.

Micropropagation
Five brand bioreactors were used for TIB trials.For each TIB, 1 liter of the nutrient medium was prepared without Gelrite.Two separate trials were set up at different times, and each trial was compared with plantlets propagated in solid media.50 plants were transferred to the TIB system and 10 plants were transferred to a solid medium.Three different trials were set up with different immersion periods and contact times in TIB trials.The

Results
The  including genotype, media content, and culture conditions (Zekai et al., 2022).Previous studies have shown diverse and sometimes conflicting results.Our study has contributed important insights and approaches to the production of banana seedlings, which hold significant importance in global agricultural production.

Conclusions
This study focused on exploring the potential of SETIS TM , a temporary immersion bioreactor system, as an innovative and high-throughput method for tissue culture, especially in the context of banana production with the 'Grand Naine' variety.The aim is to compare SETIS TM with the traditional banana growing method and to examine the effect of different immersion periods and contact times in bioreactors on the efficiency of banana production.Data obtained as a result of experimental studies revealed that SETIS TM performed better than traditional solid banana growing medium in terms of micro-propagation, rooting and adaptation to greenhouse conditions.The results achieved highlight the significant advantages offered by SETIS TM in promoting the growth and development of the banana plant.However, this study emphasizes the importance of considering the frequency of immersion and contact time in SETIS TM , as micropropagation, rooting and adaptation factors to greenhouse conditions have an impact on in vitro tissue culture studies.Therefore, it is of great importance to optimize parameters such as dipping frequency and contact time to achieve positive results in production processes.SETIS TM exhibits many desirable properties, including user-friendliness, significant surface area for plant growth, relatively low cost, and compatibility with automation.These features further increase its appeal as a viable option for large-scale commercial in vitro banana production.It is envisaged that by adopting SETIS TM and its associated benefits, the agricultural industry can unlock greater efficiency and productivity in growing banana seedlings.Overall, this study underlines the importance of using advanced techniques such as SETIS TM in in vitro tissue culture studies.Through comprehensive optimization and strategic application of immersion frequency and contact time, researchers and practitioners can achieve more successful and sustainable production results in the field of plant tissue culture.
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