An effective disinfection protocol for contamination control in vitro establishment of Mortiño (Vaccinium floribundum Kunth) and identification of endogenous microbes

Lissette  Moreno-Peña; Karen  Hidalgo-Escobar; Juan M.  Cevallos-Cevallos; Eduardo  Sanchez-Timm

doi:10.17268/sci.agropecu.2025.028

Autores/as

Lissette Moreno-Peña Centro de Investigaciones Biotecnológicas del Ecuador, ESPOL Polytechnic University, ESPOL, Campus Gustavo Galindo, km 30.5 Vía Perimetral, 090902, Guayaquil, Ecuador. https://orcid.org/0009-0002-2785-616X
Karen Hidalgo-Escobar Centro de Investigaciones Biotecnológicas del Ecuador, ESPOL Polytechnic University, ESPOL, Campus Gustavo Galindo, km 30.5 Vía Perimetral, 090902, Guayaquil, Ecuador. https://orcid.org/0009-0002-9845-6104
Juan M. Cevallos-Cevallos Facultad de Ciencias de la Vida, ESPOL, Polytechnic University, ESPOL, Campus Gustavo Galindo, Km. 30.5 Vía Perimetral, 090902, Guayaquil, Ecuador. https://orcid.org/0000-0003-4609-7998
Eduardo Sanchez-Timm Facultad de Ciencias de la Vida, ESPOL, Polytechnic University, ESPOL, Campus Gustavo Galindo, Km. 30.5 Vía Perimetral, 090902, Guayaquil, Ecuador. https://orcid.org/0000-0001-7712-3605

DOI:

https://doi.org/10.17268/sci.agropecu.2025.028

Palabras clave:

Vaccinium floribundum Kunth, micropropagation, endogenous contamination, Mortiño, Andean blueberry

Resumen

The Vaccinium genus consists of a variety of berries with high nutritious components consumed worldwide leading to the development of micropropagation protocols to supply the demand. Mortiño, the Andean Blueberry (Vaccinium floribundum Kunth) is a wild berry that grows in high-altitude grasslands with nutritious and commercial potential in Ecuador. In this study, the use of PPM^TM (Plant Preservative Mixture™) was effective controlling contamination for the in vitro establishment of Vaccinium floribundum Kunth in contrast to a conventional method using EtOH and Clorox. Stems were defoliated and cut into 1 cm segments, then immersed in liquid MS (Murashige & Skoog) supplemented with 5% v/v PPM^TM without pH adjustment for 5 hours under constant shaking. After immersion, segments were transferred to flasks containing WPM (Woody Plant Media) medium supplemented with an additional 2 mlL^-1 PPM^TM. Persistent microbial contaminants in the in vitro explants were isolated and identified through molecular methods and gene sequences analyzed using the GenBank database resulted in the identification of three bacterial species: Methylobacterium sp., Methylobacterium radiotolerans, and Bacillus pumilus. In addition, three fungal species were also discovered: Xylaria sp., Xylaria feejeensis, and Diaporthe lutecens. Additionally, a multiplication assay was made with the aseptic stems from the sterilization protocol to evaluate four different growth regulators: 2ip, kinetin, zeatin and meta-topolin. kinetin showed very low responses with a mean of 1.2 shoots per stem. The highest number of shoots per stem (9 shoots) was obtained with 5 mg L^-1 2ip. The use of zeatin and meta-topolin facilitated shoot proliferation with the following concentrations: 3 mg L^-1zeatin + 0.5 mg L^-1 NAA (1-Naphthaleneacetic Acid) and 3 mg L^-1Meta-topolin + 0.5 mg L^-1NAA. These findings demonstrate the successful establishment of an in vitro disinfection and multiplication protocol for V. floribundum.

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