A Robust Protocol for Managing Microbial Contamination of In-vitro Banana Plants

: In-vitro regeneration of banana ( Musa spp) is a crucial technique in banana improvement via modern biotechnology like virus indexing, genetic transformation, and genome editing. However, in-vitro banana plants are prone to microbial contamination from the environment, leading to the loss of important lines and germplasm. Protocols for disinfecting banana plants before their in-vitro culture have been reported; however, there is limited information on strategies for disinfecting in-vitro contaminated banana plants, which are more sensitive to most disinfectants. Thus, this study aimed to establish an efficient disinfection protocol that is effective against contaminants and safe on in-vitro plants. Contaminated in-vitro banana plants (cv. Grand naine) were subjected to commercial bleach (Jik ® ; 3.85% w/v NaClO) at different exposure times (2, 4, 6, and 8 min) with or without rinsing then reinitiated in-vitro . Jik ® exposure time of 2 min with or without rinsing preserved the plant viability by 100% but led to >75% fungal contamination. At 4 min of Jik ® exposure, the viability of the plants remained at 100%, but >33% fungal contamination was observed. No contamination was observed at 8 min of Jik ® exposure, but the plants' viability was reduced to below 85%. Notably, 6 min of Jik ® exposure preserved the viability of the plants by 100% while destroying all the contaminants and is, therefore, recommended as the most efficient treatment. This protocol can save time and other resources and should be applied in banana genetic transformation laboratories.


Introduction
Banana (Musa spp) is among the top ten most important food crops worldwide in production and consumption [1]. However, several phytopathogens threaten banana production, including viruses, fungi, bacteria, and nematodes [2][3][4][5][6][7][8]. This has necessitated its improvement via biotechnology involving in-vitro regeneration. The tissue culture of banana is particularly important in virus indexing, production of clean planting material, and genetic transformation procedures [9]. However, contamination of in-vitro banana cultures is a serious problem in the micropropagation of the crop [10]. This is especially critical in the propagation of transgenic events, in which losing one line to contamination could mean losing a year's work. In most cases, the contaminants come from the environment during the transfer of banana explants to a new medium due to improper handling, contaminated medium, or equipment failure. However, some contaminants are endogenous and may survive within the plant system for a long time [11]. Most of these contaminants include bacteria and fungi [12]. Even though most bacterial contamination can be controlled by supplementing the tissue culture medium with antibiotics [13], most fungal contaminants are difficult to eliminate and may require the plants to be removed from the culture vessel and disinfected. In-vitro plants are typically more sensitive to disinfectants than field grown plants and therefore challenging to disinfect without reducing their viability. Therefore, this study aimed to develop an efficient protocol for rescuing in-vitro banana plants from contaminants. Contaminated plants were disinfected with ethanol and commercial bleach (Jik ® ) at different exposure times to determine the optimum treatment that ensures the highest disinfection and viability of the plants. This study provides a robust protocol for rescuing invitro contaminated bananas and should be applied in laboratories dealing with banana micropropagation and genetic transformation.

Methods
In-vitro banana plants (cv. Grand naine) in mother food jars were obtained from the GTL company, Nairobi, Kenya. The lids of the bottles were opened overnight to allow contamination of the plants. Subsequently, the lids were closed, and the cultures were left to grow for seven days to allow the contaminants to establish. Next, the contaminated plants were removed from the culture vessels and immersed in 70% (v/v) ethanol for 30 s with slight agitation. After that, the plants were removed from ethanol and rinsed once with sterile distilled water then immersed in 100% Jik ® (3.85% w/v NaClO) for 2, 4, 6, and 8 min. The treatments were divided into two batches. One batch was rinsed with sterile distilled water before sub-culturing onto sterile fresh Murashige and Skoog medium [14], while the other batch was sub-cultured without prior rinsing. Control plants were rinsed with water only then, sub-cultured onto fresh sterile banana maintenance medium. The plants were cultured at 26° C under 16 h light/8h dark for 14 days. The experiment was repeated thrice, and each treatment contained four replicates. Data related to contamination and viability were recorded and subjected to one-way analysis of variance (ANOVA) and the means separated by Turkey's HSD test at p<0.05.

Results and Discussion
This is the first study to report on a protocol for disinfecting in-vitro contaminated bananas to the best of our knowledge. Ethanol and commercial bleach, particularly Jik ® have been used extensively to disinfect plants before in-vitro regeneration [15,16]. In this study, 100% Jik ® (3.85% w/v NaClO) did not affect the viability of in-vitro plants under various exposure durations except for 8 min of exposure, in which some plants bleached and underwent necrosis, reducing the viability to between 40 to 80% (Table 1). After 2 min of exposure to Jik ® , the plants were healthy with minimal or no bleaching evidence. However, contaminations rates were above 75%. Meanwhile, some contamination (less than 60%) was observed at 4 min exposure time (Table 1). Notably, this level of contamination is still considered high because contamination of a single plant in a culture vessel containing other plants eventually leads to the contamination of all the plants in the vessel. The best exposure time was 6 min without rinsing. Under this treatment, no contamination was observed, and all the plants remained viable. However, slow-growing fungal contaminants were observed in the batch that was rinsed with sterile distilled water before subculturing, indicating that rinsing reduces the disinfectant potency and should be avoided if it does not affect the viability of the plants.

Conclusion
Overall, this study shows that it is possible to save in-vitro contaminated bananas. Thus, this protocol should be applied in the laboratories dealing with banana genetic transformation and germplasm preservation.

Conflict of Interest
The authors declare no conflict of interest.