plants Article Two Advanced Cryogenic Procedures for Improving Stevia rebaudiana (Bertoni) Cryopreservation Carla Benelli 1,* , Lara S. O. Carvalho 2, Soumaya EL merzougui 3 and Raffaella Petruccelli 1 1 Institute of BioEconomy, National Research Council (CNR/IBE), 50019 Sesto Fiorentino, Florence, Italy; [email protected] 2 Department of Biology, Federal University of Lavras, Lavras 3037, Brazil; [email protected] 3 Laboratory of Biotechnology and Valorization of Natural Resources (LBVRN), Faculty of Sciences, Ibn Zohr University, 8106 Agadir, Morocco; [email protected] * Correspondence: [email protected] Abstract: Cryopreservation is a useful tool for the long-term storage of plant genetic resources, and different cryogenic procedures have recently been developed. The present study focused on the use of the Droplet-vitrification (DV) and V cryo-plate protocol for the cryopreservation of Stevia rebaudianain vitro-derived apical shoot tips and axillary shoot tips. A preliminary test showed that 90 and 120 min PVS2 (Plant Vitrification Solution 2) treatment significantly reduced the regrowth of the explants before immersion in liquid nitrogen (LN). For both procedures tested, the best osmoprotective condition for obtaining a higher regrowth of cryopreserved explants occurred when explants were PVS2 treated for 60 min. After direct immersion in LN, thawing and plating, the highest regrowth recorded was 80% with DV and 93% with V cryo-plate. Moreover, shoot tips proved to be a more suitable material for Stevia cryopreservation. A satisfactory vegetative regrowth was observed in the subcultures following cryopreservation by DV and V cryo-plate cryogenic procedures. Citation: Benelli, C.; Carvalho, L.S.O.; EL merzougui, S.; Petruccelli, Keywords: apical shoot tips; axillary shoot tips; droplet-vitrification; long-term conservation; PVS2 R. Two Advanced Cryogenic vitrification; V cryo-plate Procedures for Improving Stevia rebaudiana (Bertoni) Cryopreservation. Plants 2021, 10, 277. https://doi. org/10.3390/plants10020277 1. Introduction Stevia rebaudiana (Bertoni) is an herbaceous perennial plant of the Asteraceae family. Its Academic Editor: Gian-Pietro Di leaves produce diterpene glycosides (stevioside and rebaudiosides), and as stevioside is 300- Sansebastiano hundred-fold sweeter than sucrose, it is deemed to be a good natural sugar substitute [1,2]. Received: 29 December 2020 In addition to its sweetening properties, it has various medicinal properties and actions. For Accepted: 26 January 2021 this reason, the Stevia plant is an extremely interesting crop for breeders who select varieties Published: 31 January 2021 with high diterpene glycosides content as well as for propagators and the target market. S. rebaudiana is a self-incompatible plant and one of its limiting factors for large-scale Publisher’s Note: MDPI stays neutral cultivation is its poor seed germination [3]. Moreover, plants from seed propagation with regard to jurisdictional claims in published maps and institutional affil- have a great variability in growth, maturity, and non-uniform plants, with considerable iations. variations in the sweetening level and composition [4,5]. The recent results suggest that seed germination and stem cutting are not cost effective methods for higher biomass production, while the micropropagation can be a promising technique [3]. In vitro conservation and cryopreservation are unconventional biotechnological tools to preserve selected and valuable lines of S. rebaudiana, also taking into account the prob- Copyright: © 2021 by the authors. lems relating to its propagation by seed [2,3]. Licensee MDPI, Basel, Switzerland. A protocol for in vitro conservation of S. rebaudiana under slow growth conditions This article is an open access article distributed under the terms and and mass micropropagation after the storage period was developed by Zayova et al. [6], conditions of the Creative Commons while the long-term storage, cryopreservation of shoot tips, was carried out using the Attribution (CC BY) license (https:// vitrification method by Shatnawi et al. [7]. Cryopreservation allows the storage of plant creativecommons.org/licenses/by/ material (i.e., seeds, shoot tips, dormant buds, zygotic, and somatic embryos and pollen) ◦ 4.0/). at ultra-low temperatures in liquid nitrogen (LN; −196 C) or in the vapour phase of LN Plants 2021, 10, 277. https://doi.org/10.3390/plants10020277 https://www.mdpi.com/journal/plants Plants 2021, 10, x FOR PEER REVIEW 2 of 12 Plants 2021, 10, 277 2 of 12 vitrification method by Shatnawi et al. [7]. Cryopreservation allows the storage of plant material (i.e., seeds, shoot tips, dormant buds, zygotic, and somatic embryos and pollen) at(− ultra-low165 ◦C to temperatures−170 ◦C) [8] andin