65 How to Monitor the Acclimatization of Micropropagated Plants – from In Vitro to the Field? M.L. Osório, S. Gonçalves, N. Coelho and A. Romano Institute for Biotechnology and Bioengineering Centre of Genomics and Biotechnology (IBB/CGB) Faculty of Sciences and Technology University of Algarve Campus de Gambelas, Ed. 8 8005-139 Faro Portugal J. Osório Institute of Mediterranean Agricultural and Environment Sciences (ICAAM) Faculty of Sciences and Technology University of Algarve Campus de Gambelas, Ed. 8 8005-139 Faro Portugal Keywords: chlorophyll a fluorescence, electrolyte leakage, gas exchange, lipid peroxidation, Tuberaria major Abstract The high mortality experienced by plants during ex vitro transplantation stage is the major bottleneck in large scale application of micropropagation. The abnormal physiological and anatomical characteristics of micropropagated plantlets require that they should be gradually acclimatized to the environment of the greenhouse or field. Environmental factors, especially relative humidity and irradiance, play a critical role in both physiological and biochemical functions, namely water relations, gas exchange, photosynthetic efficiency and water oxidation, during transition from in vitro to ex vitro conditions. Throughout this period of transition and acclimatization substantial changes in those characteristics are necessary to achieve a successful micropropagation. This adaptation should be accurately monitored using relevant physiological parameters including pigment content, chlorophyll a fluorescence imaging, net photosynthetic rate, transpiration rate, and stomatal conductance. Growth traits and oxidative stress markers, such as electrolyte leakage, lipid peroxidation and hydrogen peroxide content, should be also analyzed. These parameters must be assessed during the course of ex vitro acclimatization in several periods and compared with those of plants growing in their natural habitat. The performance plants of Tuberaria major, an endangered species endemic from the Algarve region (Portugal), during their acclimatization through indoor-to-outdoor conditions was monitored using several physiological and biochemical traits as indicators. In view of the results, we concluded that the transplantation protocol described can be used for restoration purposes, contributing to the preservation of the species. INTRODUCTION Micropropagation allowing plant multiplication and germplasm conservation is important to protect endangered species. However, the high mortality experienced during the ex vitro transplantation stage becomes a major obstacle in its application. The benefit of any micropropagation system can only be fully realized by the successful transfer of plantlets from tissue culture vessels to ex vitro conditions and establishment in the field. Therefore, the ultimate success of this technology depends on a reliable acclimatization protocol, ensuring low cost and high survival rates (Hazarika, 2006). Understanding the morphological, physiological and biochemical characteristics of micropropagated plants and the changes they undergo during the acclimatization process should facilitate the development of efficient transplantation protocols (Brito et al., 2009). Plants transferred to a different environment may become more susceptible to various stresses and to endure the new conditions they require new and adequate patterns of resource allocation and morphologic and physiological features (Chaves, 1994). This is Proc. 5 th IS on Acclimatization and Establishment of Micropropagated Plants Eds.: P.E. Read and J.E. Preece Acta Hort. 988, ISHS 2013