Long-term preservation of Lotus tenuis adventitious buds
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Encapsulation-dehydration, encapsulation-vitrification, and vitrification were tested for cryopreservation of Lotus tenuis (Fagaceae) adventitious buds clusters (ABCs) obtained by a direct regeneration system from leaves cultures. Among them, the PVS3-based vitrification procedure was found to be useful for survival and regrowth of the preserved explants. For vitrification, the ABCs were dehydrated in a solution containing 2 M glycerol + 0.4 M sucrose for 25 min at room temperature, submerged in PVS3 solution for 1 h at 0 °C, then immersed in liquid nitrogen for 48 h and rapidly rewarmed. Afterword, the explants were unloaded in MS liquid medium with 1.2 M sucrose for 30 min. The washed tissues were dried superficially on filter paper and cultured in semisolid hormone-free MS medium containing 0.1 M sucrose. All cultures were maintained at 25 °C in the dark for 10 days and transferred to the light conditions. With this procedure, 79 ± 5.3% survival and more than 80% of the plantlets displaying a phenotype similar to the non-treated control after acclimatization. The data settled from ISSR showed no genetic dissimilarities between in vitro regenerants derived from cryopreserved tissues and the non-treated plants. Thus, our results indicate that the use of vitrification-based PVS3 solution offers a simple, accurate, and appropriate procedure for the cryopreservation of L. tenuis adventitious buds.
KeywordsCryopreservation PVS3 Vitrification ISSR markers
This work was supported by grants from Agencia Nacional de Promoción Científica y Técnica (PICT 2014-3718) and Secretaría General de Ciencia y Técnica-Universidad Nacional del Nordeste (PI A001/14). We extend our sincere appreciation to anonymous reviewers for their critical comments. F. Espasandin, E. Brugnoli, O. Ruiz, and P. Sansberro are members of the Research Council of Argentina (CONICET). G. Ayala and L. Ayala received a scholarship from CONICET.
FDE, OAR, and PAS conceived and designed the experiments. FDE, EAB, PGA, and LPA performed the research. FDE and PAS wrote the manuscript. All authors read and approved the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
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