Ploidy Validation of Triploid Hybrids

The ploidy of the triploid hybrids obtained from the two combinations was validated through both chromosome preparation and flow cytometry analyses. The method for chromosome preparation was that proposed by Wen et al. (2020) [75 (link)]. Briefly, root-tip tissues with a length of about 1 cm were treated with 0.002 mol/L 8-hydroxyquinoline aqueous solution for 4 h and then fixed in Carnoy’s solution overnight. Subsequently, the apical meristems were cut into the size of about 1 mm³ for the following enzymolysis. Next, the enzyme-treated apical meristems were removed from Carnoy’s solution for final chromosome preparation on a slide. The chromosomes were stained with 5% Giemsa and visualized under a microscope (Olympus, Tokyo, Japan). Flow cytometry analyses were conducted as was described by Galbraith et al. (1983) [76 (link)]. The stain method used in this study was proposed by Miller et al. (2012) [77 (link)]. A BD Accuri C5 flow cytometer (BD Bioscience, San Jose, CA, USA) was adopted for the flow cytometry analyses in this study.