Factors that Influence the Yield, Stability in Culture and Cell Wall Regeneration of Spinach Mesophyll Protoplasts

Abstract

A study has been made of factors that influence the yield, stability in culture, and ability to regenerate cell walls of isolated spinach (Spinacia olevacea L.) mesophyll protoplasts. The presence of 7 mM CaZ + or 7 mM Mg2+ in the isolation medium, which also included 1.5 % (w/v) Driselase, 0.25 % (w/v) pectinase and 0.8 M sorbitol, increased the yield and stability in culture of the protoplasts in a liquid nutrient medium. This latter medium has been used previously in the culture of spinach leaf discs, and does not support the division of spinach mesophyll cells. Protoplasts isolated in the presence of CaZ+ showed a greater capacity for cell wall regeneration compared with protoplasts isolated in the absence of ions or in the presence of Mg2+. Though darkness during culture improved the initial protoplast stability, it inhibited wall regeneration. The initial stability of protoplasts appeared to be dependent on plasma membrane stability, but after a few days in culture the most effective treatments were those which stimulated cell wall regeneration. In many cases, associated with cell wall regeneration, there was a budding off of small vesicles which sometimes contained chloroplasts. Protoplasts isolated in the presence of Ca2+ and incubated in low light had a 50% survival rate after 8 days culture and most had regenerated cell walls. Such culture of spinach protoplasts has not been shown previously and should be useful in developmental studies of spinach chloroplasts.