A cucurbit protoplast isolation protocol was established for the study of plant virus replication in vivo. This protocol is applicable to both cucumber and squash leaf tissue with significant increases in yields of viable protoplasts suitable for electroporation, compared to other published methods. A combination of Cellulase RS, Macerozyme R10 and mannitol was used as digestion enzymes and osmoticum. An average of 1.7 ?? 107 protoplasts per gram of fresh leaf tissue were obtained from cucumber cultivar Bet-??. Both cucumber cultivar Shimson and squash cultivar First Taste produced an average yield of 6.0 ?? 106 protoplasts per g of fresh leaf tissue. Electroporation of 10 ??g of Zucchini yellow mosaic potyvirus (ZYMV-S) RNA into the protoplasts resulted in virus replication and synthesis of coat protein (CP). SDS-PAGE and immunoblotting were used to detect the CP 48 h post-electroporation. This protocol is highly reproducible and will assist researchers who require cucurbit protoplasts to study virus replication. ?? 2001 Elsevier Science B.V.
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