23. Comparison of vitrification-based techniques in the efficacy of cryopreservation of Passiflora suberosa L. and P. foetida L. shoot tips

Abstract

Passiflora are used as ornamentals and in popular medicine. Passiflora suberosa is commonly used to treat hypertension, diabetes, skin diseases and as a sedative, whereas P. foetida shows analgesic and antibacterial activities. The goal of this study was the development of in vitro conservation strategies for long-term storage for both species through cryopreservation using vitrification and encapsulation-vitrification techniques. The efficacy of vitrification and encapsulation-vitrification protocols was compared, and several parameters were evaluated, including pregrowth on medium with high sucrose concentrations, type of vitrification solution (PVS2 and PVS3), exposure time to vitrification solutions, and recovery conditions. Shoot tips (0.3 cm) excised from in vitro-grown plants were precultured for 24 h on solid 1/2 MSM medium supplemented with different sucrose concentrations (0.3, 0.5 or 0.7 M), at 25 °C. Explants precultured on medium supplemented with 0.3 M sucrose were incubated in the cryoprotectant solutions PVS2 or PVS3 for different periods (0–240 min) at room temperature, and immersed into liquid nitrogen (LN) for 72 h. In the encapsulation-vitrification protocol, precultured shoot tips were encapsulated in calcium alginate beads, followed by incubation in liquid 1/2 MSM medium containing 0.75 M sucrose for 24 h, on a rotary shaker (100 rpm) at 25 ± 2°C. After this period, beads were maintained in cryovials containing PVS2 or PVS3 for 30, 60, 120 or 180 min, before immersion into LN. The materials were rapidly thawed in a warm water bath (38-40 °C) for 2 minutes and transferred to the recovery medium (1/2 MSM or MSM supplemented with 0.44, 2.2 or 4.4 μM BA). Alternatively, explants were kept in the dark for 30 or 60 days before transfer to the presence of light. Plant recovery from the shoot tips before and after cooling in LN was evaluated by assessing the percentage of shoot formation. The shoots were cultured on 1/2 MSM medium for 60–90 days for root induction, and whole plants were transferred to ex vitro conditions. The highest recovery rates for both P. suberosa and P. foetida (28% and 60%) were obtained with the encapsulation-vitrification protocol, after a pre-treatment with 0.3 M sucrose, followed by exposure to PVS2 for 60 or 120 min, respectively, and post-freezing incubation in the absence of light for 30 days on MSM medium supplemented with 0.44 μM BA. These results suggest that alginate beads provided additional protection, reducing the toxicity of the cryoprotectant solution. In addition, they demonstrate that different species from genus Passiflora display distinct responses to cryopreservation treatments.