Isolation and Characterization of Microprotoplasts from Propyzamide-treated Cell Suspension Cultures of Hemerocallis hybrida

To produce intergeneric hybrid plants with one or a few chromosomes via microprotoplast fusion in the Liliaceous ornamentals, we developed a system for isolating microprotoplasts from micronucleated cells in suspension cultures of Hemerocallis hybrida cv. Stella d'Oro (2n = 2x = 22). Micronucleation was efficiently induced with a micronucleus index up to 19.1 % by the following sequential treatment of suspension cultures: initially with 2 mM hydroxyurea for 24 h and then with 8 μM propyzamide for 60 h with the addition of 20 μM cytochalasin-B to the cultures 20 h after the initiation of the propyzamide treatment. Following the enzyme treatment of the micronucleated cells and ultra-centrifugation with a continuous iso-osmotic gradient of Percoll solution, microprotoplasts were isolated, each of which had a small nucleus surrounded by a thin rim of cytoplasm. Microprotoplasts less than 10 μm in diameter were obtained with yield of 2.9 × 10^4 cells per 1 ml packed cell volume of suspension cells through sequential filtration with nylon sieves with decreasing pore sizes (50, 20 and 10 μm). The DNA content of the microprotoplasts was less than the 2C level as indicated by flow cytometric analysis, and the relative fluorescence intensity in some of their nuclei corresponded to one or a few chromosomes