A Large-scale Isolation Procedure for Cereal Mesophyll Protoplasts

A method suitable for the large-scale isolation of cereal protoplastsfrom up to 50 g of leaf material is described. Surface-sterilizedleaves from cultivars of wheat, barley, maize, sorghum, andTriticale were diced and vacuum infiltrated with enzyme mixturecomposed of cellulysin (1 per cent w/v), hemicellulase (1 percent w/v), and macerozyme (0.5 per cent w/v). With this procedure,yields of between 10⁶ to 10⁷ protoplasts per gram of leavescan be reproducibly obtained after only 1.5–3 h of enzymatictreatment. These protoplasts were almost 100 per cent viable(as determined by fluorescein diacetate staining) and incorporationof ³H-uridine and ¹⁴C-leucine into an acid-insoluble fractionwas demonstrated. Almost one-third of the ribosomes of theseisolated protoplasts were present as polysomes. cereals, leaf mesophyll, protoplast isolation