Comparison of Different Preconditioning and Loading Treatments with Vanilla (Vanilla planifolia Jack.) Apices Cryopreserved Using the Droplet-Vitrification Procedure F. Hernandez-Ramirez, M.T. González-Arnaoa, C.A. Cruz-Cruz and M.C. Pastelin-Solano Universidad Veracruzana Facultad de Ciencias Químicas Orizaba, Veracruz Mexico
F. Engelmann IRD, UMR DIADE Montpellier France
Keywords: vanilla, apices, trehalose, sucrose, preculture, cryoprotection Abstract In order to improve post-cryopreservation survival of vanilla (Vanilla planifolia) apices, different preconditioning and loading treatments were studied comparing the effect of using sucrose or trehalose. In this work, vanilla apices were isolated from in vitro grown plants and subjected to a direct preculture on MS semisolid medium supplemented with 0.3 M sucrose or trehalose for one or seven days. Additionally, apices were also pre-conditioned after dissection by transferring them first to standard MS semisolid medium for seven days, followed by the same MS medium but containing 0.3 M sucrose or trehalose for one or seven days. After all pre-conditioning treatments, apices were loaded in solutions containing 0.4 M sucrose or trehalose mixed with 2 M glycerol, or in 0.8 M sucrose or trehalose mixed with 1 M glycerol, exposed to PVS2 or PVS3 vitrification solutios for 30 min, and then directly plunged into liquid nitrogen on droplets of PVS solution placed on aluminum foil strips. Dehydration with PVS2 solution produced the best results after cryopreservation when apices were subjected to the pre-conditioning treatments with trehalose, and loading with sucrose-glycerol solutions. Nevertheless, the highest survival (57 and 62%) rates of cryopreserved apices were achieved using trehalose both in preconditioning and in loading solutions, followed by exposure to PVS3. The droplet-vitrification protocols which allowed improvements (from 30 to 60%) in survival of cryopreserved vanilla apices comprised: preconditioning of dissected apices on standard MS semisolid medium for seven days, followed by one or seven days on MS medium supplemented with 0.3 M trehalose, loading in 0.4 M trehalose + 2 M glycerol, and exposure to PVS3 for 30 min. INTRODUCTION Vanilla planifolia G. Jack. (Orchidaceae) is an aromatic plant of great commercial importance because it is the natural source of vanillin, which is the most widely appreciated flavour compound in the world. Although vanilla is cultivated throughout the tropics, the primary gene pool of V. planifolia, considered as the most critical sources of novel genetic diversity is located in Mexico, where it is classified as threatened due to destruction of its natural habitats (Divakaran et al., 2006). In addition, many Mexican vanilla plantations are currently facing a huge problem related to the fall of immature fruits due to the climate change (Hernandez-Hernandez, 2011). Therefore, the development of efficient long term in vitro conservation techniques complementary to field germplasm collections is an urgent need. We reported for the first time cryopreservation of vanilla (V. planifolia) apices using a droplet-vitrification protocol (Gonzalez-Arnao et al., 2009). However, survival and plant regeneration percentages of cryopreserved samples were low (30 and 10%, a
[email protected]
Proc. IInd IS on Plant Cryopreservation Ed.: B.M. Reed Acta Hort. 1039, ISHS 2014
173
respectively), and not reproducible. Therefore, the droplet-vitrification protocol developed needs to be optimized before foreseeing its large scale application to a range of vanilla accessions. In order to improve the effectiveness of the procedure, the aim of the present work was to study different pre-conditioning and loading treatments and especially the effect of sucrose or trehalose on plant regeneration of cryopreserved vanilla apices. The aim of this article is to improve post-cryopreservation survival of vanilla apices studying different pre-conditioning and loading treatments composed of sucrose and trehalose. MATERIALS AND METHODS In vitro plants of V. planifolia belonging to the working collection of the Plant Biotechnology and Cryobiology Laboratory of Universidad Veracruzana, were transferred every 12 weeks to semi-solid MS (Murashige and Skoog, 1962) multiplication medium supplemented with 1 mg L-1 6-benzyl amino purine (BAP), 0.5 mg L-1 indole butyric acid (IBA), 20 g L-1 sucrose and 7 g L-1 agar. In vitro collection was maintained at 24 ± 2°C under an 16 h light/8 h dark photoperiod with a light intensity of 36 µmol m-2s-1. Vanilla apices, up to 3 mm in length, were isolated from in vitro propagated plantlets and subjected to eight preconditioning treatments on semi-solid MS medium supplemented with sucrose or trehalose, followed by the exposure for 20-30 min to four different loading solutions combining sucrose or trehalose with glycerol (Table 1). After loading, apices were exposed to PVS2 [30% glycerol (w/v) + 15% ethylene glycol (w/v) + 15% (w/v) DMSO + 0.4 M sucrose] (Sakai et al., 1990) or PVS3 [50% glycerol (w/v) + 50% sucrose (w/v)] (Nishizawa et al., 1993) vitrification solutions for 30 min, and then directly plunged into liquid nitrogen on droplets of PVS solutions placed on aluminum foil strips. Samples were stored in liquid nitrogen for at least 1 h, and then, apices were warmed by plunging the aluminium foils with tissues directly in a large volume of liquid multiplication medium enriched with 1.2 M sucrose for 15 min at room temperature. After warming, tissues were transferred to filter papers for 1-2 min to drain off excess liquid, and then apices were placed on semi-solid multiplication medium for growth recovery, and kept for two months in the dark before being transferred to standard culture conditions with illumination. After cryopreservation, survival was evaluated after 30 days by counting the number of apices which displayed formation of new shoots. The experiments were conducted with a total of at least 20 explants per treatment and were repeated three times. Data were subjected to a factorial analysis following the parametric Tukey test (P
