Growth of Tissue-Cultured Aquatic Plants: From In Vitro Multiplication to Establishment in Aquatic Systems
- นภสร ตาปะสี
- 15 hours ago
- 6 min read
Tissue-cultured aquatic plants do not all respond to culture media and plant growth regulators in the same way. Different groups have distinct structures, growth habits, and physiological requirements.
Carpeting plants such as Monte Carlo and Glossostigma often grow well on media containing low concentrations of plant growth regulators or no hormones at all. By contrast, rhizomatous and rosette-forming aquatic plants such as Anubias, Cryptocoryne, and Bucephalandra may require higher cytokinin levels to stimulate shoot proliferation. These hormone levels must then be reduced during rooting and preparation for removal from the culture vessel.
Four Main Stages of Aquatic Plant Tissue Culture
The development of tissue-cultured aquatic plants can generally be divided into four major stages:
Culture establishment
Shoot multiplication
Root development
Acclimatization after deflasking
During the establishment stage, the main challenge is reducing microbial contamination without damaging the plant tissue.
Aquatic plants are constantly exposed to water, sediment, algae, and microorganisms. Their surfaces and internal structures may therefore carry a high microbial load.
Studies involving Micranthemum and Cryptocoryne, for example, found that treatment with 0.5% sodium hypochlorite for 30 minutes provided a useful balance between contamination control and explant survival under the conditions tested. However, this treatment must still be adjusted according to tissue thickness, plant sensitivity, explant type, and the condition of the mother plant.
A sterilization formula that works well for a soft stem plant may severely damage the thicker tissues of a rhizomatous species—or may be too weak to control contamination in plants collected from sediment-rich environments.
Cytokinins Affect Both Shoot Number and Shoot Quality
During multiplication, cytokinins such as BA or BAP play an important role in stimulating bud activation and shoot proliferation.
However, a higher cytokinin concentration does not necessarily produce better-quality plants.
The carpeting plant Micranthemum tweediei ‘Monte Carlo’, for example, has produced larger and stronger clumps when cultured with low BAP concentrations or without BAP. In contrast, Cryptocoryne ‘Flamingo’ responded well to 6 mg/L BAP combined with a low concentration of NAA, producing approximately 27–28 shoots per explant under the reported experimental conditions.
These contrasting responses demonstrate that culture media must be designed around the biological growth habit of each plant rather than applying one formula to every aquatic species.
Carpeting plants naturally spread through dense lateral growth and may not require strong hormonal stimulation. Slow-growing rosette or rhizomatous plants may need more cytokinin to activate dormant buds and produce enough shoots for commercial multiplication.
Multiplication Media Are Often Unsuitable for Rooting
Media that produce the highest number of shoots are often not ideal for root formation.
Residual cytokinin can suppress root elongation, cause compact shoot clusters, and reduce the physiological quality of the plantlets. For this reason, many aquatic plants respond better after being transferred to media with reduced mineral concentrations or no plant growth regulators.
For example, Echinodorus ‘Indian Red’ developed longer, higher-quality roots on hormone-free half-strength MS medium, although IBA-containing treatments produced a greater number of roots.
Similarly, Bucephalandra ‘Wavy Dark Green’ produced approximately 7.4 roots per plantlet on quarter-strength MS medium without added auxin.
These results show that root number alone should not determine the best rooting formula. Root length, branching, structure, anchorage capacity, and post-transfer survival may be more important for successful establishment.
Explant Type Influences Multiplication and Uniformity
The type of explant affects both multiplication efficiency and the uniformity of regenerated plants.
Nodal segments, axillary buds, and young shoots generally produce better results than leaves or callus-based regeneration because new shoots arise from pre-existing meristematic tissues.
Research involving Lobelia cardinalis, Staurogyne repens, and Alternanthera reineckii found that nodal explants produced more shoots and leaves than shoot-tip explants in some culture systems.
Certain formulas were also able to stimulate both shoot and root development in the same medium. This can shorten the production process, reduce the number of transfers, and lower labor costs.
For commercial production, direct shoot development from nodes is generally preferable when the objective is to maintain stable plant form, color, and growth habit.
Callus-based regeneration may offer a higher theoretical multiplication rate, but it can also increase the risk of somaclonal variation, abnormal morphology, and inconsistent aquarium performance.
Culture Systems Influence Growth Rate and Plant Quality
The physical culture system can significantly influence the growth of aquatic plants.
Semi-solid media are relatively easy to control and generally reduce the risk of hyperhydricity. Double-layer systems and temporary immersion systems can increase nutrient availability and accelerate multiplication, but they require careful management.
In Echinodorus ‘Indian Red’, a double-layer culture system produced approximately 26.8 shoots per explant within 60 days under the conditions tested.
In Bucephalandra, temporary immersion produced favorable results when cultures were immersed approximately six times per day for around two minutes per immersion.
