Comparison of Solid vs. Temporary Immersion Bioreactor Systems for Potato Micropropagation

Table of Contents

Abstract

Solanum tuberosum (potato) is commonly micropropagated in vitro to produce disease-free seed tubers at scale [1]. Temporary immersion bioreactors (TIBs) offer a promising alternative to traditional solid media culture by periodically bathing explants in liquid nutrient media, enhancing gas exchange and nutrient uptake [1]. In this study, we compared the micropropagation efficiency of potato in hormone-free Murashige & Skoog (MS) medium under five culture conditions: traditional solid agar medium (control) versus TIB immersion frequencies of every 24 h, 12 h, 6 h, or 1 h (with corresponding immersion durations of 4 min, 2 min, 1 min, and 10 s, respectively). Each treatment (including control) had 5 replicate vessels with 5 nodal explants each, cultured for 30 days. Key growth metrics – multiplication rate (fold increase in shoots), number of shoots per explant, mean shoot length, and mean root length – were recorded and analyzed by ANOVA and Tukey’s HSD. The TIB cultures generally produced significantly more and longer shoots than the solid medium, except that an intermediate immersion schedule (12 h/2 min) yielded markedly poorer results.

After 30 days, the fastest immersion frequency (hourly 10 s) achieved the highest shoot multiplication (16.9 shoots per explant on average) and longest shoots (~24 cm), whereas the 12 h/2 min TIB condition produced only ~7.4 shoots per explant with ~9 cm shoot length, significantly inferior to all other treatments (p < 0.05). Root development was more variable and showed no statistically significant differences among treatments. These results demonstrate that TIB systems can greatly enhance potato micropropagation in hormone-free media – improving shoot proliferation and growth – but also highlight the importance of optimizing immersion frequency and duration. An inappropriate immersion regime (in this case, 12 h/2 min) can undermine the benefits of TIB, even performing worse than solid culture.

We conclude that TIBs (such as the BioTilt™ system) are a viable and efficient platform for potato micropropagation, offering higher multiplication rates and plantlet vigor without growth regulators, provided that immersion parameters are carefully tuned. Future work will explore refined immersion schedules, different cultivars, the use of growth regulators, and the impact on subsequent tuber formation and acclimatization.

Figure 1: (A) An overview of the experiment performed using the TIB system (BioCoupler® — 24 immersions/day, 10-sec duration); and (B) demonstrates significantly higher shoot production and plantlet length compared to the solid MS medium control after one month.

Introduction

Potato is one of the world’s most important food crops, and producing high-quality seed tubers is crucial for crop yield and food security [1]. In vitro micropropagation has become an essential technique for the rapid multiplication of disease-free potato plantlets, enabling the production of uniform, pathogen-free stocks year-round [1]. Traditional potato tissue culture is often done on semi-solid agar media, which supports reliable plant growth but can be labor-intensive and limited in scale. Liquid culture systems and bioreactors offer an alternative that can automate and accelerate propagation [1]. In particular, the temporary immersion bioreactor (TIB) technology periodically immerses explants in liquid media and then drains, improving nutrient availability and gas exchange around the tissues. TIBs have demonstrated a remarkable ability to promote in vitro potato growth by facilitating oxygen renewal and continuous contact of explants with nutrients [1]. Studies have reported that bioreactor-grown potato shoots can achieve higher yield and reduced hyperhydricity compared to static liquid or solid cultures [1]. However, the performance of TIB systems depends strongly on operational parameters – especially the immersion frequency and duration, as well as medium composition [1]. An optimal balance must be struck: too-frequent or prolonged immersion might cause oxygen deprivation or tissue hyperhydration, whereas infrequent immersion might limit nutrient uptake. Previous work suggests that immersions on the order of every few hours (e.g., 3 h for 2 min) are effective in potato bioreactors [1], but there is a need to systematically compare different schedules.

This study aims to evaluate the efficiency of potato micropropagation on a hormone-free MS medium using a range of TIB immersion regimes, in comparison to the conventional solid-medium method. By quantifying the multiplication rate, shoot, and root growth under each treatment, we seek to identify how immersion timing affects plantlet development. The findings will inform best practices for using TIB (in particular, the BioTilt™ TIB system) to propagate potatoes without exogenous growth regulators. We hypothesize that appropriate TIB schedules can match or exceed the solid media in producing healthy shoots, but that suboptimal immersion frequency could hinder growth.

