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

Table of Contents

1. Introduction

Ludisia discolor is a terrestrial orchid valued for its ornamental foliage and compact growth habit. Conventional micropropagation of L. discolor is commonly performed using solid agar-based tissue culture systems, which often result in relatively low multiplication rates and slow biomass accumulation. Improving propagation efficiency while maintaining plant quality is therefore of interest for both research and commercial applications.

Temporary immersion bioreactor (TIB) systems provide an alternative approach by periodically exposing plant material to liquid medium, increasing nutrient availability while allowing intervals of aeration. The BioCoupler® system, automated by BioTilt™, is a passive temporary immersion platform that does not rely on pumps or forced aeration. Instead, immersion is achieved through programmed tilting, simplifying system design and increasing space efficiency.

This study represents a preliminary pilot experiment, conducted before the formalization of a standardized operating procedure. The purpose of the experiment was to generate initial performance data for Ludisia discolor grown in BioCoupler® temporary immersion culture compared to traditional solid tissue culture, and to evaluate the effects of different immersion frequencies and durations.

2. Objective

The objective of this pilot study was to preliminarily compare the propagation performance of Ludisia discolor grown in:

• BioCoupler® temporary immersion bioreactors (BioTilt™-automated), and
• Traditional solid tissue culture,

with emphasis on:

Multiplication rate (node-based),

• Plant size,
• Root development,
• Qualitative plant robustness.

Figure 1: (A) Schematic overview of the experiment conducted with the TIB system
(BioCoupler™, 24 immersions per day with 10-second immersion cycles). (B) Growth
comparison showing Ludisia discolor cultured in BioCoupler™ displaying markedly
better development than plants grown on solid MS medium after one month.

3. Materials and Methods

3.1 Plant Material

In vitro-established Ludisia discolor plantlets were used as starting material. Each replicate was initiated with four plantlets, each containing two nodes. All plant material was already growing in tissue culture and was considered sterile; no surface sterilization was performed.

3.2 Culture Media

All treatments used the same basal formulation to isolate the effect of culture system and immersion regime:

• Murashige and Skoog (MS) basal salts
• Carbon source (30 grams of sucrose; standard laboratory concentration)
• No plant growth regulators

For solid tissue culture:

• 8 grams of Agar was added at the laboratory standard concentration.

For BioCoupler® treatments:

• Liquid medium was used without agar.

3.3 Experimental Design

The experiment was conducted from September 11, 2025, to January 23, 2026.

Traditional Solid Tissue Culture (Control)

Five solid culture replicates (Solid Media 1–5) were established in parallel with BioCoupler® treatments.

BioCoupler® Temporary Immersion Treatments

BioCoupler® units were operated using BioTilt™ automation under the following immersion schedules:

• 24 hours/4 minutes (5 replicates)
• 12 hours/2 minutes (5 replicates)
• 6 hours/1 minute (5 replicates)
• 1 hour/10 seconds (5 replicates)

Each BioCoupler® represented one replicate.

Excluded Data

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

3.4 Data Collection

Final data collected per replicate included:

• Final shoot count derived from nodes,
• Calculated multiplication rate,
• Average plant length,
• Average root length,
• Qualitative growth observations.

Photographic documentation was collected at Day 0 and at the final harvest. In this pilot experiment, images were taken with plants inside their respective culture vessels (solid containers or BioCouplers). While these images provide visual confirmation of growth and overall vigor, changes in plant orientation and overlapping tissues during culture limited their usefulness for precise size comparisons.

Initial and final fresh weight, biomass gain, and standardized monthly photographic documentation were not collected, as this experiment predates SOP standardization.

4. Results

4.1 Solid Tissue Culture

Solid tissue culture produced relatively modest multiplication and limited root development.

• Average multiplication rate: 5.19
• Final shoot counts ranged from 28 to 35
• Average plant length: approximately 5 cm
• Root systems were consistently described as smallTwo of five solid replicates were contaminated and excluded from averaging.