However, if immersion is too frequent or continues for too long, the plantlets may become hyperhydric, produce short shoots, develop abnormal tissues, or show reduced rooting.
The optimal immersion schedule must therefore balance nutrient contact with sufficient aeration. The best setting may differ among species, vessel designs, biomass densities, and developmental stages.
Deflasking Requires a Major Physiological Adjustment
After removal from the culture vessel, aquatic plants must shift from relying partly on sucrose in the culture medium to producing their own energy through photosynthesis.
They must also begin absorbing nutrients from water, sediment, aquasoil, or another substrate. This transition affects how quickly the plants recover, produce new leaves, and accumulate biomass.
Comparative studies involving Monte Carlo, Micranthemum glomeratum, and Cryptocoryne ‘Flamingo’ reported 100% survival in sand, aquasoil, and mixed substrates under the experimental conditions. However, plants grown in aquasoil developed more rapidly and produced stronger new growth than those cultivated in sand alone.
This illustrates an important distinction: survival and strong growth are not the same measurement.
A plant may remain alive in a low-nutrient substrate while growing slowly, producing few new shoots, or taking much longer to reach marketable size.
Emersed and Submerged Acclimatization
Aquatic plants may be acclimatized under emersed conditions or transferred directly into submerged systems, depending on the species and the quality of the plantlets.
Slow-growing rhizomatous plants such as Anubias, Bucephalandra, and Cryptocoryne often benefit from an initial high-humidity emersed phase. This allows them to develop stronger leaves and roots before being submerged.
Carpeting plants and well-rooted Echinodorus plantlets may sometimes be transferred directly into aquasoil or controlled aquatic systems.
Regardless of the method, agar should be washed carefully from the roots and basal tissues. Residual sugar and nutrients can promote microbial growth, algae, or tissue decay after transfer.
The plants should then be monitored for the production of new leaves and roots adapted to the final growing environment. Leaves formed in vitro may deteriorate after transfer, while new submerged or emersed leaves provide stronger evidence that the plant has successfully established.
Measuring Quality Beyond Shoot Number
The quality of tissue-cultured aquatic plants should not be assessed only by the number of shoots produced in each vessel.
A more complete evaluation should include:
Shoot length
Leaf number
Leaf color and shape
Clump area
Fresh weight or biomass
Root length and branching
Hyperhydricity
Ease of plantlet separation
Survival after transfer
Production of new leaves
Establishment speed in aquasoil or water
Final marketable plant number
For example, a medium that produces 30 small and weak shoots may generate fewer saleable plants than a formula that produces only 15 shoots if those shoots are stronger, better rooted, easier to handle, and faster to establish in an aquarium.
The most commercially useful measure is therefore the number of healthy plants that survive and continue growing under real production or aquarium conditions.
Developing Protocols by Aquatic Plant Group
Commercial laboratories should develop separate protocol strategies for different aquatic plant groups.
Carpeting Plants
Carpeting plants such as Monte Carlo and Glossostigma should generally be tested first on hormone-free or low-hormone media. Excessive cytokinin may reduce natural spreading growth and produce compact or abnormal shoots.
Stem and Nodal Plants
Stem plants such as Hygrophila, Rotala, Staurogyne, Lobelia, and Alternanthera are often well suited to nodal explants and low-to-moderate cytokinin concentrations.
In some species, shoot and root development may be combined in one culture stage, reducing production time.
Rhizomatous and Rosette Plants
Rhizomatous and rosette-forming plants such as Anubias, Bucephalandra, and Cryptocoryne may require higher cytokinin levels during multiplication because their natural growth is slower.
However, cytokinin should be reduced or removed before rooting and deflasking to improve shoot elongation, root development, and acclimatization.
Variegated Aquatic Plants
Variegated cultivars require additional quality control. Buds or nodes should be used whenever possible, callus should be minimized, and the stability of leaf color should be evaluated after transfer.
The production target should be based on the percentage of plants that retain the required pattern—not the total number of shoots produced in vitro.
Connecting Laboratory Multiplication with Aquarium Performance
Producing high-quality aquatic plants through tissue culture requires every stage to be linked together: explant selection, sterilization, culture medium, hormone concentration, culture system, rooting, and acclimatization.
For carpeting plants, hormone-free or low-hormone formulas are often the best starting point. Nodal plants generally benefit from nodal explants and low-to-moderate cytokinin levels. Rhizomatous and rosette-forming plants may require stronger cytokinin stimulation during multiplication, followed by a hormone-reduction stage before deflasking.
The true commercial goal is not the highest possible shoot count. It is the production of healthy, uniform plants that establish quickly, form new roots and leaves, and continue growing successfully after transfer into an aquatic system.
A successful protocol therefore connects what happens inside the culture vessel with how the plant performs after it enters the aquarium.
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