Abstract
This study aimed to establish a cryopreservation protocol for embryogenic cultures of A. angustifolia, enabling the ex situ conservation of the species. Embryogenic cultures were established from immature seeds and treated with variations of the cryoprotectant solutions SuDG, SoD and PVS2 prior to immersion in liquid nitrogen. Cell viability was evaluated after 30, 60 and 90 days of re-growth. The highest re-growth without morphological alterations and with normal biochemical composition was obtained with the PVS2 solution with 40 min immersion in ethanol (-20 °C). This procedure opens new horizons for the ex situ conservation of the species genetic.
Highlights
The present study intended to establish a cryopreservation protocol for embryogenic cultures of A. angustifolia, aiming to enable long-term storage
Due to the recalcitrant nature of the A. angustifolia seeds, the maintenance of ex situ seed banks is a challenge and the use of embryogenic cultures seems to be a feasible alternative for the conservation of species germplasm (Stefenon et al 2009)
Ex situ conservation approaches based on cryopreservation have been increasingly employed on plant germplasm conservation (Reed 2001), including conifers (Find et al 1998, Ford et al 2000, Touchell et al 2002, Marthur et al 2003, von Arnold et al 2005)
Summary
The present study intended to establish a cryopreservation protocol for embryogenic cultures of A. angustifolia, aiming to enable long-term storage. Suspension cells were established utilizing 0.25 g of embryogenic culture inoculated in 120 mL of liquid BM medium free of growth regulators (BM0) and incubated in the dark on a shaker at 25±2 °C. Seventeen cryoprotective treatments were employed (Table I), based on the combination of different osmotic solutions: SuDG [Sucrose: dimetil sulfoxide (DMSO):Glycerol], SoD (Sorbitol:DMSO) and the vitrification solution PVS2 (Sakai et al 1991).
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