Abstract
Transgenic papaya plants were initially obtained using particle bombardment, a method having poor efficiency in producing intact, single-copy insertion of transgenes. Single-copy gene insertion was improved using Agrobacterium tumefaciens. With progress being made in genome sequencing and gene discovery, there is a need for more efficient methods of transformation in order to study the function of these genes. We describe a protocol for Agrobacterium-mediated transformation using carborundum-wounded papaya embryogenic calli. This method should lead to high-throughput transformation, which on average produced at least one plant that was positive in polymerase chain reaction (PCR), histochemical staining, or by Southern blot hybridization from 10 to 20% of the callus clusters that had been co-cultivated with Agrobacterium. Plants regenerated from the callus clusters in 9 to13 mo.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Fitch, M.M.M., Manshardt, R.M., Gonsalves, D., Slightom, J.L., and Stanford, J.C. (1992). Virus resistant papaya plants derived from tissue bombarded with the coat protein gene of papaya ringspot virus. Nat. Biotechnol. 10, 1466–1472.
Bau, H.J., Cheng, Y.H., Yu, T.A., Yang, J.S., and Yeh, S.D. (2003). Broad-spectrum resistance to different geographic strains of papaya ringspot virus in coat protein gene transgenic papaya. Phytopathology 93, 112–120.
Bau, H.J., Cheng, Y.H., Yu, T.A., et al. (2004). Field evaluation of transgenic papaya lines carrying the coat protein gene of Papaya ringspot virus in Taiwan. Plant Disease 88, 594–599.
Chen, G., Ye, C.M., Huang, J.C., and Li, B.J. (2001). Cloning of the papaya ringsport virus (PRSV) replicase gene and generation of PRSV-resistant papayas through the introduction of the PRSV replicase gene. Plant Cell Rep. 20, 272–277.
Davis, M.J. and Ying, Z. (2004). Development of papaya breeding lines with transgenic resistance to Papaya ringspot virus. Plant Disease 88, 352–358.
Ying, Z.T., Yu, X., and Davis, M.J. (1999). New method for obtaining transgenic papaya plants by Agrobacterium-mediated transformation of somatic embryos. Proc. Fl. State Hort. Soc. 112, 201–205.
Fermin, G., Inglessis, V., Garboza, C., Rangel, S., Dagert, M., and Gonsalves, D. (2004). Engineered resistance against Papaya ringspot virus in Venezuelan transgenic papayas. Plant Disease 88, 516–522.
Zhu, Y.J., Agbayani, R., Jackson, M.C., Tang, C.S., and Moore, P.H. (2004). Expression of the grapevine stilbene synthase gene VST1 in papaya provides increased resistance against diseases caused by Phytophthora palmivora. Planta 220, 241–250.
Hood, E.E., Helmer, G.L., Fraley, R.T., and Chilton, M.D. (1986). The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J. Bacteriol. 168, 1291–301.
Murashige, T. and Skoog, K. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15, 473–497.
Fitch, M.M.M. (1993). High frequency somatic embryogenesis and plant regeneration from papaya hypocotyl callus. Plant Cell Tissue Organ. Cult. 32, 205–212.
Fitch, M.M.M., Manshardt, R.M., Gonsalves, D., and Slightom, J.L. (1993). Transgenic papaya plants from Agrobacterium-mediated transformation of somatic embryos. Plant Cell Rep. 12, 245–249.
Cheng, Y.H., Yang, J.S., and Yeh, S.D. (1996). Efficient transformation of papaya by coat protein gene of papaya ringspot virus mediated by Agrobacterium following liquid-phase wounding of embryogenic tissues with carborundum. Plant Cell Rep. 16, 127–132.
Jefferson, R.A. (1989). The GUS reporter gene system. Nature 342, 837–838.
Jefferson, R.A., Kavanagh, T.A., and Bevan, M.W. (1987). GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6, 3901–3907.
Southern, E.M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–517.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Zhu, Y.J., Fitch, M.M.M., Moore, P.H. (2006). Papaya (Carica papaya L.). In: Wang, K. (eds) Agrobacterium Protocols Volume 2. Methods in Molecular Biology, vol 344. Humana Press. https://doi.org/10.1385/1-59745-131-2:209
Download citation
DOI: https://doi.org/10.1385/1-59745-131-2:209
Publisher Name: Humana Press
Print ISBN: 978-1-58829-843-0
Online ISBN: 978-1-59745-131-4
eBook Packages: Springer Protocols