Pastos y Forrajes, Vol. 43, No. 3, 221-229, 2020 In vitro multiplication of Morus alba L. 221 In vitro multiplication of Morus alba L. Criolla variety in temporary immersion systems Jorge Liusvert Pérez-Pérez1 https://orcid.org/0000-0003-3372-7559, Maylín Fonseca-Yero2 https://orcid.org/0000-0002-9566-0751, Marisel Bahi-Arevich1 https://orcid.org/0000-0001-6822-2492, Juan José Silva-Pupo1 https://orcid.org/0000-0003-1049-5740 and Stefaan Werbrouck3 https://orcid.org/0000-0001-5195-7054 1 Centro de Estudios de Biotecnología Vegetal, Universidad de Granma. Carretera a Manzanillo, km 17,5 Peralejo, CP 85100, Bayamo, Granma, Cuba. 2 Facultad de Ciencias Agropecuarias, Universidad de Granma. Carretera a Manzanillo, km 17,5 Peralejo, CP 85100, Bayamo, Granma, Cuba. 3 Laboratory of Applied In Vitro Plant Biotechnology. Ghent University. Campus Schoonmeersen C4.52 Valentin Scientific Paper Scientific Vaerwyckweg 1, 9000 Ghent, Belgium. E-mail: [email protected] Abstract Objective: To evaluate the in vitro multiplication of Morus alba L., Criolla variety, in temporary immersion systems (SETIS™). Materials and Methods: Two trials were conducted for the evaluation of three immersion times (1,0; 3,0; 5,0 min.) and three immersion frequencies every four, six and eight hours. Afterwards, another experiment was conducted for the analysis of the morphological response of the sprouts, at 28, 45 and 60 days of cultivation. A complete randomized design was used, with three repetitions in all the experiments. Results: The results related to the immersion time showed significant differences in the evaluated variables for p < 0,05, except in the number of sprouts. In addition, it was found that the highest number of axillary buds was obtained in the immersion time of three minutes, with a value of 4,67. Meanwhile, in the length of the sprouts, the maximum value was achieved with three minutes of immersion (3,0 cm) with significant differences (p < 0,05) with regards to the other treatments. With the use of temporary immersion systems, with three minutes of immersion every eight hours, during 45 days of cultivation, the highest number of axillary buds and 1,90 sprouts were obtained; while the sprout length reached 9,82 cm. Conclusions: In the sprouts no morphophysiological changes were observed, ascribed to the cultivation conditions. It was proven that the in vitro micropropagation of mulberry, Criolla variety, in temporary immersion systems SETIS™, is possible. Keywords: bioreactors, tissue culture, Morus alba, plant growth regulators Introduction which leads to a reduced adaptability of daughter Morus alba L. (mulberry) is a forage plant that plants and restricts the cultivation of varieties specific originated in the Himalayas, which has shown ex- from each region (Wani et al., 2019). cellent qualities for feeding different animal spe- With the current biotechnological technolo- cies, whose nutritional value is one of the highest gies it is possible to obtain plants in vitro in liquid among non-legume tropical forages (Martín et culture media, considered more effective than the al., 2017). In addition, it constitutes the exclusive semisolid ones, allowing the plants higher accessi- source for feeding the silkworm (Bombyx mori L.), bility to the components of the culture media and containing unique biochemical compounds in its higher gain in biomass. The propagation time is leaves, such as morin and beta-sitosterol, which also reduced, scaling up and its automation is pos- grant it an exceptional role in silk biosynthesis sible, and no gelifiers are required, which decreases (Sarkar et al., 2018). production costs in commercial laboratories (Car- In some genotypes, propagation by seed is pos- valho et al., 2019). sible, but the main way is through the use of stakes Among the technologies that use liquid culture (Wani et al., 2019). Although the latter guarantees media are temporary immersion systems (TIS), higher homogeneity of the plantations, requires semi-automated platforms that allow during a brief from six to seven months of maturity to make the period the controlled contact of the material to be cuts in the donor plants, and its use limits the feed propagated with a liquid medium in an aseptic availability for the animals (Vijayan et al., 2014). environment (Georgiev et al., 2014). In some Besides, it reduces the vigor of plants in the next gen- species, the physiological response of the plants has erations, with lower development of the root system, been improved and the multiplication indexes have Received: October 31, 2019 Accepted: September 08, 2020 How to cite this paper: Pérez-Pérez, J. L.; Fonseca-Yero, Maylín; Bahi-Arevich, Marisel; Silva-Pupo, J. J. & Werbrouck, S. In vitro multiplication of Morus alba L. Criolla variety in temporary immersion systems. Pastos y Forrajes. 