PLANT TISSUE CULTURE Improving of rooting and ex vitro acclimatization phase of Agave tequilana by temporary immersion system (BioMINT™) Kelly M. Monja-Mio 1 & Diego Olvera-Casanova 1 & Gaston Herrera-Herrera 1 & Miguel Ángel Herrera-Alamillo 1 & Felipe L. Sánchez-Teyer 1 & Manuel L. Robert 1 Received: 10 February 2020 /Accepted: 21 July 2020 / Editor: Yong Eui Choi # The Society for In Vitro Biology 2020 Introduction In Mexico, the production of tequila is an important agro- industry that uses an agave species (Agave tequilana Weber var. Azul) as the raw material to produce this well-known spirituous drink. Tequila cultivation faces serious problems due to an extreme susceptibility to some pests and diseases that severely reduce productivity. The basis of these appears to be a lack of genetic variability due to the fact that the crop is almost entirely propagated by asexual means through the pro- duction of rhizomes (Arizaga and Ezcurra 1995; Robert et al. 2004). Conventional genetic improvement is difficult in this crop due to several biological characteristics, such as its long- life cycle (8–12 yr), and the shape of the plant and its inflo- rescence, that make it difficult to implement controlled polli- nation programs (CRT 2004; Robert et al. 2004). Given this circumstance and the exponential growth of the tequila indus- try, an alternative for the genetic improvement of these agaves is imperative. In vitro micropropagation of selected “elite” individuals with better agronomic and industrial characteris- tics is the best available option (Robert et al. 1992, 2004, 2006a; Monja-Mio et al. 2019). However, the cost of in vitro micropropagated plants is higher than those of naturally propagated rhizomes, so very efficient and low-cost methods are needed for large-scale sys- tems to be applied to the production of elite plants for com- mercial planting. Efficient systems have been developed and used for tequila and several other economically important agave crops in Mexico, such as henequen and mezcal (Robert et al. 2004, 2006a; Monja-Mio and Robert 2016) but they are all susceptible of improvements to reduce time and diminish costs. An option for this is the use of temporary immersion systems (TIS) during the preadaptation stages pre- vious to transplantation to ex vitro conditions. Currently, there are several types of TIS for the micropropagation of plants of commercial interest (Robert et al. 2006b; Georgiev et al. 2014; Monja-Mio et al. 2016). Properly designed, the TIS can be rapid, economical systems that generate high-quality plants that adapt more efficiently when transplanted to soil (Berthouly and Etienne 2005; Robert et al. 2006b). The culture conditions in TIS need, how- ever, to be optimized for the species being cultured, the mor- phogenic pathway, and the phase of the micropropagation process used (multiplication or rooting). Ex vitro acclimatization is a key stage of the micropropagation process, in which plants leave the protected environment of in vitro culture (sterile environment, low irra- diation, and high relative humidity), to grow and form new leaves and roots, exposed to different types of stress present in ex vitro conditions (Rescalvo-Morales et al. 2019). Water stress and photoinhibition, often accompanying the acclimati- zation, lead to the overproduction of reactive oxygen species (ROS) and consequently to oxidative stress (Carvalho et al. 2006; Batková et al. 2008; Faisal and Anis 2009; Osório et al. 2010; Dias et al. 2011; Goncalves et al. 2017). Depending on the species, many plants may die at this stage due to abnormal stomatal development, absence of epicuticular waxes, and few functional roots. Adequate pre-adaptation conditions are nec- essary to achieve high levels of survival and therefore effec- tive micropropagation protocols. Preparing the vitroplants for their subsequent exit to ex vitro conditions is necessary to succeed in the acclimatization phase. Changes to the environ- mental conditions of the in vitro culture, generally by reducing sugar levels, increasing light intensity, raising levels of CO2, reduction of humidity, etc., make plants less dependent on * Kelly M. Monja-Mio kellymabel@hotmail.com * Manuel L. Robert robert@cicy.mx 1 Unidad de Biotecnología, Centro de Investigación Científica de Yucatán, A.C. Calle 43 No. 130, Chuburná de Hidalgo, 97200 Mérida, Yucatán, Mexico In Vitro Cellular & Developmental Biology - Plant https://doi.org/10.1007/s11627-020-10109-5