VIEW ARTICLE

Techniques

Formation and Purification of Protoplasts from Rhizoctonia solani. T. Hashiba, National Institute of Agricultural Sciences, Yatabe, Ibaraki-ken 305, Japan; M. Yamada, National Institute of Agricultural Sciences, Yatabe, Ibaraki-ken 305, Japan. Phytopathology 72:849-853. Accepted for publication 24 November 1981. Copyright 1982 The American Phytopathological Society. DOI: 10.1094/Phyto-72-849.

A high yield of protoplasts from Rhizoctonia solani was obtained with a combined enzyme system containing cellulase “onozuka” R-10 from Trichoderma viride, macerozyme R-10 from Rhizopus sp., and β-glucuronidase extracted from gut juice of Helix pomatia. When 1 g (fresh weight) of 15-hr-old mycelium was incubated with this enzyme mixture in 0.6 M mannitol at pH 5.2, 6.0 × 10⁷ protoplasts were obtained within 3 hr. The age of the mycelium strongly affected the yield of protoplasts. Intact protoplasts were separated from mycelial fragments and cell debris in an aqueous two-phase system, which consisted of 0.6 M sucrose-0.6 M mannitol. Purified protoplasts produced colonies after 48–72 hr at 25 C.