Root protoplasts of Quercus rubra L. were isolated from 12 day old seedlings with an enzyme mixture containing Cellulase R1O + Rhozyme HP150 + Macerozyme R1O, supplemented with cysteine and bovine serum albumin.Protoplasts were purified by a Ficoll density gradient centrifugation and cultured at low density in a liquid medium. The modified woody plant medium, containing 2.2 μM benzyladenine + 1.8; μM zeatin + 5.3 μM α-naphthaleneacetic acid + 2.2 μM dichlorophenoxyacetic acid, allowed sustained divisions and formation of microcalluses.Protoplast - derived microcallus developed into green and compact callus when transferred to an agarose solidified medium, supplemented with casein hydrolysate and indole 3-acetic acid (devoid of 2,4 dichlorophenoxyacetic acid) and placed under low illumination.