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

Abstract

Isolation and culture of cotton ovule epidermal protoplasts (prefiber cells) and analysis of the regenerated wall 47 Abstract. Culture protocols were developed and characterization of the regenerated cell walls was performed for protoplasts of cotton (Gossypium hirsutum L., L., var. Acala SJ-2) ovule epidermal cells. This work was undertaken in order to extend studies concerning nutritional effects and regulation of nucleotide sugar incorporation into/3-1,3-and/3-1,4-glucan components of cotton fiber cell walls. Protein and carbohydrate polymers and recovered from the culture medium. Analysis of a cellular fraction indicated that the majority of 1'C incorporated from [l*C]glucose was present in the hot-watersoluble fraction of the cells. The majority of label incorporated into cell wall material could be solubilized with acetic-nitric reagent, indicative of noncellulosic material, and characterized as #-l,3-1inked glucans. Only 5 to 15% of the regenerated cell wall could be characterized as/3-1,4-1inked glucose indicative of cellulose.

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

“…Fifty ml of enzyme solution was prepared according to Gould et al, [16], with the substitution of 28 000 units (0.7 g/50 ml) "CEL," purified cellulase (Worthington/Cooper Biomed) for Cellulysln, and the addition of 0.1 M CaC12"2H20. The concentrated enzyme mixture (llOOmOs/kg, or 0.9M mannitol equivalent), was diluted with culture medium, which adhered to the ovule fibers, to yield a final solution of 740mOs/kg, or the equivalent of 0.6 M mannitol equivalents.…”

“…This layer was removed and the floatation process repeated until most of the cytoplasts had been transferred from the 3% ficoll layer to culture medium. Cytoplasts were diluted to a hnal density of 2 × 10Scytoplasts/ml and cultured according to Gould et al [16]. Glucose and/or UDPglucose were supplied, l mM;[14C]glucose and/or UDP[14C] glucose (ICN) were supplied as described for each experiment.…”

“…Viability and cell wall formation were determined as previously de~,qh-~ [16]. The presence of nuclei was determined using a 0.0005% solu ....... 6-diamidino-2-phenylindole (DAPI, Sigma) fluorescence.…”

“…Cultures were pooled and diluted once with water and centrifuged 1000g, 10min. The pellet was washed 3 times with water and extracted in one of two ways: (a) extraction with hot water, chloroform/methanol and acetic/nitric reagent (A/N) was followed as outlined previously [11,16] ; (b) for analyses of cell wall composition, cultures were pooled, pelleted and extracted with water as described above and separated by f'lltration through a mlllipore 0.2/a filter into a 'hot water soluble' fraction and a pellet. Prior to acetylation and periodation analysis, the cellular pellet was treated with a-amylase to remove starch [21 ].…”

“…4 cm for boll-grown fibers and 2 cm for fibers grown in culture. A protoplast system from pre-fiber ovule epidermal cells isolated during primary cell wall synthesis on the day of anthesis has been developed [16]. A similar system to study precursor utilization during active secondary wall synthesis was desired; however, because of the elongated nature of the fiber cells at the onset of secondary wall synthesis, intact, nucleated protoplasts could not be obtained.…”