Preliminary Comparison of BioCoupler® Temporary Immersion Bioreactors (BioTilt™-Automated) and Traditional Solid Tissue Culture for Agave parrasana

Table of Contents

1. Introduction

Micropropagation of Agave parrasana is of interest for both conservation and commercial production. Traditional solid tissue culture systems are widely used but can be labor-intensive and space-limited. Temporary immersion bioreactor (TIB) systems offer potential advantages, including improved nutrient uptake, enhanced gas exchange, and higher propagation density per unit area.

The BioCoupler® system, automated by BioTilt™, is a passive temporary immersion system that does not rely on pumps or forced aeration. Instead, immersion is achieved through programmed tilting, simplifying operation, and increasing space efficiency. However, optimal immersion frequency and duration can differ from actively aerated TIB systems described in the literature.

This study represents a preliminary pilot experiment conducted prior to SOP finalization. The objective was to compare BioCoupler® performance against traditional solid tissue culture for Agave parrasana under hormone-free conditions and to explore the effects of different immersion schedules.

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

2. Objective

To preliminarily evaluate the performance of BioCoupler® temporary immersion bioreactors (BioTilt™-automated) compared to traditional solid tissue culture for Agave parrasana, focusing on:

• Shoot multiplication
• Plant size
• Root development

This experiment was conducted as a feasibility study and does not include all

measurements defined in the finalized SOP.

3. Materials and Methods

3.1 Plant Material

Agave parrasana plantlets previously established in tissue culture were used. Each replicate was initiated with three small shoots (plantlets) derived from a compact clump. No surface sterilization was performed, as the material was already sterile.

3.2 Culture Media

All treatments used the same basal formulation:

• Murashige and Skoog (MS) basal salts
• Sucrose: 30 g/L
• Plant Preservative Mixture (PPM™): 2 mL/L
• No plant growth regulators (PGRs)

For solid culture:

• Agar: 8 g/L

For BioCoupler® treatments:

• Liquid medium only (no agar)

3.3 Experimental Design

The experiment was conducted from September 11, 2025, to January 6, 2026 (117 days).

BioCoupler® Treatments (BioTilt™-Automated)

BioCouplers were operated using the following immersion programs:

• 24 hours / 4 minutes
• 6 hours / 1 minute
• 1 hour / 10 seconds

Each BioCoupler represented one replicate.

Solid Tissue Culture Control

Solid MS medium was used as the traditional control.

Each treatment consisted of five replicates, except where noted due to contamination or exclusion.

Excluded Data

• The 12-hour/2-minute BioCoupler treatment was excluded from analysis due to a BioTilt™ operational issue that prevented proper immersion. These replicates were classified as discarded rather than failed.

3.4 Data Collection

Due to the pilot nature of the experiment, data collection was limited to final measurements:

• Final shoot count per replicate
• Estimated multiplication rate
• Average plant length
• Average root length

Initial fresh weight, final fresh weight, and biomass gain were not recorded, as the SOP had not yet been finalized at the time of the experiment.

4. Results

4.1 Multiplication

Solid tissue culture produced relatively low multiplication rates, typically yielding 4–8 shoots per replicate, corresponding to multiplication rates ranging from 0.24 to 1.2.

BioCoupler® treatments showed greater variability depending on the immersion schedule:

• 24 h/4 min: moderate multiplication, generally comparable to solid culture.
• 6 h/1 min: the highest multiplication observed in the study, with one replicate producing 39 shoots, corresponding to a multiplication rate of 8.64.
• 1 h/10 sec: consistently higher multiplication than solid culture in surviving replicates, with multiplication rates up to 3.12.

4.2 Plant Size and Root Development

Average plant length across treatments ranged from approximately 2 to 6 cm. BioCoupler®-grown plants generally exhibited comparable or greater plant length than solid controls.

Root length ranged from 3 to 11 cm, with the longest roots observed in BioCoupler® treatments, particularly under the 6 h/1 min immersion schedule.

4.3 Contamination

Contamination occurred in several replicates across different treatments, including solid culture and BioCoupler® treatments. Because explants were introduced sequentially and not randomized, contamination was attributed to the initial explant condition rather than the treatment effect. No conclusions regarding contamination frequency by immersion schedule were drawn.

Figure 2: Initial Setup of Solid Media and TIB Systems (Day 0, September 11, 2025), showing the five replicate jars used for the solid-medium control treatment, each containing three Agave shoot explants on hormone-free MS agar; and the Tissue Immersion Bioreactor (TIB) systems, shown at various immersion frequencies and durations, containing three Agave shoot explants in hormone-free liquid MS media (without agar).

Figure 3: Representative solid media cultures at Day 117 (January 6, 2026), showing growth in the five replicate jars used for the solid-medium control treatment and TIB systems (at different frequencies and immersion durations) on the final day of the experiment.

5. Discussion

Despite the absence of plant growth regulators, BioCoupler® temporary immersion culture supported higher multiplication than traditional solid tissue culture for Agave parrasana in this pilot study.

The 1 h/10 sec immersion schedule performed consistently well, supporting the hypothesis that short, frequent immersion pulses are advantageous in passive TIB systems, where gas exchange occurs primarily during the non-immersed phase.

The 6 h/1 min schedule also performed strongly, producing the highest multiplication observed, suggesting that intermediate immersion frequencies may balance tissue wetting and aeration effectively for Agave-type plants.

The lack of biomass measurements limits conclusions regarding plant robustness. However, visual observations and root length measurements suggest that BioCoupler®-grown plants were not inferior in quality to solid-grown controls.

6. Limitations

This study predates the finalized SOP and therefore lacks:

• Initial and final fresh weight measurements
• Biomass gain calculations
• Monthly photographic documentation
• Strict replicate uniformity

As a result, conclusions should be interpreted as preliminary.

7. Conclusions

This pilot experiment indicates that BioCoupler® temporary immersion bioreactors automated by BioTilt™ can outperform traditional solid tissue culture for Agave parrasana in terms of shoot multiplication and root development, even in hormone-free media.

Short, frequent immersion schedules (1 h/10 sec) and intermediate schedules (6 h/1 min) appear promising for Agave propagation in passive TIB systems.

8. Future Work

Future experiments will strictly follow the finalized SOP and will include:

• Initial and final fresh weight measurements
• Biomass gain calculations
• Monthly photographic documentation
• Use of optimized multiplication and rooting media with PGRs
• Replication across additional Agave species

These improvements will allow for more robust statistical comparisons and support protocol optimization.