A Large-scale Isolation Procedure for Cereal Mesophyll Protoplasts

A method suitable for the large-scale isolation of cereal protoplasts from up to 50 g of leaf material is described. Surface-sterilized leaves from cultivars of wheat, barley, maize, sorghum, and Triticale were diced and vacuum infiltrated with enzyme mixture composed of cellulysin (1 per cent w/v), hemicellulase (1 per cent w/v), and macerozyme (0.5 per cent w/v). With this procedure, yields of between 106 to 107 protoplasts per gram of leaves can be reproducibly obtained after only 1.5–3 h of enzymatic treatment. These protoplasts were almost 100 per cent viable (as determined by fluorescein diacetate staining) and incorporation of 3H-uridine and 14C-leucine into an acid-insoluble fraction was demonstrated. Almost one-third of the ribosomes of these isolated protoplasts were present as polysomes.