Micropropagation of Nymphaea pubescens: An Efficient Protocol for Tuber-Derived Tissue Culture, Shoot Multiplication, and Field Establishment

Waterlilies (Nymphaea spp.) are ecologically and ornamentally important aquatic plants, yet their micropropagation remains challenging due to endophytic contamination and the recalcitrant nature of tuber explants. This study established an efficient in vitro protocol for Nymphaea pubescens using field-collected tubers. Surface sterilization with 0.2% mercuric chloride (HgCl2) for 15 min achieved complete sterility with 80% explant viability, and an “escape technique” of excising newly emerged shoots from the maternal tuber reduced latent bacterial contamination. Liquid Murashige and Skoog (MS) medium with 1.0 mg/L 6-benzylaminopurine (BAP), 0.1 mg/L thidiazuron (TDZ), and 0.5 mg/L 1-naphthaleneacetic acid (NAA) produced greater shoot multiplication (17.20 ± 2.53 shoots/explant) and elongation (1.03 ± 0.22 cm) than semi-solid medium (9.70 ± 2.11 shoots/explant; 0.70 ± 0.13 cm) (p < 0.01). Transfer to semi-solid maturation medium with glutamine and casein hydrolysate produced the highest survival rate (100%) and significantly improved shoot quality (p < 0.01), mitigating hyperhydricity from prolonged liquid culture. Rooting on semi-solid medium with 0.5 mg/L NAA yielded 90% rooting, and over 80% of acclimatized plantlets established successfully in the field within two weeks. These results provide a practical, reproducible protocol for conservation and mass propagation of N. pubescens and a foundation for future in vitro mutation breeding in ornamental waterlilies.