Vol.:(0123456789) 1 3 Plant Cell Tiss Organ Cult DOI 10.1007/s11240-017-1202-y ORIGINAL ARTICLE Epichloë occultans enhances micropropagation efciency in Lolium multiflorum J. J. Regalado 1,3  · M. V. Vignale 2,3  · M. V. Novas 2,3  · S. I. Pitta‑Alvarez 1,3  · L. J. Iannone 2,3   Received: 1 February 2017 / Accepted: 12 March 2017 © Springer Science+Business Media Dordrecht 2017 micropropagation. The use of endophyte-infected (E+) seeds can be a solution to make this grass more amena- ble to diferent biotechnological tools, such as the genetic transformation. Keywords Callus culture · Fungal endophytes · Grasses · Italian ryegrass · Organogenesis · Plant regeneration Introduction Grasses are considered recalcitrant to plant tissue culture techniques, thus hindering genetic manipulation (Giri and Praveena 2015). The existence of a robust in vitro plant regeneration protocol is necessary for the propagation of grasses and the optimization of other biotechnological techniques, such as genetic transformation (Li et al. 2010; Zhang et al. 2010a; Song et al. 2012; Ran et al. 2014). In the last years, there have been reports on the infuence of diferent factors on the micropropagation of grasses, such as genotypes, explant origin, nutritional components, plant growth regulators or culture environment (Głowacka et al. 2010; Chai et al. 2011; Chen et al. 2012; Zhao et al. 2013; Carloni et al. 2014). However, the possible efect of symbi- ont microorganisms has not been examined. The efect of symbionts on micropropagation has been mostly studied in others species such as medicinal and aro- matic plants (Thomas et al. 2010; Prasad et al. 2013; Ben- son et al. 2014; Sharma et al. 2014; Verma et al. 2015), ornamentals (mainly orchids) (Ovando-Medina et al. 2007; Ye et al. 2014; Larraburu and Llorente 2015a, b), Prunus (Quambusch et al. 2016) or oil palm (Azlin et al. 2007). Symbiont microorganisms improve micropropagation of their hosts in several ways (Rödel et al. 2016), for exam- ple, by increasing plant biomass (Rai 2001; Kapoor et al. Abstract Italian ryegrass (Lolium multiflorum) is an annual grass considered as one of the most important tem- perate forage grasses in the world. However, it is recalci- trant to plant tissue culture techniques hindering its genetic manipulation. Epichloë occultans is an endophytic fun- gus associated with L. multiflorum. This symbiosis causes improvements in physiological and ecological traits of the host plants. The objective of this work was to study the efect of E. occultans on L. multiflorum micropropaga- tion. We compared the response of endophyte-infected (E+) and endophyte-free (E−) seeds in diferent micro- propagation stages. The E+ seeds were more successful than E− seeds in in vitro germination (83 ± 5 vs. 63 ± 6%), callus induction (78 ± 5 vs. 57 ± 6%), callus proliferation (average diameter of 21.5 ± 1.3 mm in two subcultures vs 17.3 ± 0.8 mm in three subcultures) and plant regenera- tion from callus (83 ± 7 vs. 30 ± 8%). These results indi- cate that E. occultans enhances signifcantly L. multiflorum * J. J. Regalado jjrg@eelm.csic.es S. I. Pitta-Alvarez sandrapitta-alvarez@conicet.gov.ar; spitta1959@gmail.com 1 Laboratorio de Cultivo Experimental de Plantas y Microalgas, Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, No. 68, Ciudad Autónoma de Buenos Aires, Argentina 2 Laboratorio de Micología y Fitopatología, Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, No. 69, Ciudad Autónoma de Buenos Aires, Argentina 3 Instituto de Micología y Botánica (INMIBO), Universidad de Buenos Aires-CONICET, Ciudad Autónoma de Buenos Aires, Argentina