Jaime A. Teixeira da Silva
Faculty of Agiculture, Kagawa University, Japan
ABSTRACT
This study provides detailed reproducible methods for the transformation of Dendranthema X grandiflora Shuhou-no-chikara (standard) and Lineker (spray) stem internode Thin Cell Layers (TCLs) and conventional stem internode explants, using intron-containing and intron-less plasmids. Discussed methodologies and results have used GUS reporter, nptII selector genes, as well as pac1/2,5-A/RNaseL genes coding for virus/viroid resistance. Transformation efficiencies are reported at the kanamycin selection (callus formation and plantlet rooting), GUS, PCR, Southern and Western levels. Notes on regeneration improvement through the use of TCLs, as well as the effective use of sonication for both regeneration and generation of transformants, stimulation of Agroinfection and elimination of Agrobacterium, are included. Protocols are provided for the use of scanning electron microscopy to confirm developmental processes, as well as flow cytometry to check for cell competence and division stages. The difficulties still experienced in the genetic transformation of chrysanthemum globally by researchers require a detailed protocol that exposes the subtleties in methodology. This study provides that information source.
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How to cite this article
Jaime A. Teixeira da Silva, 2005. Effective and Comprehensive Chrysanthemum (Dendranthema X grandiflora)
Regeneration and Transformation Protocols. Biotechnology, 4: 94-107.
DOI: 10.3923/biotech.2005.94.107
URL: https://scialert.net/abstract/?doi=biotech.2005.94.107
DOI: 10.3923/biotech.2005.94.107
URL: https://scialert.net/abstract/?doi=biotech.2005.94.107
REFERENCES
- Jong, J., W. Rademaker and M.F. Wordragen, 1993. Restoring adventitious shoot formation on chrysanthemum leaf explants following cocultivation with Agrobacterium tumefaciens. Plant Cell Tissue Organ Cult., 32: 263-270.
Direct Link - Da Silva, J.A.T. and S. Fukai, 2003. Chrysanthemum organogenesis through thin cell layer technology and plant growth regulator control. Asian J. Plant Sci., 2: 505-514.
CrossRefDirect Link - Murashige, T. and F. Skoog, 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant., 15: 473-497.
CrossRefDirect Link - Vancanneyt, G., R. Schmidt, A. O'Connor-Sachez, L. Willmitzer and S.M. Rocha, 1990. Construction of an intron-containing marker gene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol. Gen. Genet., 220: 245-250.
Direct Link - Mishiba, K. and K. Mii, 2000. Polysomaty analysis in diploid and tetraploid Portulaca grandiflora. Plant Sci., 156: 213-219.
Direct Link - Jefferson, R.A., T.A. Kavanagh and M.W. Bevan, 1987. GUS fusions: Beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J., 6: 3901-3907.
Direct Link - Murray, M.G. and W.F. Thompson, 1980. Rapid isolation of high molecular weight plant DNA. Nucl. Acids Res., 8: 4321-4326.
CrossRefPubMedDirect Link - Silva, Da J.A.T. and D.T. Nhut, 2003. Control of Plant Organogenesis Genetic and Biochemical Signals in Plant Organ Form and Development. In: Thin Cell Layer Culture System Regeneration and Transformation Applications, Nhut, D.T., K.T. Van Thanh, B.V. Le and T. Thorpe (Eds.). Kluwer Academic Publishing, Dordrecht, The Netherlands, pp: 135-190.