Implementación de una Técnica para la Produción de Plantas Transgénicas de Clavel Dianthus caryophyllus L. Variedad Bagatel
Resumen
El objetivo de este trabajo era establecer un protocolo para la obtención de plantas transgénicas de clavel Dianthus caryophyllus L. variedad Bagatel mediado por Agrobacterium tumefaciens, que pueda eventualmente ser utilizado por el programa de mejoramiento de la UMNG. Se generaron las condiciones de regeneración partiendo de entrenudos de plántulas propagadas in vitro y se determinó la concentración del agente seleccionante. Los ensayos de transformación se hicieron con el plásmido pBI121, el cual es portador de los genes NPTII que confiere resistencia al antibiótico kanamicina y GUS que codifica para la enzima -glucuronidasa, dentro de su T-DNA. Se evaluaron dos protocolos de transformación, con los cuales se logró la obtención de plántulas transformadas completas. La presencia de los transgenes fue detectada en plántulas transformadas por PCR con cebadores específicos para los genes NPTII y GUS, y la actividad de estos fue comprobada mediante la evaluación de la resistencia a kanamicina y por ensayos histoquímicos con un sustrato para la enzima -glucuronidasa.Descargas
Agencias de apoyo:
Dianthus caryophyllus, Agrobacterium tumefaciens, clavel, plantas trangénicas, transformaciónReferencias bibliográficas
- An G, Ebert PR, Mitra A. Ha SB. 1988. Binary vectors. In Plant Molecular Biology Manual. Kluwer Academy Publishers. A3/1.1 y A3/7.
- Beijersbergen A., Dulk-Ras A., Schilperoor, R, Hooykaas, P. 1992. Conjugative Transfer by the Virulence System of Agrobacterium tumefaciens. Science 256: 1324-1327.
- Bevan M. 1984. Binay Agrobacterium vectors for plant transformation. Nucleic Acids Research 12: 8711-8721.
- Brencic A, Winans S. 2005. Detection of and response to signals involved in host-microbe interactions by plant-associated bacteria. Microbiology and Molecular Biology Reviews 69: 155-194.
- De Block M, Botterman J, Vandewiele M, Dockx J, Theoden C, Dossele V, Rao Movva N, Thompson C. Van Montagu M, Leemmans J. 1987. Engineering herbicide resistance in plants by expression of a detoxifying enzyme. EMBO Journal 6: 2513-2518.
- Doyle J, Doyle J. 1990. Isolation of plant DNA from fresh tissue. American Journal of Botany 75: 1238-1240.
- Escudero J, Neuhaus G, Hohn B. 1995. Intracellular Agrobacterium can transfer DNA to the cell nucleus of the host plant. Proceedings of the National Academy of Sciences USA 92: 230-234.
- Estopà M, Marfà V, Melé E., Messeguer J. 2001. Study of different antibiotic combinations for use in the elimination of Agrobacterium with kanamycin selection in carnation. Plant Cell, Tissue and Organ Culture 65: 211–220.
- Fillati JJ, Kiser J, Rose R, Comai L. 1987. Efficient transfer of a glyphosate tolerance gene into tomato using a binary Agrobacterium tumefaciens vector. Bio/Technology 5: 726-730.
- Frey L, Janick J. 1991. Organogenesis in carnation. Journal of the American Society for Horticultural Science 116: 1108-1112.
- Gelvin S. 2003. Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiology and Molecular Biology Reviews 67: 16-37.
- Hoeckema A, Hirsch PR, Hooykas PJ, Schilperoot, R.A. 1983. A binay plant vector strategy based on separation of vir and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: 179-180.
- Hoekema A, Roelvink P, Hooykaas P, Schilperoort R. 1984. Delivery of T-DNA from the Agrobacterium tumefaciens chromosome into plant cells. The EMBO Journal 3: 2485 -2490.
- Iwazaki Y, Kosugi Y, Waki K, Yoshioka T, Satoh S. 2004. Generation and ethylene production of transgenic carnations harboring ACC synthase cDNA in sense or antisense orientation. Journal of Applied Horticulture 6:67-71.
- James C. 2007. Resumen ejecutivo No .37 ISAAA International service for the acquisition of the agribiotech applications: Situación mundial de la comercialización de cultivos biotecnológicos/transgénicos en 2007. http://www.agbios.com/docroot/articles/2000192-A.pdf.
