Cucurbit protoplast isolation for the study of plant virus replication

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....

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Bibliographic Details
Published in:Journal of virological methods 2001, Vol.91 (1), p.21-27
Main Authors: Lee, Kian-Chung, Koh, Adeline, Loh, Chiang-Shiong, Wong, Sek-Man
Format: Article
Language:English
Subjects:
Biological and medical sciences
Cell Separation
Cucurbit
Cucurbitaceae - cytology
Cucurbitaceae - virology
Electroporation
Fundamental and applied biological sciences. Psychology
Immunoblot
Microbiology
Plant Viruses - physiology
Potyvirus
Potyvirus - physiology
Protoplast
Protoplasts - virology
Virus Cultivation
Virus Replication
Zucchini yellow mosaic virus
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Summary: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 1.7×10 7 protoplasts per gram of fresh leaf tissue were obtained from cucumber cultivar Bet-α. Both cucumber cultivar Shimson and squash cultivar First Taste produced an average yield of 6.0×10 6 protoplasts per g of fresh leaf tissue. Electroporation of 10 μg 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.
ISSN:0166-0934
1879-0984
DOI:10.1016/S0166-0934(00)00237-8