Abstract
The family Passifloraceae contains many species exploited in the food, pharmaceutical, and ornamental plant industries. The routine culture of isolated protoplasts (naked cells) followed by reproducible plant regeneration, is crucial to the genetic improvement of Passiflora spp. by somatic cell technologies. Such procedures include somatic hybridization by protoplast fusion to generate novel hybrid plants, and gene introduction by transformation. Seedling leaves are a convenient source of totipotent protoplasts. The protoplast-to-plant system developed for Passiflora edulis fv. flavicarpa is summarized in this chapter. The procedure involves enzymatic degradation of leaf tissue using commercially-available Macerozyme R10, Cellulase R10, and Driselase. Isolated protoplasts are cultured in Kao and Michayluk medium, semi-solidified with agarose. The medium containing the suspended protoplasts is dispensed as droplets or thin layers and bathed in liquid medium of the same composition. Shoot regeneration involves transfer of protoplast-derived tissues to Murashige and Skoog-based medium. The protocols developed for P. edulis are applicable to other Passiflora spp. and will underpin the future biotechnological exploitation of a range of species in this important plant family.
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References
Oliviera, J. C. (1987) Melhoramento genético in Cultura do maracujazeiro, vol. 1, (Ruggiero, C., ed.), Editora Legis Summa, Ribeirão Petro, Brazil, pp. 218–246.
Vanderplank, J. (1991) Passion flowers, Cassel Publishers, London.
Payan, F. R. and Martin, F. W. (1975) Barriers to the hybridization of Passiflora species. Euphytica 24, 709–716.
Otoni, W. C., Blackhall, N. W., d’Utra Vaz, F. B., Casali, V. W., Power, J. B., and Davey, M. R. (1995) Somatic hybridization of the Passiflora species, Passiflora edulis fv. flavicarpa Degener. and P. incarnata L. J. Exp. Bot. 46, 777–785.
Dozier, Jr W. A., Rodriguez-Kabana, R., Caylor, A. W., Himelrick, D. G., McDaniel, N. R., and McGuire, J. A. (1991) Ethephon hastens maturity of passion fruit grown as an annual in a temperate zone. Hort. Sci. 26, 146–147.
Dornelas, M. C., Tavares, F. C. A., Oliviera, J. C., and Vieira, M. L. C. (1995) Plant regeneration from protoplast fusion in Passiflora spp. Plant Cell Rep. 15, 106–110.
Davey, M. R., Power, J. B., and Lowe, K. C. (2000) Plant protoplasts, in Encyclopedia of Cell Technology (Spier, R. E., ed.), Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 1090–1096.
Davey, M. R., Anthony, P., Power, J. B., and Lowe, K. C. (2004) Protoplast applications in biotechnology, in Encyclopedia of Plant and Crop Science (Goodman, R. M., ed.), Marcel Dekker Inc., New York, USA, pp. 1061–1064.
Power, J. B., Davey, M. R., Anthony, P., and Lowe, K. C. (2004) Protoplast culture and regeneration, in Encyclopedia of Plant and Crop Science (Goodman, R. M., ed.), Marcel Dekker Inc., New York, USA, pp. 1065–1068.
d’Utra Vaz, F. B., dos Santos, A. V. P., Manders, G., Cocking, E. C., Davey, M. R., and Power, J. B. (1993) Plant regeneration from leaf mesophyll protoplasts of the tropical woody plant, passionfruit (Passiflora edulis fv. flavicarpa Degener.): the importance of the antibiotic cefotaxime in the culture medium. Plant Cell Rep. 12, 220–225.
Dornelas, M. C. and Vieira, M. L. C. (1993) Plant regeneration from protoplast cultures of Passiflora edulis var. flavicarpa Deg., P. amethystina Mikan. and P. cincinnata Mast. Plant Cell Rep. 13, 103–106.
Kao, K. N. and Michayluk, M. R. (1975) Nutritional requirements for growth of Vicia hajastana cells and protoplasts at a very low population density in liquid media. Planta 126, 105–110.
Anthony, P., Lowe, K. C., Davey, M. R., and Power, J. B. (1997) Synergistic effects of haemoglobin and Pluronic® F-68 on mitotic division of cultured plant protoplasts. Adv. Exp. Med. Biol. 428, 477–481.
Lowe, K. C., Anthony, P., Davey, M. R., and Power, J. B. (1999) Culture of cells at perfluorocarbon-aqueous interfaces. Artif. Cells Blood Substit. Immobil. Biotechnol. 27, 255–261.
Frearson, E. M., Power, J. B., and Cocking, E. C. (1973) The isolation, culture and regeneration of Petunia protoplasts. Dev. Biol. 33, 130–137.
Gilmour, D. M., Golds, T. J., and Davey, M. R. (1989) Medicago protoplasts: Fusion, culture and plant regeneration, in Biotechnology in Forestry and Agriculture, vol. 8, Plant Protoplasts and Genetic Engineering I (Bajaj, Y. P. S., ed.), Springer-Verlag, Heidelberg, Germany,. 370–388.
Widholm, J. (1972) The use of FDA and phenosafranine for determining viability of cultured plant cells. Stain Technol. 47, 186–194.
Murashige, T. and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 56, 473–497.
Andras, S. C., Hartman, T. P. V., Marshall, J. A., et al. (1999) A drop-spreading technique to produce cytoplasm-free mitotic preparations from plants with small chromosomes. Chromosome Res. 7, 641–647.
Raccuia, S. A., Mainolfi, A., Mandolino, G., and Melilli, M. G. (2004) Genetic diversity in Cynara cardunculus revealed by AFLP markers: comparison between cultivars and wild-types from Sicily. Plant Breed. 123, 280–284.
Nybomb, H. (2004) Comparison of different nuclear DNA markers for estimating intraspecific genetic diversity in plants. Mol. Ecol. 13, 1143–1155.
Dziechciarkova, M., Lebeda, A., Dolezalova, I., and Astley, D. (2004) Characterization of Lactuca spp. germplasm by protein and molecular markers—a review. Plant Soil Environ. 50, 47–58.
Li, W., Sun, G., Lui, J., et al. (2004) Inheritance of plant regeneration from maize (Zea mays L.) shoot meristem cultures derived from germinated seeds and the identification of associated RAPD and SSR markers. Theor. Appl. Genet. 108, 681–687.
Weiland, J.J. and Yu, M.H. (2003) A cleaved amplified polymorphic sequence (CAPS) marker associated with root-knot nematode resistance in sugarbeet. Crop Sci. 43, 1814–1818.
Bardini, M., Lee, D., Donini, P., et al. (2004) Tubulin-based polymorphism (TBP): a new tool, based on functionally relevant sequences, to assess genetic diversity in plant species. Genome 47, 281–291.
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Davey, M.R., Anthony, P., Power, J.B., Lowe, K.C. (2006). Isolation, Culture, and Plant Regeneration From Leaf Protoplasts of Passiflora . In: Loyola-Vargas, V.M., Vázquez-Flota, F. (eds) Plant Cell Culture Protocols. Methods in Molecular Biology™, vol 318. Humana Press. https://doi.org/10.1385/1-59259-959-1:201
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DOI: https://doi.org/10.1385/1-59259-959-1:201
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