18 Feb 2026

The Science of Better Multiplication and Rooting in Tissue Culture

Anjali Singh, MS

As a content and community manager, I leverage my expertise in plant biotechnology, passion for tissue culture, and writing skills to create compelling articles, simplifying intricate scientific concepts, and address your inquiries. As a dedicated science communicator, I strive to spark curiosity and foster a love for science in my audience.

Table of Contents

Every tissue culture hobbyist or professional eventually hits a plateau. You have a protocol that works, you have your medium recipe dialed in, and your explants are clean—yet, the growth feels sluggish.

You find yourself wondering: Why does it take months to see a significant increase in biomass, and why do so many plantlets fail to develop a robust root system before they are ready for soil?

If we want to move past "surviving" and into "thriving," we have to look deeper into the physiology of the plant.

Success in the multiplication and rooting stages isn't just about what is in the agar; it is about how we manipulate the plant’s internal signaling and its physical environment to trigger a growth explosion.

The Hormonal Dialogue: Multiplication and Shoot Quality

The multiplication stage is governed by the Skoog-Miller model, which dictates that the ratio of cytokinins to auxins determines the path of organogenesis.

While the goal is to break apical dominance and encourage axillary budding, it is critical to recognize that the "best" hormone is entirely dependent on the species' unique biosynthetic pathways and its sensitivity to exogenous regulators.

Calibrating Cytokinin Selection

For decades, 6-Benzylaminopurine (BAP) has been the industry standard for multiplication. It is effective and inexpensive, but it is not without its drawbacks. In many species, BAP can lead to high rates of hyperhydricity—a physiological disorder where tissues become excessively hydrated and brittle.

For plants that are recalcitrant or prone to "glassiness," many researchers are turning to aromatic cytokinins like Meta-Topolin (mT). Unlike the more stable BAP, Meta-Topolin is often metabolized more cleanly by the plant’s own enzymes, aligning more closely with the plant's natural metabolic rate. This reduces the "carry-over effect" into the rooting stage, which is a frequent cause of rooting failure.

The Role of Phenolic Synergists

We often overlook non-hormonal additives that can drastically change how a plant responds to hormones. Phloroglucinol (1,3,5 {-trihydroxybenzene}), for example, acts as a precursor to lignin biosynthesis. At concentrations of 135-160 mg/L, it can strengthen cell walls and act as an auxin-protector. By preventing the premature degradation of internal auxins, Phloroglucinol helps maintain a more balanced growth profile, reducing the "stunting" often seen when using high concentrations of cytokinins.

The Rooting Transition: Beyond Constant Exposure

Rooting is frequently the bottleneck of micropropagation. A plantlet that looks perfect in the jar is useless if it cannot survive the transition to the greenhouse. This stage requires a radical shift in the plant's internal chemistry, moving from a shoot-producing state to a root-initiating one.

The "Auxin Pulse" Technique

A common mistake in rooting protocols is the constant exposure of plantlets to auxin-rich media. While auxins like Indole-3-butyric acid (IBA) are necessary to initiate root primordia, their continued presence can actually inhibit the elongation of those same roots once they have formed.

The "Quick Dip" or Auxin Pulse is a scientifically superior method for many species. By exposing the base of the shoot to a high concentration of IBA (500-1000 mg/L) for a very short duration and then transferring the tissue to a hormone-free (HF) medium, we provide the "start" signal without the long-term inhibitory effects. This leads to a more natural, branched root architecture that is far more efficient at nutrient uptake once the plant is moved to soil.

Managing the Micro-Atmosphere

In a sealed container, the plant is at the mercy of its own metabolic byproducts. Ethylene, a gaseous plant hormone, can build up to toxic levels, leading to leaf abscission and the inhibition of root growth. Incorporating Activated Charcoal (0.5-2.0 g/L) can help by adsorbing inhibitory phenols and residual hormones. Furthermore, for ethylene-sensitive species, the addition of silver thiosulfate (STS) or silver nitrate (AgNO3) can block ethylene receptors, allowing the plant to focus its energy on rooting rather than stress responses.

Why Agar is the Limiting Factor

Most of our understanding of tissue culture is built on semi-solid media (agar). While agar provides excellent support, it creates a "diffusion-limited" environment. The plant can only consume the nutrients and hormones in the immediate "depletion zone" around its base. Furthermore, the stagnant air inside a traditional jar creates a "boundary layer" that slows down gas exchange.

In industrial scaling, agar becomes a significant cost and a labor burden. From a bioprocess perspective, the manual labor of "plugging" every explant into gel is the primary bottleneck. Research indicates that labor for manual subculturing on agar can account for 43-70% of total production costs.

Evidence in Practice: The Martin Study on Rotula aquatica

To see how these variables translate into real-world results, we can look at the landmark study by K. P. Martin (2002) titled "Rapid in vitro multiplication and ex vitro rooting of Rotula aquatica Lour., a rare rhoeophytic woody medicinal plant." This research provides a masterclass in balancing efficiency with economic viability.

Martin’s work established a highly efficient protocol for Rotula aquatica by utilizing Murashige and Skoog (MS) medium. The study found that the most effective medium for axillary bud proliferation was MS fortified with 1.0 mg/L BAP and 0.5 mg/L indole-3-butyric acid (IBA), which induced shoots at a rate of 15 per node.