4.2 BioCoupler® – 24 Hours/4 Minutes

This immersion schedule resulted in a substantial increase in multiplication compared to solid culture.

• Average multiplication rate: 15.28
• Final shoot counts ranged from 42 to 119
• Average plant length: 5–8 cm

Plant morphology varied among replicates. Some produced smaller, clumpier plants with very high multiplication, while others produced significantly larger individual plants with fewer shoots.

4.3 BioCoupler® – 6 Hours/1 Minute

The 6-hour immersion interval produced high multiplication with increased variability.

• Average multiplication rate: 13.78
• Final shoot counts ranged from 38 to 136
• Average plant length: 5–7 cm

One replicate exhibited extremely high multiplication associated with dense clumping and smaller individual plants. One replicate was contaminated and excluded.

4.4 BioCoupler® – 1 Hour/10 Seconds

The hourly short-immersion treatment produced consistently robust plants.

• Average multiplication rate: 12.13
• Final shoot counts ranged from 39 to 76
• Average plant length: 6–8 cm
• Root systems were consistently described as healthy and well-developed

Plants in this treatment were frequently noted as compact, vigorous, and well-balanced between shoot multiplication and root growth.

Figure 2: Initial experimental setup (Day 0; September 11, 2025) of Ludisia discolor. All
vessels contain four plantlets (two nodes each) cultured on hormone-free MS media.
The image displays five replicate jars for the solid-medium control (agar) alongside
Temporary Immersion Bioreactor (TIB) systems operating at varying immersion
frequencies and durations in liquid medium.

Figure 3: Representative cultures at Day 134 (January 23, 2026), showing plant growth in the five replicate jars used for the solid-medium control treatment and in the TIB systems operating at different immersion frequencies and durations on the final day of the experiment.

5. Discussion

All BioCoupler® treatments substantially outperformed traditional solid tissue culture for Ludisia discolor, even in the absence of plant growth regulators. Average multiplication rates in BioCoupler® systems were more than double those observed in solid culture.

Differences among immersion schedules revealed trade-offs between multiplication intensity and plant morphology.

• The 24 h/4 min schedule favored maximum multiplication but occasionally produced clumpier plants.
• The 6 h/1 min schedule yielded the highest individual replicate multiplication but showed greater variability.
• The 1 h/10 sec schedule produced slightly lower multiplication while consistently generating robust plants with superior root development.

These outcomes are consistent with expectations for passive temporary immersion systems, where frequent short immersions promote aeration while ensuring adequate nutrient exposure.

6. Limitations

As a preliminary study, this experiment lacked:

• Initial and final fresh weight measurements,
• Biomass gain calculations,
• Standardized monthly photographic documentation.

Photographic documentation was limited by the use of in-vessel imaging, which made accurate visual comparison of plant size difficult due to plant movement, overlap, and variable orientation during growth. Despite these limitations, the dataset clearly demonstrates system-level differences between BioCoupler® temporary immersion culture and traditional solid tissue culture.

7. Conclusions

This pilot study demonstrates that BioCoupler® temporary immersion bioreactors automated by BioTilt™ significantly enhance the micropropagation performance of Ludisia discolor compared to traditional solid tissue culture.

Among the tested immersion schedules:

• 24 h/4 min maximized multiplication,
• 1 h/10 sec provided the best balance between multiplication, plant size, and root quality.

The BioCoupler® system shows strong potential as a scalable and efficient propagation platform for Ludisia discolor.

8. Future Work

Future experiments will follow the finalized SOP and will include:

• Initial and final fresh weight measurements,
• Biomass gain calculations,
• Monthly photographic documentation,
• Standardized grid-based imaging of explants outside culture vessels at both Day 0 and final harvest,
• Evaluation of optimized multiplication and rooting media.

These improvements will enable more rigorous quantitative analysis and support further optimization of immersion parameters.