Isolation of microprotoplasts from a partially synchronized suspension culture of Citrus unshiu

The effect of hydroxyurea (HU) and amiprophos-methyl (APM) on the synchronization of suspension cultures of Satsuma mandarin (Citrus unshiu) and micronucleation of the suspension cells sequentially treated with both, HU and APM, were investigated. When suspension cultures in early-log phase were treated with 4 or 10 mM HU for 24 h, the number of cells in the S-phase and the mitotic index (MI) increased significantly. Exposure of the early-log phase suspension culture to 32 μM APM led to a marked increase in the MI 12 and 24 h after treatment, while higher as well as lower concentrations (16, 24 and 48 μM) had no effect. Suspension cultures subjected to sequential treatment, e.g. pretreatment with 10 mM HU for 24 h followed by treatment with 32 μM APM for 24 h, also showed a considerably increased MI. Furthermore, 61.5% of the protoplasts isolated from the sequentially treated suspension cells were micronucleated, whereas only 3.6% of the control protoplasts, isolated from untreated cells, showed micronucleation. Ultra-centrifugation of the micronucleated protoplasts generated microprotoplasts of different sizes, most of them below 5 μM in diameter, with 1 or few chromosomes. The potential application of microprotoplasts in citrus genetic improvement is discussed.