Callus formation from root protoplasts of Quercus rubra L. (red oak).

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.