Citation: Trasar-Cepeda, C.; Sánchez, C.; Casalderrey, M.; Bello, D.; Vielba, J.M.; Rico, S.; Aldrey, A.; Vidal, N. Effect of Soil Type and In Vitro Proliferation Conditions on Acclimation and Growth of Willow Shoots Micropropagated in Continuous Immersion Bioreactors. Plants 2023, 12, 132. https://doi.org/ 10.3390/plants12010132 Academic Editor: Mikihisa Umehara Received: 1 December 2022 Revised: 20 December 2022 Accepted: 21 December 2022 Published: 27 December 2022 Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). plants Article Effect of Soil Type and In Vitro Proliferation Conditions on Acclimation and Growth of Willow Shoots Micropropagated in Continuous Immersion Bioreactors Carmen Trasar-Cepeda, Conchi Sánchez , Mar Casalderrey, Diana Bello, Jesús María Vielba , Saleta Rico , Anxela Aldrey and Nieves Vidal * Misión Biológica de Galicia Sede Santiago de Compostela, Consejo Superior de Investigaciones Científicas, Apdo 122, 15780 Santiago de Compostela, Spain * Correspondence: nieves@mbg.csic.es Abstract: Salix viminalis L. is a species with high capacity for micropropagation and acclimation and could therefore be used to evaluate emergent techniques in the field of plant propagation. The aims of this study were to propagate willow in liquid medium with a continuous immersion system, to explore the application of photoautotrophic conditions and to investigate the adaptation of willow plantlets to different soils that could be used as alternatives to commercial peat. For proliferation, we used 3% sucrose or sugar-free medium, and as substrates, we used commercial peat, a soil from an oak forest with high organic matter content and a crop soil with low organic matter content. The effect of sugar supplementation during proliferation and the soil characteristics during acclimation and growth were evaluated on the basis of aerial and root growth and the hydrolytic and dehydrogenase enzymatic activities of the soils. The results indicate that under photoautotrophic conditions, the supplementation of sucrose during micropropagation did not affect the subsequent growth of the plantlets. All plants acclimated without loss, but the type of soil influenced the height and vigor. Plants produced the highest shoots in peat, whereas the most root development occurred in crop soil. Soil enzyme activities were more influenced by the type of soil than by the presence of plants. Keywords: liquid medium; photoautotrophic growth; photomixotrophic growth; soil enzyme activities; sucrose; soil organic matter 1. Introduction The acclimation process is a major issue for the micropropagation of many plants [1]. Plants are usually micropropagated photomixotrophically, meaning they obtain their energy and biomass from the medium supplied with sugar, grown in small, air-tight vessels with high humidity and low gas exchange. Shoots experience CO 2 depletion during most of the photoperiod and are exposed to relatively high ethylene concentrations and relatively low photosynthetic photon flux density (PPFD), contributing to disturbances in plant development and photosynthetic performance [2]. After transfer to ex vitro conditions plants have to correct the abnormalities, and aerial and root sections have to adapt to the new environments in the greenhouse or in the field [3,4]. The use of liquid media by continuous (CIS) or temporary immersion (TIS) in bioreac- tors with forced ventilation has been proposed as a means of improving the physiological status of the explants, enhancing the photosynthetic ability and making them more com- petent to undergo rooting and acclimation [5–11]. In our laboratory, we have used CIS to culture Castanea spp. [12,13] and TIS to culture Alnus glutinosa [14], Prunus domestica [15], Cannabis sativa [16], Castanea spp. [13,17] and Salix viminalis [18,19]. Salix spp. and hybrids have been micropropagated in semisolid medium [20–26]. For acclimation, these authors reported a first step in which plantlets were maintained under high humidity conditions followed by a second step in which humidity was gradually decreased to adapt the plantlets Plants 2023, 12, 132. https://doi.org/10.3390/plants12010132 https://www.mdpi.com/journal/plants