43 (3):221-229, 2020. This is an open access article distributed in Attribution NonCommercial 4.0 International (CC BY-NC4.0) https://creativecommons.org/licenses/by-nc/4.0/ The use, distribution or reproduction is allowed citing the original source and authors. Pastos y Forrajes, Vol. 43, No. 3, 221-229, 2020 222 Jorge Liusvert Pérez-Pérez been increased (Businge et al., 2017; Sarkar et al., variety, according to the methodology proposed by 2018; Gianguzzi et al., 2019). Salas et al. (2005). Although the in vitro regeneration of M. alba Culture medium. For the multiplication in has been described by diverse authors (Salas et al., temporary immersion systems, the culture medium 2005; Gogoi et al., 2017), in this species the infor- proposed by Sales et al. (2011) was used. For such mation about the use of methodologies with the purpose, the whole salts were used, including utilization of temporary immersion systems, which vitamins in Murashige and Skoog medium allow scaling up at commercial level, is limited. (Murashige and Skoog, 1962) 4,40 g L-1 (Duchefa Until the present, there is the antecedent of the work Biochemie B.V.), which contains myo-inositol 100 conducted by Salas et al. (2011), at the Plant Bio- mg L-1 and glycine 2,0 mg L-1; besides sucrose 30 technology Institute (IBP, for its initials in Spanish) g L-1, 6-benzylaminopurine (6-BAP) 0,5 mg L-1 of Santa Clara, Cuba. These authors used a bioreac- and naphthaleneacetic acid (NAA) 0,5 mg L-1. The tor of temporary immersion (BIT®), constituted by pH was adjusted to 5,8 with NaOH (0,1N) and HCl two twin containers, connected between them by a (0,1N) solutions, before sterilization. silicon hose. The silicon hose, hydrophobic filters (0,22 µm, Today there is a commercial variant, known as MIDISART), SETIS™ flasks and culture medium, TIS, called SETIS™ (Vervit, 2016). It is a modification were sterilized in vertical autoclave (BK75) at 121 ºC of the systems based on flow and reflow, which and 1,2 kg cm2 of pressure during 20 min. After- constitutes a simplification of the system of twin flasks wards, in the laminar flow cabinet Vitrofural® (116 (Georgiev et al., 2014). It is composed by two horizontal mg L-1) was added when the culture medium had an polypropylene rectangular-shaped recipients, coupled approximate temperature of 80-90 ºC. At the end, it one over the other, which allows higher utilization of the was agitated until achieving homogenization. surface (Vervit, 2016). This type of system has proven Cultivation conditions. The cultivated explants in to be an efficacious tool for the micropropagation of temporary immersion systems SETIS™, were placed different economically important plant species from in growth chamber with indirect sunlight (Salas et al., the genera Saccharum, Musa, Solanum, Dioscorea, 2011), with photoperiod duration between 11 and 12 h, Phalenopsis, Stevia and of forestry species such as density of the flow of photosynthetic photons of 60-70 Eucaliptus and Paulownia, among others (Balogun et µmol m-2 s-1 and temperature of 26 ± 2,0 °C. al., 2017; Rosales et al., 2018). Each temporary immersion system consists in Nevertheless, the non-optimum cultivation two overlapping flasks, one for plant growth and the conditions in TIS can lead to the formation of other as reservoir of the culture medium (below). hyperhydric plants. This type of plant develops a degree They are both coupled with a silicon hose, of 6,0 mm of morphoanatomic and physiological disorder that diameter and 18,0 cm of length, from the connectors affects its regeneration capacity, in vitro multiplication located in the front lower part of each flask. In ad- and survival under certain environmental conditions dition, hydrophobic filters of 0,2 µm were placed to (Quiala, 2012). This requires the study of the main guarantee air sterility within the flasks (figure 1). indicators that influence the micropropagation and its The cultivation medium circulated from the optimization in each genotype, to achieve its effective lower to the top flask through a hose, due to the use at commercial scale. pressure of air emitted from a compressor and From the above-mentioned elements, the objec- regulated by a nanometer at 0,1 bar. When the tive of this work was to evaluate the in vitro multi- air pressure within the flasks is concluded, the plication of M. alba, Criolla variety, in temporary culture medium returns due to gravity to the lower immersion systems SETIS™. recipient. Between immersions of culture medium, Materials and Methods the air is pumped to the recipient that contains the Location. The research was conducted at the plant tissues to renovate the gaseous atmosphere Center of Studies on Plant Biotechnology (CEBVEG) inside it (Vervit, 2016; Balogun et al., 2017), reduce of the University of Granma, between December, humidity, prevent hyperhydricity and accumulation 2018, and May, 2019. of toxic gasses (Rocano et al., 2017). Plant material. Nodal segments of 1,0 cm Three trials were conducted, with consecutive length, with one axillary bud, were taken from in sequence, which allowed to fix the studied factors vitro plants in second multiplication subculture, in each case and were significant in the trials that in semisolid culture medium of mulberry, Criolla are described below: Pastos y Forrajes, Vol.