Materials and Methods

Plant Material and Medium: Nodal stem explants of Solanum tuberosum (potato) were used as the starting material. Explants were approximately 5 cm in length and contained three axillary buds. All cultures were grown on basal Murashige and Skoog (MS) medium without any added plant growth regulators (hormone-free). The medium was supplemented with 30 g/L sucrose as a carbon source and 2 mL/L Plant Preservative Mixture (PPM™) to suppress microbial contamination [2]. For solid medium cultures, 0.8% agar (w/v) was used as the gelling agent. The pH of the media was adjusted to 5.8 before autoclaving. All work was conducted under sterile conditions in a laminar flow hood.

Culture Systems and Treatments: Five propagation treatments were compared, differing in whether explants were grown on solid medium or in a temporary immersion bioreactor (TIB) with various immersion schedules:

Culture Systems and Treatments: Five propagation treatments were compared, differing in whether explants were grown on solid medium or in a temporary immersion bioreactor (TIB) with various immersion schedules:

1. Solid medium (Control): Explants cultured on solidified MS agar in standard culture jars (no immersion).
2. BioTilt™ – 24 h immersion cycle: Explants in a BioCoupler® immersed in liquid MS for 4 minutes every 24 hours (daily immersion).
3. BioTilt™ – 12 h immersion cycle: Explants in BioCoupler® immersed for 2 minutes every 12 hours (twice daily immersion).
4. BioTilt™ – 6 h immersion cycle: Explants BioCoupler® immersed for 1 minute every 6 hours (4 - 5 times daily).
5. BioTilt™ – 1 h immersion cycle: Explants in the BioCoupler® immersed for 10 seconds every hour (hourly frequent immersion).

For the BioCoupler® treatments, the BioTilt™ temporary immersion system (Plant Cell Technology) was used.

This system automatically tilts culture vessels to periodically bathe the explants in liquid medium and then drains it away, without manual intervention. All BioCouplers® had the same working volume of liquid medium and headspace. During the “dry” intervals between immersions, explants were exposed to humid air inside the vessel, allowing gas exchange.

Experimental Design: Each treatment consisted of 5 replicate culture vessels. In the solid medium treatment, each jar (replicate) contained 5 3-nodal explants inserted into the agar (a total of 25 explants for the treatment). In each BioCoupler® replicate, 5 3-nodal explants were placed on one of the BioCoupler® jars; when the BioTilt™ tilted, the liquid medium covered the explants for the set duration, then drained. Thus, each BioCoupler® replicate also started with 5 explants (the equivalent of 5 plants, comprising roughly 15 total nodal buds across those explants). All cultures were incubated in a growth room at 22 ± 1 °C under a 16 h light / 8 h dark photoperiod (cool-white fluorescent lighting). The experiment was initiated on September 3, 2025 (Day 0 for all cultures), and ran for 30 days without subculturing or medium exchange.

At the start of the experiment, the explants in all treatments were similar in size and appearance. Figure 1 shows the Day 0 setup for the solid medium control, with five representative culture jars each containing 5 potato nodal explants. (The BioCoupler® similarly started with comparable explant material on Day 0.) All cultures remained contamination-free throughout the 30-day period, indicating the effectiveness of sterile technique and PPM™ addition.

Figure 2: Initial Setup of Solid Media and TIB Systems (Day 0, September 3, 2025), showing the five replicate jars used for the solid-medium control treatment, each containing five potato nodal explants on hormone-free MS agar; and the Tissue Immersion Bioreactor (TIB) systems, shown at various immersion frequencies and durations, containing five potato nodal explants in hormone-free liquid MS media (without agar).

