Contents lists available at ScienceDirect Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop Miscanthus × giganteus: Regeneration system with assessment of genetic and epigenetic stability in long-term in vitro culture Sandra Cichorz a, ⁎ , Maria Gośka a , Dariusz R. Mańkowski b a Department of Genetics and Breeding of Root Crops, Plant Breeding and Acclimatization Institute – National Research Institute, Research Division in Bydgoszcz, Powstańców Wielkopolskich Ave. 10, 85-090 Bydgoszcz, Poland b Department of Seed Science and Technology, Plant Breeding and Acclimatization Institute – National Research Institute, Radzików, Błonie, Poland ARTICLE INFO Keywords: Bioenergy crop Embryogenic callus Immature inflorescence Micropropagation MS-ISSR RAPD ABSTRACT Species of Miscanthus Anderss., C 4 perennial rhizomatous grasses with low input requirements, have become representative crops for lignocellulosic bioenergy and material resources in temperate climates. Most of the commercial cultivars appropriate for cropping within Europe and North America are based on Miscanthus × giganteus species. Because of their triploid hybrid nature, micropropagation of these species using in vitro cultures is a common method both of plant multiplication on a commercial scale and of long-term preservation of their valuable genotypes. The somaclonal variation that can occur during in vitro culture may be useful for strain improvement during plant breeding, but is undesirable for long-term genotype preservation; therefore, the genetic and epigenetic stability of regenerated plants needs to be assessed. The objectives of our study were (1) to develop an effective system for Miscanthus × giganteus plantlet production, starting from callus induction, and shoot and root regeneration, and ending with plant acclimatization; and (2) to evaluate any genetic and epigenetic instability induced by long periods of in vitro cultivation using ISSR, RAPD and MS-ISSR molecular markers on selected M. × giganteus clones. We present an efficient and stable method of M.× giganteus regeneration and preservation by long-term in vitro culture. Both morphological and molecular marker analyses revealed no major genetic or epigenetic changes among clonally propagated M.× giganteus that had been maintained by long-term shoot culture. However, when screening for genetic variation, particular attention should be paid to genomic regions associated with GACA, GATA, AG and CTC repeats. 1. Introduction Bioenergy has significant potential to ensure local energy supply security and to contribute to climate change mitigation. Over the past 25 years, perennial species selected as dedicated biomass crops have received much attention as potential renewable feedstocks for bioe- nergy and material usage (Clifton-Brown et al., 2017). Species of Mis- canthus Anderss. are C 4 perennial rhizomatous grasses with low input requirements that originate from diverse climates ranging from tropical Africa and South-East Asia up to Siberia. They have become re- presentative crops for lignocellulosic bioenergy and material resources in temperate climates (Robson et al., 2013). Although results depend on location and climate conditions, field trials in Europe have demon- strated that Miscanthus × giganteus (Greef et Deu. Ex Hodkinson et Renvoize) (2n = 3x = 57) is suitable for combustion in terms of feed- stock supply, quality, conversion efficiency and ecological benefits (Clifton-Brown et al., 2008; Heaton et al., 2010; Robson et al., 2013; Iqbal et al., 2015; Clifton-Brown et al., 2017). In the climate of north- eastern Poland, M. × giganteus is more energy-efficient than maize, Amur silver grass, sweet sorghum and alfalfa or timothy grass (Jankowski et al., 2016). This allotriploid hybrid appeared as a natural cross between two Miscanthus species, diploid M. sinensis (2n = 2x = 38) and allote- traploid M. sacchariflorus (2n = 4x = 76). Abnormal development of both male and female gametophytes and a strong postzygotic barrier attributed to total or partial sterility of triploid M. × giganteus that re- sults in a very low frequency of seed production (Linde-Laursen, 1993; Lafferty and Lelley, 1994; Słomka et al., 2012; Tamura et al., 2016). https://doi.org/10.1016/j.indcrop.2018.02.055 Received 12 November 2017; Received in revised form 20 January 2018; Accepted 15 February 2018 ⁎ Corresponding author. E-mail addresses: s.cichorz@ihar.bydgoszcz.pl, sandra.cichorz@interia.pl (S. Cichorz). Abbreviations: ABA, abscisic acid; BAP, 6 benzylaminopurine; CPA, chlorophenoxyacetic acid; DIC, dicamba 3,6-dichloro-o-anisic acid; 2,4-D, 2,4-dichlorophenoxyacetic acid; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; 2-IP, 2-isopentenyl adenine; ISSR, inter simple sequence repeat; KIN, kinetin; MS-ISSR, methylation-sensitive inter simple sequence repeat; MS, Murashige and Skoog medium; NAA, α-naphthalene acetic acid; PGR(s), plant growth regulator(s); PMS, proliferation MS; RAPD, random amplified polymorphic DNA; RMS, rooting MS; RP, recovery percentage; TIBA, 2,3,5-triiodobenzoic acid; TDZ, thidiazuron Industrial Crops & Products 116 (2018) 150–161 0926-6690/ © 2018 Elsevier B.V. All rights reserved. T