liquid it is becoming nitrogen a(LN; widely −196 practised °C) or in method the vapour for the phase long-term of LN (storage−165 °C ofto plant−170 °C) genetic [8] and resources it is becoming [9–12]. Ana widely advantage practised of the method cryopreservation for the long-term is that storageplant germplasm of plant genetic can theoretically resources [9–12]. be kept An indefinitely advantage inof verythe cryopreservation little space and is at that low plantcost, excludinggermplasm the can initial theoretically investment. be kept Over indefinitely the last 30in years,very little various space cryopreservation and at low cost, excludingtechniques the have initial been developedinvestment. using Over conventional the last slow30 years, freezing various methods cryopreservation [13–15] as well techniquesas several vitrification-basedhave been developed cryopreservation using conventional procedures slow freezing (encapsulation-dehydration; methods [13–15] as wellvitrification; as several encapsulation-vitrification vitrification-based cryopreservation [16–18], and more recently,procedures droplet-vitrification (encapsulation- dehydration;(DV) [19]. Two vitrification; recent novel encapsulation-vitrification cryopreservation techniques [16–18], have and been more identified recently, and droplet- have vitrificationresulted in V (DV) cryo-plate [19]. Two [20] recent and D novel cryo-plate cryopr [21eservation]. The DV techniques uses aluminum have foilbeen strips, identified while andthe mosthave recentresulted cryogenic in V cryo-plate procedure [20] (D and or VD cryo-plate)cryo-plate [21]. uses The aluminum DV uses cryo-plates. aluminum foil strips,The while regrowth the most rate recent obtained cryogenic in Stevia procedure with the vitrification(D or V cryo-plate) protocol uses by Shatnawi aluminum et cryo- al. [7] plates.was 68%. Continuous research and technological evolution have markedly improved the cryogenicThe regrowth methodologies, rate obtained allowing in Stevia to enhance with th thee vitrification recovery percentage protocol by of Shatnawi the species, et al. as [7]has was occurred 68%. Continuous over the years, research for example, and technological in Vitis spp. [evolution22,23] and have potato markedly [24,25]. Theimproved aim of thethis cryogenic study was methodologies, to assess the efficiency allowing of to the enha novelnce procedures,the recovery Droplet-vitrification percentage of the species, and V ascryo-plate, has occurred in order over to the improve years, the forS. example, rebaudiana in cryopreservationVitis spp [22,23] protocol.and potato [24,25]. The aim of this study was to assess the efficiency of the novel procedures, Droplet-vitrification 2. Results and V cryo-plate, in order to improve the S. rebaudiana cryopreservation protocol. 2.1. Evaluation Plant Vitrification Solution 2 (PVS2) Tolerance 2. ResultsIn this study, a preliminary experiment on apical shoot tips (AST) and axillary shoot tips (AxST) to evaluate the effect of exposure duration and optimize temperature of PVS2 2.1. Evaluation Plant Vitrification Solution 2 (PVS2) Tolerance treatment showed that exposure to PVS2 induced time-dependent regrowth in both of the explantsIn this assessed study, a and preliminary that the temperature experiment ofon the apical treatment shoot tips can (AST) influence and regrowth axillary shoot rates tips(Figure (AxST)1). After to evaluate 28 days the of effect culture, of theexposu bestre regrowth duration rates and wereoptimize obtained temperature for the explants of PVS2 treatmenttreated with showed PVS2 that with exposure exposure to times PVS2 ranging induced from time-dependent 20 min to 60 minregrowth at 0 ◦C, in andboth similar of the explantstrends were assessed observed and forthat AST the andtemperature AxST. AST of treated the treatment with PVS2 can solution influence for regrowth 60 min at rates both (Figuretemperatures 1). After had 28 90%days regrowth of culture, (Figure the best1a), regrowth while in rates the AxST, were theobtained regrowth for the was explants 90% at treated0 ◦C and with 85% PVS2 at 25 with◦C (Figure exposure1b). times PVS2 ranging treatments from markedly 20 min to affected 60 min regrowthat 0 °C, and potential similar trendsafter 60 were min, observed when a significant for AST and drop AxST. was AST observed, treated suggesting with PVS2 a toxicsolution response for 60 tomin long- at bothterm temperatures exposure to PVS2, had 90% furthermore regrowth regrowth (Figure was1a), alsowhile affected in the byAxST, treatment the regrowth temperature. was 90%As regards at 0 °C theand longer 85% at exposure 25 °C (Figure time (120 1b). min), PVS2 the treatments regrowth markedly AST decreased affected significantly regrowth potentialup