Data Collection: After 30 days of growth (harvest on October 3, 2025), each replicate was evaluated for the following parameters: (a) Number of shoots per explant – the total count of distinct shoots produced from the initial explants in that vessel, divided by the number of explants (5) to give an average shoots per explant; (b) Multiplication rate, defined here as the fold increase in shoot number per explant relative to the initial state. (Since each explant initially had three main shoots, this is essentially the same as the number of shoots per explant at Day 30, including the original shoots and new branches); (c) Average shoot length –the mean lengthof shoots in the replicate, measured from base to apex in centimeters (cm). For each replicate, all individual shoots (primary and any branches that were ≥1 cm) were measured, and their average is reported; (d) Average root length – the mean length of roots per plantlet in the replicate, in cm. Many explants produced at least some roots by Day 30; all well-developed roots were measured for each plantlet and averaged per replicate. If an explant had no roots, a length of 0 was recorded for that plant in computing the average.

Statistical Analysis: Data from the 5 replicates per treatment were used for statistical comparisons. One-way analysis of variance (ANOVA) was performed for each growth parameter (shoot count, shoot length, root length, etc.) to test for significant differences among the five treatments. When the ANOVA indicated a significant effect of treatment (p < 0.05), a Tukey’s Honest Significant Difference (HSD) post-hoc test was applied to determine which pairwise differences were significant. Differences were considered statistically significant at the 95% confidence level (α = 0.05). Results are reported as mean ± standard deviation (SD). In tables, treatments that do not share a letter were significantly different according to Tukey’s test.

Results

After 30 days of culture, substantial differences were observed in shoot multiplication and growth among the treatments. Table 1 summarizes the growth outcomes for each treatment, including the mean multiplication rate, mean number of shoots per explant, average shoot length, and average root length. Statistical groupings from Tukey’s HSD are indicated by letter notation for each variable.

Shoot Multiplication: All BioCoupler® treatments except the 12 h cycle yielded as many or more shoots per explant than the solid-medium control. The control (solid agar) produced on average ~14.7 shoots per explant, corresponding to a 2.94-fold multiplication rate (since each explant started as a single shoot). TIB with 24 h immersions and TIB with 6 h immersions showed very similar results to the control in terms of shoot numbers (around 15.1 shoots/explant, 3.0× multiplication). The most frequent immersion (1 h cycles) achieved the highest proliferation, with 16.9 shoots per explant on average, equivalent to a 3.38× multiplication – slightly higher than the control, though this difference was not statistically significant. In contrast, the intermediate 12 h/2 min immersion treatment performed significantly worse than all other groups: only about 7.4 shoots per explant on average (1.47× multiplication). Statistical analysis confirmed that the 12 h TIB treatment’s shoot production was significantly lower than the control and every other TIB regime (ANOVA p = 0.0059; Tukey HSD p < 0.05). The other four treatments (solid, TIB 24 h, 6 h, 1 h) showed no significant differences among each other for shoot number (Table 1). Thus, aside from the poorly performing 12 h schedule, the TIB systems were able to match or modestly exceed the solid medium in propagating new shoots without any plant growth hormones.

Shoot Growth (Length): The quality of shoots, as indicated by their length and vigor, differed markedly among treatments. Notably, the bioreactor-grown shoots in the 24 h, 6 h, and 1 h immersion treatments were much taller on average than those grown on solid medium. The average shoot length in the control jars was ~11.1 cm, and the 12 h TIB treatment was similarly low at ~9.2 cm (no significant difference between these two). In contrast, shoots in the TIB with 24 h immersions reached about 22.2 cm on average, and those in the 6 h and 1 h immersion treatments were about 22.9 cm and 24.4 cm long, respectively (Table 1). These three BioCoupler® conditions produced shoots roughly twice as long as the solid-medium plants. According to Tukey’s HSD, the 24 h, 6 h, and 1 h TIB groups all formed a homogeneous subset that was significantly different (p < 0.05) from the subset containing the solid and 12 h treatments. In other words, any TIB regime with daily or more frequent immersions resulted in significantly greater shoot elongation than the non-immersed agar culture (or the suboptimal 12 h regime). The BioCoupler® shoots were not only longer but also appeared to have larger, healthier leaves (qualitative observation), suggesting improved overall vigor in liquid culture when conditions were optimal.

