The isolated protoplasts were filtered through a 100 μm nylon mesh (Millipore, Durham, UK) and centrifuged at 1,000 rpm for 5 min. Pellets were resuspended in 0.5 M sucrose with 1 mM MES, overlaid with 2 ml of W5 salt solution (Menczel et al. 1981), and centrifuged for 10 min at 1,200 rpm. Protoplasts, now localized in the interphase between two solutions, were collected into a new tube with addition of W5 solution, and centrifuged at 1,000 rpm for 5 min. The protoplast pellet was then resuspended in culture medium, and protoplast yield was counted using a hemocytometer. Density of cultured protoplasts was adjusted to 8× 105 per milliliter of culture medium. The protocol of Damm and Willmitzer (1988) with modification of Grzebelus et al. (2012) for protoplast immobilization in a calcium alginate layer was employed. A suspension of protoplast and alginate solution consisting of 2.8% (w/v) Na-alginate and 0.4 M mannitol was prepared by gentle mixing in equal volumes to obtain a final density of protoplasts in the culture of 4×105 ml−1. Approximately 0.5 ml of protoplast–alginate mixture was gently spread onto 1% (w/v) agar containing
266 KIEŁKOWSKA AND ADAMUS
20 mM CaCl2. After 1 h incubation at room temperature, alginate disks with immobilized protoplasts were formed. The disks were transferred to the 6 cm Petri dish containing 4 ml of culture medium.