Interploidy crosses between diploid daylily (Hemerocallis) cultivars (2n = 22) and tetraploid cultivars (2n = 44) were performed via in vivo hybridization aiming to produce triploid hybrid germplasm (3n = 33). Plant growth regulator-free MS based medium containing 5 levels of sucrose-1, 2, 3, 4 or 5% was used to optimize the embryo rescue medium. It was determined that the medium supplemented with 3% sucrose gave highest rate of immature hybrid embryo rescue. Thirty-seven hypothetically triploid genotypes were subjected to verification of ploidy status by root tip chromosome counting and flow cytometry. Thirty-one of them were confirmed triploids. These genotypes were in vitro propagated, acclimatized to ex vitro conditions, and planted for future field performance evaluation and environmental testing. The newly developed triploid genotypes could open new horizons for further polyploidy breeding.

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