Development of High-Efficiency in-Vitro Protocols in Amaryllis (Hippeastrum Hybridum) Cv. Pusa Suryakiran

Abstract

The present study investigates various treatments for surface sterilization, callus induction, shoot multiplication, and root regeneration in the in-vitro culture of hippeastrum hybridum. For surface sterilization, the treatment comprising 10% v/v Calcium hypochlorite (CaOCl2) and 70% v/v Ethanol, demonstrated superior efficiency in terms of higher survival rates, lower mortality, and minimal microbial contamination compared to treatment that contained 70% Ethanol and 0.05% v/v Mercuric chloride. Although mercuric chloride effectively reduced microbial contamination, it also adversely affected plant health, resulting in higher mortality rates among cultured explants. Optimal callus induction was achieved with a treatment containing MS Media supplemented with 2,4-D (1.0 mg L-1) and Kinetin (0.5 mg L- 1), facilitating high-quality callus initiation. Nutrient medium strength was found critical for overall growth and biomass accumulation. With treatment combination, MS Media + 2,4-D (1.0 mg L-1) + Kinetin (0.5 mg L-1) yielded the highest fresh callus weight. Regarding shoot multiplication, indirect organogenesis was identified as a rapid and effective method for mass production, leveraging callus induced from various plant parts for shoot proliferation. However, direct organogenesis exhibited a higher response percentage for shoot initiation (91.05±1.438%) than indirect organogenesis (71.22±3.482%), as evidenced in treatment containing 6 mg L-1 SNP and 0.5 mg L-1 NAA. Combining SNP with NAA, outperformed other treatments for shoot regeneration. Nonetheless, a gradual increase in SNP concentration did not correlate with improved outcomes. Observations indicated variability in shoot initiation parameters, suggesting optimal SNP concentrations are crucial. For root regeneration in in-vitro raised shoots, treatment combination having Full MS + IBA 1.0 mg L-1 + IAA 1.0 mg L-1 + NAA 0.5 mg L-1) facilitated rapid and robust root development per plant. The highest in-vitro rooting response rate was observed in treatment containing (Full MS Media + IBA 1.5 mg L-1), while treatment having (Full MS + IBA 1.5 mg L-1 + IAA 0.5 mg L-1) yielded the highest number of roots per explant