Effective in vitro bulbification and alkaloid dereplication by GC-MS of Chilean Zephyranthes splendens, an endemic species of the Andes Mountains

The indigenous Chilean Zephyranthes splendens is a representative of the subfam. Amaryllidoideae (Amaryllidaceae) and is endemic to the South-Central foothills of the Andes Mountains. The plants of this subfamily are well known for producing bioactive alkaloids from the isoquinoline-type skeleton. For the first time it is established an in vitro micropropagation and bulbification protocol for Z. splendens and the alkaloid pattern of its in vitro cultures are characterized. To optimize the plant in vitro growth on the MS medium, sucrose concentrations and combinations of growth regulators (BAP and NAA) were evaluated. It was observed that the combination 2 mg/L BAP - 0.2 mg/L NAA promoted highly efficient direct organogenesis, while 90 g/L sucrose favoured bulb proliferation. Kinetin concentration and culture time were also evaluated for bulb development. Higher kinetin concentrations resulted in a significant increase in bulb volume over time, while sucrose promoted shoot development when modulated by kinetin. Dereplication of alkaloids by GC-MS has shown different amounts of alkaloids between wild bulbs and bulbils, the latter showing reduced metabolic activity with lower variability of alkaloids possibly due to their juvenile phase. The juvenile vegetative period along with a diminished photosynthetic process may have played a role on the metabolic processes in in vitro bulblets. Altogether, the results highlight the potential of Z. splendens bulblets to produce alkaloids under controlled in vitro conditions, providing a methodological basis for their biotechnological exploitation.