Root Development: Unlike the clear trends seen in shoot multiplication and length, the differences in rooting among treatments were less pronounced. Somewhat surprisingly, the longest average roots were observed in the TIB 24 h treatment (9.1 cm), followed by the solid control (7.0 cm). The 6 h and 12 h TIB treatments had intermediate mean root lengths around 5.6–6.4 cm, and the 1 h frequent immersion group had the shortest mean roots (~5.4 cm). However, variability was high – many explants in all treatments produced only short roots or none at all by Day 30 (reflected in large standard deviations; see Table 1). Statistical analysis confirmed no significant differences in root length across the five treatments (ANOVA p = 0.57; all pairwise Tukey comparisons n.s.). In practical terms, this indicates that the TIB system did not greatly enhance or inhibit root formation relative to solid media under these hormone-free conditions. The slight trend toward longer roots in the 24 h immersion and solid media treatments might suggest that less frequent immersion (or a more static environment) favors root elongation, whereas very frequent immersion (1 h cycles) may discourage extensive root growth. Nonetheless, any such trend was not statistically reliable here. It was observed that many plantlets in the BioCoupler® treatments developed thick callus at the base and only short adventitious roots, possibly due to the constant high moisture and lack of rooting hormones.

Contamination and Plant Health: Throughout the 30-day culture period, no contamination was observed in any vessel, which can be attributed to the inclusion of PPM™ in the medium and strict sterile technique.

All surviving explants remained green and viable. In the 12 h BioCoupler® treatment, in addition to having fewer shoots, some explants showed signs of stress such as slight tissue browning at cut ends – potentially due to suboptimal immersion timing (discussed below). The other BioCoupler® treatments and control showed healthy, green shoots. By Day 30, the biomass (total shoot mass) was visibly greater in the successful BioCoupler® treatments (1 h, 6 h, 24 h) compared to the solid media jars, primarily due to the longer shoots and more numerous branches.

Table 1: Growth metrics after 30 days of in vitro culture of potato under different culture treatments. Values are mean ± SD (n = 5 replicates per treatment). Different letters within a column indicate significant differences between treatments for that metric (Tukey HSD, p < 0.05). No letters are displayed for root length since differences were not significant.

(Note: Multiplication rate is the fold increase in shoot number per explant relative to the initial. “Shoots per explant” is the average count of shoots produced from each initial explant after 30 days. Letters a/b denote statistical groupings within each column; treatments sharing the same letter are not significantly different for that variable. All treatments share “a” in the Root Length column, reflecting no significant differences.)

Figure 3: Representative solid media cultures at Day 30 (October 3, 2025), showing the five replicate jars used for the solid-medium control treatment and TIB systems (at different frequencies and duration of immersion) at the final day of the experiment.

Discussion

This experimental comparison demonstrates that temporary immersion bioreactors can substantially improve the growth and multiplication of potato explants in vitro, even in the absence of exogenous hormones. The BioCoupler® and BioTilt™ system provided a more dynamic culture environment that stimulated potato buds to break dormancy and elongate into shoots more effectively than the traditional static agar medium in our study. In particular, BioCoupler® treatments with very frequent short immersions (hourly 10 s) or infrequent longer immersions (daily 4 min) both achieved high proliferation (approximately 15–17 shoots per explant) and produced tall, vigorous shoots roughly double the length of those on solid media. These findings align with the known benefits of liquid immersion: improved nutrient delivery and aeration around the explants can accelerate growth [1]. By periodically submerging the tissue in fresh medium, the BioCoupler® likely ensured that even inner orotherwise dormant buds received nutrients and were induced to sprout. Additionally, liquid immersion may reduce apical dominance by flushing away inhibitory compounds, thus allowing more axillary buds to develop (a phenomenon often exploited with cytokinin in conventional micropropagation, but here achieved to some extent without added hormones).

