Spontaneous appearance of polyploida in plants regenerated from embryogenic calli derived from seedling-meristems of ruzigrass (Brachiaria ruzizienbsis Germain et Everard

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. Embryogenic 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