I've been having some trouble initiating cell suspension cultures of Arabidopsis following the protocol of May and Leaver 1993. I have no problem getting callus to grow on plates. When transferring the callus to liquid medium however one of two things usually happen:
A) The callus quickly grows into a giant mass of cells not small individual cells (There is some differentiation, can see roots growing out of the large clumps of cells).
B) The callus does not grow very well and eventually dies off despite subculturing regularly.
Is there some special step to do when transferring the callus to liquid media? Do you filter the callus through a small filter? Do you add something to make the cells stay apart? Is there a special subculture routine following transfer to liquid media? Something else?
Any help would be appreciated