Cryopreservation of shoot tips excised from zygotic embryos of Araucaria angustifolia (Bertol.) Kuntze

Abstract

Araucaria angustifolia (Bertol.) Kuntze is the only species of the genus Araucaria naturally occurring in Brazil. The species has recalcitrant seeds, and is in the vulnerable category of the IUCN endangered list. This work presents a protocol for the in vitro conservation of A. angustifolia through the cryopreservation of apical shoot tips excised from zygotic embryos. In vitro regrowth of shoot tips was assessed in half-strength MS medium supplemented with 0.0, 1.25, 2.5, 5 and 10 µM of 6-benzylaminopurine (BAP). For the toxicity test, shoot tips were exposed to the loading solution for 20 min, after exposure to PVS2 by 0, 15, 30, 60 and 120 min before immersion in liquid nitrogen (LN). Exposure to loading solution supplemented with phloroglucinol (0, 0.2, 1 and 5%) was also tested (20 min) before immersion in PVS2 for 15 min. The explants were also plunged into LN, using the vitrification method and the rewarming was performed in a water bath at 40°C for 3 min. After, the explants were treated with unloading solution for 15 min followed by post-culture on 0.3 M sucrose for 24 h in the dark. The survival of the explants was assessed after 30 days in a growth chamber with a 16 h photoperiod at 25±2°C. The highest regrowth percentage (35.3%) of shoot tips was obtained in half-strength MS medium with 2.5 µM BAP. The exposure to PVS2 for 15 min produced the highest survival (53.2%) of the explants. The addition of phloroglucinol in the loading solution was not effective as a cryoprotectant in embriogenic apexes, since the control (0% phloroglucinol) promoted the same percentage survival (41.7%) after cryopreservation, using the method of vitrification.