Oxidative stress during the cryopreservation of Passiflora suberosa L. shoot tips using the V-Cryo-plate technique: determination of the critical stages of the protocol
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Passiflora suberosa L. is a wild species of Passiflora, with great agronomic, ornamental and medicinal potential. In spite of this, there are few biotechnological studies aiming at its in vitro propagation and conservation. Thus, the development of cryopreservation protocols is considered of great relevance for this species. However, cryopreservation may be associated with oxidative damages, which cause injuries and may result in low recovery frequencies. The goal of this work was the establishment of a cryopreservation protocol for P. suberosa shoot tips with the V-Cryo-plate technique, evaluating the influence of the age of the explant and exposure to the vitrification solutions PVS2 and PVS3. In addition, the occurrence of oxidative stress at the different stages of the protocol was evaluated by monitoring lipid peroxidation and the activity of antioxidant enzymes. Plant recovery from cryopreserved shoot tips occurred at distinct frequencies, according to explant age and exposure to the vitrification solutions. Highest post-freezing recovery was observed in 40-day old shoot tips treated with PVS2 for 60 min (45%) or PVS3 for 45 to 90 min (50–60%). The occurrence of oxidative stress was evaluated by the quantification of lipid peroxidation through malondialdehyde detection, total protein content, and activity of the antioxidant enzymes superoxide dismutase, catalase and ascorbate peroxidase. These assays revealed that oxidative stress mainly occurred at the osmoprotection and PVS3 dehydration stages, which were considered as the most critical of the V-Cryo-plate protocol for the cryopreservation of P. suberosa shoot tips.
Establishment of a cryopreservation protocol for Passiflora suberosa shoot tips with the V-Cryo-plate technique, and assessment of the oxidative stress for the determination of the critical stages of the protocol.
KeywordsCryoinjury In vitro conservation Malondialdehyde Passion fruit Vitrification solution
The authors acknowledge Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq – Programa Ciências sem Fronteiras), for research fellowships and financial support.
MV, EM, FE and GP conceived and designed research. MV and RG conducted experiments. EM and GP contributed with reagents or analytical tools. MV, EM, FE and GP analyzed data. All authors wrote, read and approved the manuscript.
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001. Funding was also provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico (Grant Nos. 305265/2014-8 and A054-2-13) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (Grant No. E-26/010.001631/2014).
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