The findings offer several critical insights for those looking to scale:

• Hormonal Synergy: The excision of node segments and their subsequent culture on media with balanced BAP and IBA concentrations facilitated enhanced axillary bud proliferation.

• The Rooting Transition: Shoots rooted best on half-strength MS medium supplemented with 0.5 mg/L naphthaleneacetic acid (NAA).

• Innovative Rooting Techniques: When the basal ends of shoots were dipped in an NAA (0.5 mg/L) solution for 25 days (a variation of the "Quick Dip" method), it resulted in a mean of 5.6 roots per shoot with a 75% survival rate upon transfer to pots.

• Economic Advantage: Remarkably, Martin found no significant difference in growth when using commercial sugar and tap water compared to tissue-culture grade sucrose and distilled water. This simple shift makes the protocol much more economically advantageous for large-scale operations.

The ultimate success of this protocol was staggering: About 750 plantlets were procured in a 3-month period starting from a single node explant.

The Role of the Physical Environment

While the chemistry provides the instructions, the physical environment provides the means. Better growth in vitro is often determined by factors that have nothing to do with what is in the agar.

Light Quality and Intensity

Light is a signal that tells the plant how to grow. During multiplication, a moderate intensity (30–50 µmol/m²/s) with a higher Blue light ratio helps keep shoots compact. As we move to rooting, increasing the intensity and shifting the spectrum toward Red signals the plant to begin the transition to autotrophy—developing its own photosynthetic machinery.

The Gas Phase and Ethylene

In a sealed jar, ethylene (C2H4)—a gaseous plant hormone—can build up to toxic levels, causing leaf yellowing and stunted roots. This is why gas exchange is vital. Allowing the plant to "breathe" by venting ethylene and introducing fresh CO2 is often more effective than adding more hormones.

Osmotic Potential

Sugar (sucrose) is more than just food; it determines the osmotic "pull" of water. Lowering the sugar concentration in the rooting stage (as seen in Martin’s use of half-strength MS) can "force" the plant to become more photosynthetic and makes it easier for developing roots to draw up moisture, reducing transplant shock.

Scaling with Temporary Immersion Systems (TIS)

To break through the scaling wall, we move from diffusion to Mass Flow. This is achieved through Temporary Immersion Systems (TIS). In a TIS setup, the plants are kept in a dry, well-ventilated chamber and are periodically bathed in liquid medium.

The Science of the "Dry Phase"

TIS works because it respects the plant's need for both nutrients and oxygen. During the immersion phase, the liquid medium shatters the boundary layer and allows every part of the plant—not just the base—to absorb nutrients via mass flow.

However, the "Dry Phase" is where the physiological hardening occurs. Once the liquid drains, the plant is left with a thin film of moisture but is fully exposed to air. This promotes:

1. Gas Exchange: Ethylene is vented out, and CO2 is more easily absorbed.

2. Hardening: The slight water stress during the dry phase encourages the development of the cuticle (the waxy layer on leaves).

3. Photosynthetic Competence: Plants in TIS often develop more functional stomata compared to those grown in the high-humidity, stagnant air of agar jars.

Scaling Made Simple: The BioCoupler and BioTilt

While the benefits of TIS are clear, older systems were often too complex, relying on air compressors, miles of tubing, and high risks of contamination. Plant Cell Technology addressed these mechanical hurdles with the BioCoupler™ and BioTilt™.

The BioCoupler: Contamination-Free Liquid Culture

The BioCoupler is a gravity-based TIS device that connects two standard glass jars via a specialized vane and a liquid-transfer system. By eliminating the external pumps and tubes found in traditional bioreactors, the BioCoupler effectively removes the most common entry points for pathogens. It is a closed, autoclavable system that allows for rapid liquid transfer with minimal mechanical complexity.

The BioTilt: Automating the Labor

The real breakthrough in scaling comes from the BioTilt™ system. This is an automated rack that holds multiple BioCoupler units and tilts them at programmable intervals.

The shift in production economics is significant. By using the BioTilt to automate the liquid immersion process, labor costs can be slashed to as low as 24%. You are no longer limited by how many jars a technician can manually fill; you are limited only by the number of racks you can fit in your growth room.

The BioTilt allows you to customize the immersion frequency to suit the specific needs of your species. You can pulse the nutrients more frequently during multiplication to drive biomass, and then reduce the frequency during rooting to encourage the plantlets to "harden" and develop more robust root systems.

Conclusion: A Synergy of Biology and Engineering

Getting better growth at the multiplication and rooting stages requires us to move beyond the "recipe" and start thinking about the system. It is about choosing the right hormonal tools—like Meta-Topolin or IBA pulses—and delivering them through an engineered environment that maximizes the plant's physiological potential.

By moving from the stagnant environment of agar to the dynamic, oxygen-rich environment of the BioCoupler and BioTilt, you aren't just growing plants faster; you are growing them better. You are producing plantlets that are physiologically superior, more resilient to transition, and significantly cheaper to produce at scale.

Revolutionize Your Lab Today

Are you ready to stop fighting the limitations of traditional media and start scaling your production with confidence? At Plant Cell Technology, we provide the advanced tools and biological expertise to help you master every stage of the tissue culture process.

Discover the future of micropropagation. Explore the BioCoupler™ and BioTilt™ systems and our full range of laboratory-grade supplies to take your plant production to the next level.