Lotus Tissue Culture: A Review of Research and Practical Approaches for Producing High-Quality Plantlets
Lotus (Nelumbo nucifera Gaertn.) is an economically and culturally important aquatic plant valued as an ornamental species, a food crop, a medicinal plant, and a symbol of cultural heritage. As a result, it has attracted considerable research interest in countries such as China, Japan, India, and Malaysia. In recent decades, tissue culture research has focused on rapid propagation, disease-free plant production, germplasm conservation, and providing plant materials for future breeding programs. Consequently, plant tissue culture has become an important tool for advancing lotus propagation from laboratory research to commercial-scale production.
Although lotus can be propagated conventionally through rhizome division or seeds, these methods have several limitations. Rhizome propagation is relatively slow, requires substantial cultivation space, and carries a higher risk of transmitting soil-borne pathogens. In addition, rhizomes are bulky and susceptible to mechanical damage during transportation. Tissue culture offers an efficient alternative by enabling the large-scale production of uniform, high-quality planting materials within a shorter period while preserving the desirable characteristics of elite cultivars. For example, when producing specialty ornamental lotus cultivars with unique flower colors, tissue culture can generate large numbers of genetically consistent plantlets.
Numerous studies have demonstrated that explant selection is one of the most critical factors influencing culture success. The most commonly used explants include shoot apical meristems, plumules from immature seeds, and embryos, as these tissues generally exhibit lower contamination rates and higher regeneration potential than leaves or petioles, which are more prone to microbial contamination and tissue browning.
The establishment of aseptic cultures typically begins by thoroughly washing the explants under running water, followed by treatment with a surfactant, 70% ethanol, and surface sterilization using disinfectants such as sodium hypochlorite or mercuric chloride. The explants are then rinsed several times with sterile distilled water to remove residual sterilizing agents. Because lotus is an aquatic plant, its tissues often harbor relatively high populations of bacteria and fungi. Therefore, using shoot tips or embryos as starting materials is generally more effective in minimizing contamination during culture initiation.
For the multiplication stage, most studies have reported that Murashige and Skoog (MS) medium provides the best results when supplemented with appropriate concentrations of cytokinins and auxins. For example, Mahmad et al. (2014) reported that MS medium supplemented with 0.5 mg/L BAP and 1.5 mg/L NAA produced an average of approximately 16 shoots per explant. Likewise, Yu et al. (2015) found that MS medium containing 2.22 μM BA generated up to 21.33 shoots per explant within four weeks. These findings clearly demonstrate that optimizing plant growth regulator concentrations plays a crucial role in improving shoot multiplication efficiency.
Once healthy shoots have been obtained, the next stage is root induction. Previous studies have shown that MS medium supplemented with 0.54 μM NAA successfully induced rooting in all regenerated shoots within approximately four weeks. After rooting, plantlets are removed from the culture vessels, the agar is carefully washed from the roots, and the plantlets are transplanted into well-drained substrates such as clay soil mixtures or loamy clay. High humidity is maintained during the initial acclimatization period before gradually exposing the plants to normal environmental conditions. Several studies have reported survival rates of up to 97% following acclimatization.
Another promising approach is the double-layer medium system, which combines liquid and solid media at an approximate ratio of 2:1. Research has shown that this culture system can enhance shoot proliferation, improve root development, and promote shoot elongation more effectively than solid medium alone. Consequently, it has considerable potential for commercial micropropagation, particularly in laboratories aiming to maximize production efficiency.
Despite these advances, lotus tissue culture still presents several technical challenges. Common problems include microbial contamination, tissue browning caused by phenolic compound oxidation, hyperhydricity, and poor root development when cytokinins are maintained at excessively high concentrations. For example, prolonged exposure to high levels of BA often results in dense shoot clusters that root poorly. Therefore, many laboratories transfer regenerated shoots to cytokinin-free medium before the rooting stage to improve shoot quality and root formation.
Current research trends in lotus tissue culture are increasingly focused on improving multiplication efficiency, developing callus-based regeneration systems for plant breeding and genetic engineering, producing valuable bioactive compounds through cell culture, and conserving rare lotus germplasm. Although several efficient protocols have already been reported, responses remain highly genotype-dependent. Consequently, any published protocol should be regarded as a starting point rather than a universal standard. Before adopting a protocol for commercial production, laboratories should optimize culture conditions for each specific cultivar to achieve high multiplication rates, superior plant quality, and maximum survival during acclimatization.
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