ISOLATION AND CHARACTERIZATION OF ALEURONE PROTOPLASTS FROM A MALTINGVARIETY OF BARLEY (HORDEUM-VULGARE L)

A procedure has been developed to isolate protoplasts from mature aleu rone layers of the malting variety Alexis and four other barley genoty pes. II combines induction of endogenous cell wall degrading enzymes t ogether with use of Onuzuka cellulase R 10 and driselase and results i n better yields for two varieties than can be obtained with the huskle ss variety Himalaya. The viability of the freshly isolated protoplasts is greater than 90% and in spite of the presence of gibberellic acid during isolation procedures, most of the protoplasts are at an early d evelopmental stage, as judged by ultrastructure. Gibberellic acid-indu ced changes in protoplast structure resemble those reported for Himala ya protoplasts. The protoplasts secrete both alpha-amylase (EC 3.2.1.1 ) and (1-3, l-4)-beta-glucanase (EC 3.2.1.73) into the surrounding med ium. Transfection studies using a low pi a-amylase promoter to direct chloramphenicol acetyltransferase expression in aleurone protoplasts f rom Alexis and Himalaya revealed significant differences in their horm one responsiveness. In the absence of hormones, low levels of expressi on of the reporter enzyme were obtained in Alexis protoplasts, while h igh levels were characteristic for Himalaya protoplasts. An 8-fold inc rease in the expression of the reporter gene was induced by supplying the transfected Alexis protoplasts with gibberellin A(3), whereas expr ession in Himalaya protoplasts remained unchanged. When Himalaya proto plasts were isolated from aleurone layers that had not been incubated with GA(3) during the initial stages of protoplasting (the classical p rocedure), the hormone response of the promoter was 2.5-fold. It is th us possible to optimize the aleurone protoplast isolation procedure fo r different barley genotypes and mutants of interest in studies of tra nsgenic gene expression and hormone induced secretion of proteins from this unique secretory plant tissue.