Notably, the superiority of the BioCoupler® was most evident in shoot elongation. Shoots in the 24 h, 6 h, and 1 h TIB regimes grew to ~22–24 cm in 30 days, whereas on solid medium, they only reached ~11 cm on average. This suggests that the liquid culture provided a more favorable environment for stem growth – possibly due to better nutrient uptake (unhindered by agar diffusion limits) and improved gas exchange leading to vigorous metabolism. The constant high humidity in the closed BioCoup ler® vessels, combined with regular immersion, might have also reduced water stress and encouraged continuous growth. Interestingly, despite the increased shoot length in TIB, we did not observe excessive hyperhydricity (waterlogged, glassy tissues) in the successful BioCoupler® treatments. The shoots, though larger, appeared morphologically normal – a positive outcome that indicates the immersion durations used (1–4 min) were short enough to avoid prolonged anoxic conditions that cause hyperhydricity. This corroborates literature reports that temporary immersion systems, when properly managed, can minimize hyperhydricity while boosting growth [1].

One of the most striking results was the failure of the twice-daily (12 h/2 min) immersion schedule to support good growth. Explants under the 12 h TIB regimen produced roughly half the number of shoots as all other treatments and very short shoots, performing even worse than the solid medium control. There are several possible reasons for this outcome. It appears that immersion frequency and duration must be carefully balanced – the 2-minute immersion every 12 hours may have been an unfavorable combination. One hypothesis is that 2 minutes was insufficient to fully rehydrate and nourish the explants for the subsequent 12-hour dry interval. In the 24 h treatment, a longer 4 min immersion might have saturated the tissue enough to last an entire day, whereas in the 12 h case, the shorter immersion might not have delivered adequate moisture/nutrients to sustain growth for the next 12 hours. Essentially, the explants could have experienced a mild nutrient/water deficit repeatedly, stunting their growth. Conversely, compared to the more frequent immersions, the 12 h interval might have allowed metabolic waste (like ethylene or phenolics) to build up around the explants for half a day without flushing, potentially inhibiting bud growth. The more frequent 6 h and 1 h cycles would refresh the medium and gas environment more often, preventing such buildup. It is also possible that the 12 h schedule led to some subtle physiological stress – for example, partial drying of tissues followed by immersion can create stress conditions if not optimized. Overall, this result underscores that not all BioTilt™ schedules are beneficial; an intermediate frequency that one might assume to be moderate turned out to be the worst in this scenario. This finding is valuable for protocol optimization: it suggests that when using a given immersion duration, one should either immerse often or ensure the immersion is sufficiently thorough to cover longer intervals. A suboptimal regime can negate the advantages of TIB or even impede growth relative to solid culture.

In terms of root development, the experiment did not find a clear advantage for TIB systems. The lack of significant differences in root length across treatments may be partly due to the hormone-free medium – in potato tissue culture, robust root formation often requires an auxin or occurs during a separate rooting phase. None of the treatments here received any auxin (like IBA or NAA), so rooting was minimal and somewhat erratic in all cases. There was a hint that the daily immersion (24 h) might have yielded slightly longer roots (averaging 9 cm) compared to the more frequently immersed explants (~5–6 cm), which could be explained by the roots experiencing more aeration time in between feedings. Frequent immersion (especially the 1 h cycle) keeps roots constantly wet, which might limit their need to elongate in search of nutrients and oxygen, and could even suppress root extension due to lower oxygen in the liquid phase. Meanwhile, the solid medium provided a semi-solid support and a gentle gradient that allowed moderate root growth (~7 cm). However, given the variability and lack of statistical significance, we cannot draw firm conclusions on rooting differences. Importantly, none of the TIB treatments outright prevented rooting – a few roots did form in most vessels – indicating that, at least for short-term culture, the bioreactor environment is not detrimental to root initiation per se. For applications where rooted plantlets are desired, it may be beneficial to include a dedicated rooting step after the multiplication phase, or to incorporate a low-dose auxin into the later stage of culture.

From a practical perspective, the results highlight that a TIB system like BioTilt™ can achieve comparable or greater propagation rates than traditional agar, with the added advantages of automation and potentially lower labor. In our study, the best BioTilt™ condition (hourly immersion) produced ~3.4-fold multiplication in 4 weeks without hormones. While many conventional protocols use cytokinin (e.g., BAP or kinetin) to induce multiple shooting, here the physical stimulation of immersion alone yielded a significant proliferation of shoots. This suggests that for potato, which naturally has strong apical dominance 10, the TIB’s enhanced nutrient delivery and microenvironment may partially substitute for the need for cytokinin, at least in the short term. That said, the absolute multiplication factors observed (around 3–3.5× per month) are modest compared to what can be achieved with hormonal stimulation in some cases (often 5–10× is reported with optimal cytokinin levels). The main takeaway is that TIB can boost growth even in hormone-free conditions, and it provides a baseline that could be further improved by medium additives.

