Development of High-Efficiency in-Vitro Protocols in Belladonna Lily (Amaryllis bellodonna)

Abstract

This study focuses on developing high-efficiency in-vitro protocols for Belladonna lily (Amaryllis belladonna), aimed at minimizing microbial contamination, and optimizing phytohormones for callus induction, shoot multiplication, and root regeneration. Conducted in the Plant Tissue Culture Lab at the College of Horticulture, SVPUA&T, Meerut, the research identifies the most effective surface sterilant and phytohormone combinations. The study found that the treatment with 70% ethanol for 5-10 minutes followed by 0.1% HgCl2 for 2 minutes resulted in the lowest microbial contamination (49.02%) and mortality rates (16.63). Conversely, the control treatment with deionized water showed the highest contamination (76.31%) and mortality rates (58.64). The highest survival rates (51.93%) were observed with a combination of 70% ethanol and 10% CaOCl2. For callus induction, the optimal treatment was MS Media supplemented with 2,4-D (1.0 mg/L) and Kinetin (0.5 mg/L), which also showed the fastest callus initiation (76.24%), most profuse growth, and highest biomass accumulation (141.87 mg). In contrast, ½ MS Media showed the poorest performance. In terms of shoot multiplication (direct organogenesis), MS Media with BA 2 mg/L and NAA 0.5 mg/L demonstrated the quickest shoot initiation (4.85 days), while MS Media with KIN (2 mg/L) and NAA (0.5 mg/L) achieved the highest response percentage (91.99 %), shoot proliferation (5.67 shoots), shoot length (12.27 cm.), and leaf number per explant (5.33). Indirect organogenesis, MS Media with BA 6 mg/L demonstrated the quickest shoot initiation (8.25 days), while MS Media with KIN (6 mg/L) and NAA (0.5 mg/L) achieved the highest response percentage (71.08%). In case of shoot proliferation (3.67 shoots), shoot length (26.42 cm.), and leaf number per callus (4.33) the MS Media supplemented with KIN (2 mg/L) and NAA (0.5 mg/L) showed best results. For root regeneration, the fastest initiation (23.89 days), number of roots per culture (12.67) and maximum length of root (6.74 cm.) were achieved with full MS Media containing IBA (1.5 mg/L) and IAA (0.5 mg/L). Overall, this study establishes effective sterilization methods and phytohormone treatments, enhancing the micropropagation efficiency of Amaryllis belladonna, ensuring rapid, viable, and disease-free plant material.