Open AccessComparative Study of Three Vitrification Solutions for an Effective Droplet-vitrification Cryopreservation Procedure for Pfaffia Glomerata (Spreng.) Pedersen
Author(s) -
Daniela Sousa Oliveira,
Antonieta Nassif Salomão,
Ildeu Soares Martins,
I. R. I. Santos
Publication year - 2021
Publication title -
european journal of medicinal plants
Language(s) - English
Resource type - Journals
ISSN - 2231-0894
DOI - 10.9734/ejmp/2021/v32i730403
Subject(s) - vitrification , cryopreservation , liquid nitrogen , factorial experiment , horticulture , chemistry , zoology , andrology , botany , biology , embryo , mathematics , medicine , statistics , organic chemistry , microbiology and biotechnology
Aims: The objective of this research was to establish a cryopreservation protocol for shoot tips (ST) of in vitro P. glomerata using the droplet-vitrification technique.
Study Design: The experimental design was a factorial, with four factors, arranged in a completely randomized design. Three vitrification solutions (PVS2, PVS3, PVS4), three times (20, 40, 60 min) and two temperatures (25 ± 2 °C and 0 °C) of treatment with the solutions, followed by freezing (LN+) or not (LN-) with liquid nitrogen (LN) were tested. All tests were performed using six replicates and the results analysed using Two-way ANOVA and Tukey’s tests and expressed as the mean ± the standard error of the means (SEM) deviation.
Place and Duration of Study: Laboratory of Plant Cryobiology, Embrapa Genetic Resources and Biotechnology, over a two-year period.
Methodology: ST excised from in vitro plantlets were pre-cultured overnight (19h), treated with a loading solution (LS) and three different vitrification solutions (PVS2, PVS3, PVS4) prior to freezing in LN. Treatment with the vitrification solutions was carried out at 0 or 25°C, for 20, 40 or 60 min. For freezing, drops of the vitrification solutions containing a single ST were dispensed on aluminum foil strips and the strips were submerged in LN (-196°C). For thawing, foil strips were submerged into unloading solution (US) at 40 ± 2°C, for three min. Thawed ST were transferred to regeneration medium and cultured in vitro.
Results: Highest regeneration percentages after cryopreservation were 82% for ST treated with PVS3, at 0°C, for 60 min; 32% for ST treated with PVS4 at 25°C for 60 min or 0°C for 40 min and 22% for those treated with PVS2 at 0°C for 60 min.
Conclusion: Droplet-vitrification is a suitable technique to ensure survival of P. glomerata ST after cryopreservation. This procedure can be applied to establish germplasm collections of this medicinal species in gene banks.