It is also instructive to compare the 1 h and 6 h immersion schedules with literature recommendations. A frequency of every 3 h for 2 min is noted as effective in other potato TIB studies 8 – this lies between our tested 1 h and 6 h intervals. Our data showed that 6 h (with 1 min) and 1 h (with 10 s) were both effective and not significantly different in outcomes. It is possible that a 3 h/2 min regime would perform similarly or even slightly better, as it provides a middle ground – enough frequency to keep nutrients flowing, and enough duration to soak thoroughly. The fact that 1 h immersions did not out-yield 6 h immersions in shoot count suggests there may be a saturation point beyond which increasing frequency returns diminishing benefits. In practical terms, more frequent immersions also mean the automation runs more often, which could increase wear or energy use, so if 6 h gives the same result as 1 h, one might choose the less frequent. On the other hand, the hourly cycle did tend to produce the absolute longest shoots (24.4 cm) and the highest (though not significantly highest) shoot count. Therefore, for maximal top growth in a given time, very frequent feeding seems helpful. This could be because the rapidly expanding shoots in the 1 h treatment never entered a nutrient-depleted state; they always had fresh sucrose and minerals each hour to support continuous growth. In contrast, in 6 h or 24 h treatments, nutrients around the explant might be consumed or localized between immersions, slowing growth until the next medium renewal.

Limitations: One anomaly in the data was the high variability in root length, which made it difficult to detect differences. Some replicates, especially in the 12 h and 1 h treatments, had one or two jars where almost no roots formed, skewing the average. This could be due to slight differences in explant condition or microenvironment. Additionally, the experiment was conducted with a single potato variety under a specific set of conditions; results could vary with different cultivars or if the explant type (e.g. single-node vs multi-node cuttings) were different. We also note that the multiplication rates reported include all shoots present after 30 days, counting secondary branches. In a few cases, TIB-grown shoots had begun to branch further (producing second-order shoots), which contributed to the count. On solid medium, branching was minimal. If subculturing were to continue beyond 30 days, the differences might become even more pronounced in favor of the TIB, as those secondary buds would continue to develop.

Conclusion

In summary, the temporary immersion bioreactor approach proved to be an effective method for potato micropropagation under hormone-free conditions. Compared to the conventional solid agar method, the TIB systems (BioTilt™) produced equal or greater numbers of shoots and significantly taller, more vigorous plantlets within the same 30-day period. These advantages stem from the improved cultural environment in TIBs – periodic immersion delivers nutrients and oxygen more efficiently to the explants, which in turn accelerates growth 2. However, this study also clearly demonstrates that the success of a TIB system is highly dependent on the immersion schedule. While very frequent immersions (every hour) and very infrequent immersions (once a day) both yielded strong results, an intermediate schedule (every 12 hours) was detrimental, underscoring that suboptimal timing can negate the benefits of automation. Users of TIB systems should thus carefully optimize the frequency and duration parameters for their specific plant material. When tuned correctly, TIBs can achieve high propagation rates without the need for plant growth regulators, simplifying the protocol and reducing chemical inputs.

For potato seed production programs, adopting TIB technology could significantly increase throughput and reduce labor, as dozens of explants can be grown in a single automated vessel with minimal handling. The hormone-free success observed here is particularly attractive for maintaining genetic stability (avoiding potential somaclonal variation sometimes associated with prolonged hormone use) and for cost reduction. The results indicate that even if growth regulators are omitted, one can still attain a 3–4 fold multiplication per month in TIB, which can be invaluable for rapid bulking of virus-free seed stock. Furthermore, the enhanced shoot vigor in TIB-grown plantlets might translate to better performance during acclimatization and transplanting to soil, especially if coupled with a robust root system [1]. That said, the root development issue will need to be addressed to take full advantage of these plantlets.

