High efficiency protoplast isolation from in vitro cultures and hairy roots of Maesa lanceolata

Abstract

In vitro cultures of the medicinal plant Maesa lanceolata were established to enable the cultivation of
plant material for the production of protoplasts. Callus cultures were initiated using leaves collected
from shoot cultures and the root tips from hairy root cultures obtained upon Agrobacterium rhizogenes
transformation. For the isolation of protoplasts, the different explant material of M. lanceolata was
exposed to an enzyme mixture consisting of 1.5% cellulase, 0.5% macerozyme R-10 and 0.5 M mannitol.
About 6 x 10⁶ protoplasts g^-1 fresh weight were obtained from leaf material and 5 x 10⁵ protoplasts g^-1
fresh weight from callus. To obtain high amounts of hairy root protoplasts, the cultures were pretreated
with the auxin indole-3-butyric acid (IBA) that stimulated the formation of novel root tips. Using
the dissected root tips as starting material, 8 x 10⁵ protoplasts g^-1 fresh weight were obtained per
preparation. The protoplast isolation method will enable further studies on the transformation and
fusion of protoplasts from M. lanceolata.

Key words: Maesa lanceolata, in vitro conservation, tissue culture, protoplast isolation.