ISHS Acta Horticulturae 425: II International Pineapple Symposium
PROTOPLAST ISOLATION OF ANANAS COMOSUS (L.) MERR. CV ‘PEROLERA’ |
| Authors: | N.M. Pinho Guedes, J. Maria, L. Zambolim, J. Ventura |
| DOI: | 10.17660/ActaHortic.1997.425.29 |
| Abstract:
Somatic hybrids show great potential to obtain new resistant genotypes of pineapple. Protoplasts isolation was obtained from the 3d and 4th leaves of in vitro plantlets of the cv. ‘Perolera’, a major source of resistance to fusariosis in Brazil, after maintaining them 24h under dark or light conditions. Leaf ribs were scratched on the abaxial surface with a scalpel, pre-plasmolyzed with CPW9M, CPW11M or CPW13, and incubated 4, 14 or 21h in an enzymatic mixture (EM) to digest the cellular wall: EM1 (cellulysin 2.0% + hemicellulase 0.5% + macerozyme R10 0,2%), EM2 (cellulase onozuca 2,0% + hemicellulase 0,5% + macerozyme R10 0,5%), EM3 (cellulase RS 0,5% + pectolyase 0,5% + macerozyme R10 0,1%) or EM4 (meicelase 2,0% + rhozyme 2,0% + macerozyme 0,3%) prepared with CPW13M. EM1, EM2 and EM4 were supplemented with 400 mg 1-1 ampicilin, 10 mg 1-1 gentamycin and 10 mg 1-1 tetracyclin. Then the EM was changed for CPW11M and the leaves were slightly crushed followed by filtration through 64 mesh screen. Leaf debris were removed from the obtained protoplasts by different centrifugation periods (4, 5 and 10 min), velocities (500 and 800 rpm) and washing numbers (3 and 4, the last one always in sucrose gradient). The protoplasts were resuspended in 7,0 ml of CPW15S, 50 ml CPW21S and 1,0 ml of CPW9M. The best results were obtained with 10 min pre-plasmolysis, 14 h incubation in EM4 and 3 washings, yielding 21.103 protoplasts per ml. | |
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