* Corresponding author: Humberto Estrella-Maldonado Copyright © 2024 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. In vitro establishment from mature nodal segments of Persian Lime (Citrus latifolia Tan.) Humberto Estrella-Maldonado 1, * , Julio Ventura-Bello 2 , Ricardo Santillán-Mendoza 1 , Cristian Matilde- Hernández 1 , Arianna Chan-León 3 and Felipe Roberto Flores-de la Rosa 1 1 National Institute for Research on Agriculture, Forestry and Livestock (INIFAP). Ixtacuaco Experimental Field, Km 4.5. Martínez de la Torre-Tlapacoyan Street, Cong. Rojo Gómez, Tlapacoyan, Veracruz, Mexico. 2 University for Wellbeing “Benito Juárez García. Street University Loc. 1, Aguardientera Section Second, C.P. 73956, Chignautla, Puebla, Mexico. 3 National Technological Institute of Mexico, Campus Úrsulo Galván, Km 4.5 Street City Cardel-Chachalacas, Úrsulo Galván C.P. 91667, Veracruz, Mexico. International Journal of Science and Research Archive, 2024, 12(01), 854–861 Publication history: Received on 02 April 2024; revised on 15 May 2024; accepted on 18 May 2024 Article DOI: https://doi.org/10.30574/ijsra.2024.12.1.0868 Abstract In vitro propagation is a technique that has been used to produce large-scale, healthy, pathogen-free plants. However, the presence of endophytic microorganisms in explant tissues is one of the limiting factors for the in vitro establishment of any species. In this sense, we studied the effectiveness of PPM ® biocide (Plant Preservative Mixture) and Chlorothalonil fungicide to control and/or reduce microbial and fungal contamination in mature nodal segments of different Persian lime clones under in vitro conditions. The results showed that explants cultured in MS medium (Murashige and Skoog 1962) supplemented with 0.2 mL L -1 of PPM biocide + 1.0 mL L -1 of Chlorothalonil were adequate to achieve a higher percentage of disinfection (44%) in mature nodal segments in the Persian lime clones. Furthermore, it was observed that PPM concentrations above 0.3 mL L -1 resulted in greater phytotoxicity in mature nodal segments at 60 days. Keywords: Biocide; Citrus latifolia; Contamination; Disinfection; Endophytic microorganisms 1. Introduction In vitro propagation is a reliable technique to produce great number of pathogen-free plants with high uniformity in a short period of time [1]. However, a limiting factor for in vitro propagation is the severe contamination of explants during in vitro establishment phase by endophytic microorganisms [2-4]. Microbial and fungal microorganisms are the most common contaminants during the in vitro establishment phase because, being systemic, they are difficult to detect and eliminate in disinfection protocols [5-6]. Therefore, a protocol must be in place that ensures the disinfection of microbial and fungal microorganisms in the first phase of the in vitro establishment, this because the culture medium provides a favorable environment for these microorganisms, causing the mortality of the introduced plant material [7-8]. Consequently, the use of antibiotics, fungicides, and biocides is necessary during the in vitro introduction phase in order to control the development of these microorganisms. In this sense, PPM is a broad-spectrum biocide for in vitro tissue culture that reduces fungal and bacterial contamination of explants and enables aseptic establishment of mature nodal segments [9-10]. PPM has been used to disinfect in vitro explants of species such as bamboo, strawberry, red ginger, etc. [11-14]. Likewise, Chlorothalonil is a broad-spectrum fungicide with a polychlorinated aromatic component that delays mycelial growth