OPTIMIZATION OF CONDITIONS FOR DNA UPTAKE AND TRANSIENT GUS EXPRESSION IN PROTOPLASTS FOR DIFFERENT TISSUES OF WHEAT AND BARLEY

Conditions have been optimized for the routine isolation and transfection of protoplasts from developing endosperm, aleurone, leaves and roots from barley (Hordeum vulgare L.) and wheat (Triticum aestivum L.). Transient expression of the beta-glucuronidase (GUS) reporter gene under the control of the cauliflower mosaic virus 35S promoter was used to evaluate the best procedure for each tissue. Electroporation was similar to 300% more efficient than polyethyleneglycol (PEG) mediating 35S-GUS transient expression in leaf and root protoplasts while endosperm protoplasts had to be PEG transfected since even the mildest electroporation parameters tested (250 V/cm, 200 mu F, 10 ms) were lethal for them. Protoplasts isolated from dry aleurone showed the highest viability (approximately 90%), but GUS activity after PEG transfection was 50% of that obtained with endosperm protoplasts. Conditions optimized for a given tissue of a barley cultivar such as Bomi, were readily applied to other barley cultivars and to other cereal species.