Partial Synchronization of Cell Division and Micronucleation in Suspension-cultured Cells of Hemerocallis hybrida: The effects of Hydroxyurea and Various Spindle Toxins.

Abstract

For the production of intergeneric hybrid plants with partial alien genome via microprotoplast fusion in the Liliaceous ornamentals, the effects of the DNA synthesis inhibitor, hydroxyurea (HU), and the spindle toxins, colchicine (COL), oryzalin (ORY), amiprophos-methyl (APM), butamiphos (BUT), isopropyl N-(3-chloro-phenyl)carbamate (CIPC) and propyzamide (PRO) on the metaphase index (MI) and the percentage of micronucleated cells (micronucleus index; MNI) were examined in cell suspension cultures of Hemerocallis hybrida cv. Stella d’Oro. Suspension cells were subcultured every three days in MS medium containing 10mgl-1 picloram. Although MI was only 2-3% in the asynchronous control cultures, it increased up to 8.9% and 9.7% by treatment of the cultures with HU and COL, respectively. In addition, MI was further increased by using the sequential treatments of the cultures with HU and each spindle toxin: the highest MI of 30.5% was obtained by treatment with 2mM HU for 24h followed by that with 250μM COL for 20h. COL and ORY were more effective for synchronizing cell division than the other four spindle toxins. The effects of various spindle toxin treatments on the micronucleation of suspension cultures were also examined by combining the HU pre-treatment for 24h. Among the six spindle toxins, COL and ORY induced few micronuclei, whereas APM, BUT, CIPC and PRO induced micronucleation in cells to various extents. The most effective treatment for micronucleation was that with 8μM PRO for 66h, where MNI was 14.7% and the number of micronuclei per cell ranged from 1-7 were obtained.