دانلود 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  protoplast,  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.