Cryopreservation and effect of lighting conditions and cytokinins on in vitro multiplication of Miconia ligustroides (DC.) Naudin

Abstract

Miconia ligustroides is a species that is native to Brazil and has medicinal and ecological importance. However, the species shows a lack of uniformity and delay in ex vitro germination. Thus, this study aimed to establish in vitro propagation for the species and to develop a protocol for the cryopreservation of seeds. For in vitro germination, activated charcoal (0.0, 0.5, 1.0, and 2.0 g L-1) was tested in Murashige and Skoog (MS) culture medium. Lateral buds excised from the plants were germinated in vitro and were encapsulated in an alginate matrix supplemented with 6-benzylaminopurine (BAP), kinetin, and thidiazuron (TDZ: 0.0, 2.0, 8.0, and 16.0 µM). Shoots derived from encapsulated units were inoculated in MS culture medium supplemented with different concentrations of BAP (0.0, 2.5, 5.0, and 10.0 µM) under white or Gro-Lux fluorescent lamps for multiplication. For cryopreservation, the toxicity of the cryoprotectant solution PVS2 (0, 15, 30, 60, 120, and 180 min) was evaluated before the seeds were immersed in liquid nitrogen. The MS culture medium supplemented with 1.0 g L-1 of activated charcoal yielded the highest percentage of germination (78%). The encapsulated units presented the largest percentages of regeneration (75%) with 8.0 µM BAP, which assisted in the formation of shoots that were 8.03 cm in length. For shoot production, the highest mean number (3.03 shoots) was obtained in the MS medium containing 5.41 µM BAP. When seeds were subjected to cryopreservation, the immersion time in the PVS2 did not affect the survival of the seeds, which was satisfactory (70%). The protocols developed are considered viable alternatives for use in the conservation of the species, production of seedlings for commercialization purposes, and use in programs of reintroduction in degraded environments.