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#m X 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: ~3-1,3-1inked glucans were predominantly synthesized when l mM UDP[14C]glucose was supplied; /3-1,4-1inked glucans were predominantly synthesized when 1 mM [14C]-glucose was supplied. Thus the composition of the regenerated cell wails formed by the anucleate protoplasts was similar to the secondary cell wall synthesized by intact cotton fibers under the same culture conditions.

Isolation and culture of cotton ovule epidermal protoplasts (prefiber cells) and analysis of the regenerated wall

“…This stage of development was chosen because: (a) The rapid elongation occurring in the epidermal-fiber cells in the days following anthesis, resulted in protoplasts that were large (greater than 80/~) and fragile; (b) Isolation of protoplasts from fibers 3 days or older, yielded anucleate cellular vesicles or 'subprotoplasts' [14].…”

“…This low level of incorporation of label into cellulose has been encountered in protoplast [20], in plasma membrane preparations in higher plants [11,12], as well as in intact cotton fibers [18]. In all in vitro systems designed to study cellulose synthesis, other polymers, notably /3-1,3-glucans are formed (see discussion in accompanying manuscript [14]). …”

“…If developmental stage of the isolated cells can influence the composition of the regenerated cell walls [28], protoplasts derived from cells actively synthesizing secondary wall material may produce more of the ~ 1,4-1inked glucans. This approach is reported in an accompanying manuscript [ 14].…”