Eustoma grandiflorum is an ornamental species used worldwide in floriculture. Eustoma exaltatum and E. grandiflorum are the only two species in the Eustoma genus. They differ with respect to post-harvest behavior, edaphoclimatic requirements, and morphology. Genetic improvement programs have focused on hybridizing them to produce individuals with outstanding characteristics. Twelve hybrid lines resulting from E. grandiflorum × E. exaltatum crosses were evaluated with respect to in vitro germination of seeds, adventitious shoots induction, and rooting. The existing genetic variability between four outstanding hybrid lines selected based on phenotypic characteristics was quantified using SSR markers developed for Gentianaceae family. In vitro organogenic behavior differed between genotypes. The germination varied between 18 and 67%, and the number of induced adventitious shoots was highest in hybrids 1512095 (16.9 ± 2.4) and 1512096 (18 ± 2.7).
Root regeneration also differed from 25 to 85% between the hybrids. Analysis of molecular variance (AMOVA) indicated that a larger proportion of genetic variation (66.9%) was due to differences between, than within hybrid lines (33.0%). The observed heterozygosity (Ho = 0.7) was higher than the expected one (He = 0.4). The in vitro protocol used in this study produced Eustoma hybrids viable for commercial floriculture, and genetic variation found in the hybrids confirmed the potential for future genetic improvement programs for the genus Eustoma.