ORIGINAL ARTICLE Spontaneous appearance of polyploids in plants regenerated from embryogenic calli derived from seedling-meristems of ruzigrass (Brachiaria ruziziensis Germain et Everard) Genki Ishigaki 1 , Takahiro Gondo 2 , Mohammad M. Rahman 3 , Nafiatul Umami 4 and Ryo Akashi 1 1 Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan 2 Frontier Science Research Center, University of Miyazaki, Miyazaki, Japan 3 Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia 4 Faculty of Animal Science, Gadjah Mada University, Yogyakarta, Indonesia Keywords Brachiaria ruziziensis; embryogenic callus; polyploidy; somaclonal variation. Correspondence Ryo Akashi, Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan Email: rakashi@cc.miyazaki-u.ac.jp Received 19 June 2013; accepted 7 December 2013. doi: 10.1111/grs.12040 Abstract Ruzigrass (Brachiaria ruziziensis Germain et Everard cv. Kennedy) is an important forage grass in tropical and sub-tropical areas. Previously, we reported transgenic ruzigrass plants generated by our transformation system were sterile and tetraploid in spite of beginning with diploid plants. This study analyzed ploidy variation in embryogenic calli and the regenerants of diploid ruzigrass. The morphological traits and fertility were also investigated to develop a methodology for the production of stable transgenic lines. Embryo- genic calli at different stages (2, 4, 6 and 12-month-old) were regenerated via somatic embryogenesis. An approach of flow cytometry (FCM) analysis was used to determine the ploidy level of embryogenic calli and regenerants of ruzigrass. FCM analysis revealed that embryogenic calli were spontaneously reduplicated at a high frequency and resulting regenerants were polyploids (tetraploid or octoploid), including 15 tetraploid regenerants (68%) and seven octoploid regenerants (32%) derived from 12-month-old embryogenic calli. These regenerants exhibited the morphological variations among different ploidy levels. The viability of pollen grains was significantly (P < 0.01) decreased in tetraploid and octoploid regenerants. Our findings indicated that clarification and resolution of ploidy variation in ruzigrass combined with ploidy level checking using FCM analysis before transformation steps is crucial for plant regeneration in transformed ruzigrass. Introduction Ruzigrass (Brachiaria ruziziensis Germain et Everard cv. Kennedy) is a cross-fertilizing sexual diploid species (Fergu- son and Crowder 1974) that is widely cultivated in tropical and sub-tropical regions worldwide as a forage crop (Keller-Grein et al. 1996). In Brachiaria breeding programs, it is very difficult to conduct traditional crosses among Brachiaria species because most important species, such as B. brizantha (A. Rich.) Stapf cv. Marandu, B. decumbens Stapf cv. Basilisk, are predominantly facultative apomictic tetraploids (Miles and do Valle 1996; do Valle and Savidan 1996). Recently, transformation techniques have been developed in many crops since the techniques can intro- duce new characteristics into plants directly. In the trans- formation process, many researchers observed that embryogenic callus produced many fertile transgenic plants in wheat (Triticum aestivum L.) (Cho et al. 2000), barley (Hordeum vulgare L.) (Cho et al. 1998), pearl mil- let (Pennisetum glaucum (L.) R. Br.) (Goldman et al. 2003) and bahiagrass (Paspalum notatum Fl€ ugg e) (Gondo et al. 2003, 2005). Previously, we reported in vitro plant regeneration in ruzigrass (Ishigaki et al. 2009a) via multi- ple shoot formation or somatic embryogenesis from seed- derived apical meristems of ruzigrass. As an application © 2014 Japanese Society of Grassland Science, Grassland Science, 60, 24–30 24 Japanese Society of Grassland Science ISSN1744-6961 Japanese Society of Grassland Science