72. Cryopreservation of Passiflora pohlii – Vitrification and encapsulation-vitrification

Autor: Elisabeth Mansur, T.S.M. Merhy, Georgia Pacheco, M.G. Vianna, Renata Garcia
Rok vydání: 2012
Předmět:
Zdroj: Cryobiology. 65:362-363
ISSN: 0011-2240
DOI: 10.1016/j.cryobiol.2012.07.073
Popis: Passiflora pohlii is a wild tropical species of the genus Passiflora , it is native to Brazil and found in Cerrado, Atlantic Forest and Pantanal. This species has a potential agronomic interest due to its tolerance to soil-borne pathogens of Phytophthora sp, which causes damage to passionfruit culture. P. pohlii occurs in regions that are impacted by human actions, with the possibility of disappearance of many genotypes that could be used in breeding. Considering these facts, the establishment of different conservation strategies for this species is of great importance. The goal of this work was to develop cryopreservation protocols for P. polhii using vitrification-based techniques. Nodal segments (0.3 cm) excised from in vitro -grown plants were precultured for 24 h on solid MSM (Monteiro et al. In vitro Cell Dev Biol – Plant 36, 527–531) medium supplemented with 0.7 M sucrose, at 25 °C. For vitrification, the precultured explants were placed into 2.0 ml cryovials and incubated in the cryoprotectant solution PVS2 for different periods (0, 30, 60 or 120 min) at room temperature. After this, the cryovials were plunged into liquid nitrogen (LN 2 ) and stored for 72 h. For encapsulation-vitrification, the precultured explants were suspended in calcium-free MS medium supplemented with 3% sodium alginate. The mixture, including the nodal segments, was dispensed with a sterile pipette into MS medium containing 100 mM CaCl 2 , and held for 20 min at 25 °C. Beads, each containing one explant, were precultured in liquid 1/2 MSM medium containing 0.75 M sucrose for 24 h on a rotary shaker (100 rpm) at 25° ± 2 °C, and then placed into 2 ml cryovials containing PVS2 for 0, 30, 60 or 120 min. After these periods, cryovials were immersed into liquid nitrogen (LN 2 ) and stored for 72 h. Thereafter, the cryovials were removed from LN 2 and rapidly re-warmed in a water bath (40 °C) for 2 min, and transferred to the MSM supplemented with 31 μM BA. The explants were maintained in the dark for 30 or 60 days in a growth chamber at 30° ± 2 °C. After these periods, the explants were exposed to total irradiance (46 μ mol m −2 s −1 ). When using the vitrification technique, the highest recovery rate (40%) was obtained after exposure to PVS2 for 60 or 120 min. On the other hand, a very low survival rate was attained when explants were submitted to the encapsulation-vitrification protocol. In summary, in the present study we established a protocol for long-term conservation of P. pohlii through cryopreservation. We demonstrated that the vitrification protocol resulted in higher recovery rates when compared to the results obtained with the encapsulation-vitrification technique. In addition, the high multiplication capacity of nodal segments was not affected by cryostorage.
Databáze: OpenAIRE
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