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A protocol for the isolation of protoplasts from leaf mesophyll, leaf and root calluses of Prunus cerasus L. "Montmorency" was developed. The experimental factors investigated were : the age and the physiological status of the source materials, the pre-treatment of source tissues, the enzymatic treatment (source, concentration and specific activity of enzymes), the incubation time, and the isolation protocol. Bacterial contaminations were revealed after protoplast isolation. These bacteria were formed from endophytic contaminants present as wall-free prokaryote cells, as observed in the micropropagated plants by electron microscopy, or after grinding leaf tissues in solutions with high osmotic pressure. After synthesis of a cell wall and subsequent development on nutritive media, four contaminating bacterial strains were identified as Pseudomonas syringae and Agrobacterium rhizogenes species. The amplification by PCR of the gene coding for a receptor protein of siderophores in fluorescent species of Pseudomonas, confirmed the presence of those wall free prokaryote contaminants in mesophyll tissues of apparently healthy micropropagated plants. In presence of antibiotics, leaf callus protoplasts remained alive for 20 to 25 days on MHABZ medium. During this period, the synthesis of a new cell wall was observed in 43 % of leaf callus protoplasts after 10 to 20 days of incubation, and first divisions were observed in 17.6 % after 15 to 20 days of incubation.

Prunus cerasus --- protoplasts --- Tissue culture --- Pseudomonas syringae --- Agrobacterium --- genetic code --- antibiotics --- Contrôle des maladies --- Agrobacterium rhizogene