Future Work

Several avenues of future research and development are suggested by this study:

• Refining Immersion Protocols: Additional intermediate frequencies and immersion durations should be tested to pinpoint the optimal schedule for potato explants. For instance, a 3 h immersion interval (as noted in literature) or a 2 h interval could be evaluated, and the effect of immersion duration (e.g. 30 s vs 1 min vs 2 min) at each frequency could be studied. This would help generate a response surface for immersion parameters, guiding users to the best settings for maximum shoot proliferation with minimal stress.
• Cultivar Variability: The experiment can be repeated with different potato cultivars or clones. Some genotypes may respond differently to immersion – for example, some might tolerate longer intervals or might be more prone to hyperhydricity. Understanding genotype-specific responses will be important for the practical deployment of TIBs in potato micropropagation, since commercial seed programs handle multiple varieties.
• Inclusion of Growth Regulators: While this study deliberately avoided hormones to isolate the effect of the physical culture method, it would be worthwhile to examine how adding a cytokinin (such as 6-BAP or kinetin) into the TIB medium impacts results. We anticipate that a low dose of cytokinin could further boost the number of shoots per explant (by breaking apical dominance more strongly) [1]. It would be interesting to see if the combination of hormonal stimulation and optimal immersion yields multiplicative benefits or if the effects plateau. Care must be taken to adjust immersion parameters if hormones accelerate growth (to ensure nutrient supply keeps up).
• Nutrient Medium Optimization: The use of alternative basal salt formulations or enriched media (for example, E3P/ETP01 basal salts medium, which has a modified macroelement profile) could be explored in TIB versus solid culture. A more concentrated or optimized medium might better support the rapid growth in TIB and possibly improve root development. Future tests could compare standard MS with other formulations in the bioreactor to see if even higher growth rates or improved rooting can be achieved.
• Microtuber Induction in TIB: An important next step for potato propagation is the production of microtubers (in vitro tubers) in liquid systems. Given the promising shoot growth in the TIB, one could introduce tuberization conditions (such as higher sucrose concentration, growth regulators like gibberellin inhibitors, and a dark period) to see if these shoots can form microtubers in situ. Temporary immersion bioreactors have been reported to produce more and larger microtubers than solid media [1, 3], so adapting our system for tuber induction could further enhance its utility for seed tuber programs.
• Scaling and Economic Analysis: Finally, it would be valuable to evaluate the scalability of the BioTilt™ TIB system for commercial production. This includes analyzing how many propagules can be handled per vessel and per machine, the labor savings from automation, and any differences in hardening-off (greenhouse acclimatization) success between TIB-derived plantlets and agar-derived plantlets. Previous studies indicate TIB-grown potato plantlets (or microtubers) can have high survival and performance ex-vitro [1], but confirming this under our specific conditions would be important. An economic comparison factoring in medium costs (liquid vs agar), labor, and equipment costs would help determine the break-even point at which switching to bioreactors becomes advantageous for a given production scale.

In conclusion, temporary immersion bioreactors represent a powerful tool for improving potato micropropagation efficiency. This study provides proof-of-concept that even without growth regulators, an optimized TIB schedule can yield robust potato shoots ready for downstream applications. By continuing to refine the technique and address remaining challenges (like rooting and tuberization), TIB systems could play a key role in the large-scale production of seed potatoes and possibly other clonally propagated crops, combining biological efficacy with automation to meet the growing demands of agriculture.

References

1. Jácome Sarchi, G. A., Coronel Montesdeoca, N. T., Hernández, F., & Martínez, R. T. S. (2025). In Vitro Techniques for Seed Potato (Solanum tuberosum L.) Tuber Production: A Systematic Review. Plants, 14(17), 2777. https://doi.org/10.3390/plants14172777
2. Plant Preservative Mixture (PPM™). https://plantcelltechnology.com/products/plant-preservative-mixture-ppm
3. Gautam, S., Solis-Gracia, N., Teale, M. K., Mandadi, K., da Silva, J. A., & Vales, M. I. (2021). Development of an in vitro Microtuberization and Temporary Immersion Bioreactor System to Evaluate Heat Stress Tolerance in Potatoes (Solanum tuberosum L.). Frontiers in plant science, 12, 700328. https://doi.org/10.3389/fpls.2021.700328