Isolation and culture of anucleate protoplasts from cotton fiber; assessment of viability and analysis of regenerated wall polymers

Abstract

Procedures were developed for the isolation and culture of an anucleate protoplast system from cotton fibers actively undergoing secondary wall synthesis. Because the fibers at this stage are elongated single cells (30 μm × 1–2 cm), most of the cellular vesicles released in the process of isolation are anucleate. After purification, the protoplast population was nuclei-free. When transferred to culture medium, the anucleate protoplasts (cytoplasts) synthesized starch, hydrolyzed fluorescene diacetate for up to 9 days and formed cell wall material for at least 7 days. The composition of the regenerated cell walls was dependent upon the substrate supplied in the medium: β-1,3-linked glucans were predominantly synthesized when 1 mM UDP[14C]glucose was supplied; β-1,4-linked glucans were predominantly synthesized when 1 mM [14C]-glucose was supplied. Thus the composition of the regenerated cell walls formed by the anucleate protoplasts was similar to the secondary cell wall synthesized by intact cotton fibers under the same culture conditions.