- Jefferson R. 1989. The GUS reporter gene system. Nature 342: 837-838
- Jefferson R, Kavanagh T, Bevan M. 1987. GUS fusions: b-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. The EMBO Journal 6: 3901 -3907.
- Jen G, Chilton M. 1986. Activity of T-DNA borders in plant cell transformation by mini-T plasmids. Journal of Bacteriology 166: 491-499.
- Jeyaramraja P, Meenakshi S. 2005. Agrobacterium tumefaciens-mediated transformation of embryogenic tissues of tea (Camellia sinensis (L.) O. Kuntze). Plant Molecular Biology Reporter 23: 299a–299i.
- Kinouchi T, Endo R, Yamashita A, Satoh S. 2006. Transformation of carnation with genes related to ethylene production and perception: towards generation of potted carnations with a longer display time. Plant Cell Tissue and Organ Culture 86:27–35.
- Kikuchi R, Sage-Ono K, Kamada H, Ono M. 2005. Efficient transformation mediated by Agrobacterium tumefaciens with a ternary plasmid in Pharbitis nil. Plant Biotechnology 22: 295–302.
- Krasnyanski S, Sandhu J, Domier L, Buetow D, Korban S. 2001. Effect of an enhanced CAMV 35S promoter and a fruit-specific promoter on UIDA gene expression in transgenic tomato plants. Society for In Vitro Biology 37: 427-433.
- Murashige T, Skoog F. 1962. A revised médium for rapid growth and bioassays with tobacco tissue cultures. Physiología plantarum 15: 473-497.
- Nontaswatsri C, Fukai S, Goi M. 2004. Revised cocultivation conditions produce effective Agrobacterium mediated genetic transformation of carnation (Dianthus caryophyllus L.). Plant Science 166: 59-68.
- Sankhla D, Davis T, Sankhla N, Upadhyaya A. 1995. In vitro regeneration of the heat-tolerant ‘german red’ carnation through organogenesis and somatic embryogenesis. Gartenbauwissenschaft 60: 228-233.
- Shen W, Escudero J, Schlappi M, Ramos C, Hohn B, Koukolikova-Nicola Z. 1993. T-DNA transfer to maize cells: histochemical investigation of glucuronidase activity in maize tissues. Proceedings of the National Academy USA 90:1488-1492.
- Sheng J, Citovsky V. 1996. Agrobacterium-plant cell DNA transport: have virulence proteins, will travel. The plant cell 8:1699-1710.
- Teixeira Da Silva J, Fukal S. 2002. Increasing transient and subsequent stable transgene expression in chrysanthemum following optimization of particle bombardment and agroinfection parameters. Plant Biotechnology, 19: 229–240.
- Taskin K, Ercan A, Turgut K. 1999. Agrobacterium tumefaciens mediated transformation of sesame (Sesamum indicum L.). Journal of Botany 23: 291-295.
- Tzfira T, Rhee Y, Chen M, Kunik T, Citovsky V. 2000. Nucleic acid transport in plant-microbe interactions: the molecules that walk through the walls. Annual Reviews Microbiology 54: 199-209.
- Tzfira T, Citovsky V. 2006. Agrobacterium-mediated genetic transformation of plants: biology and biotechnology. Current Opinion in Biotechnology 17:147–154.
- Van Altvorst A, Bruinsma T, Koehorst H, Dons J. 1992. Regeneration of carnation (Dianthus caryophyllus) using leaf explants. Acta Horticulturae 307: 109-116.
- Venkatachalam P, Geetha N, Khandelwal A, Shaila M, Lakshmi G. 2000. Agrobacterium-mediated genetic transformation and regeneration of transgenic plants from cotyledon explants of groundnut (Arachis hypogaea L.) via somatic embryogenesis. Current Science 78: 1130-1136.
- Vergunst A, Jansen L, Hooykaas P. 1998. Site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana mediated by Cre recombinase. Nucleic Acids Research 26: 2729–2734.
- Zhang S, Zhu L, Li X, Ahlman A, Welander M. 2005. Infection by Agrobacterium tumefaciens increased the resistance of leaf explants to selective agents in carnation (Dianthus caryophyllus L. and D. chinensis). Plant Science 168: 137–144.
- Zuker A, Chang P, Ahroni A, Cheah K, Woodson W, Bressan R, Wated A, Hasegawa P, Vainstein A. 1995. Transformation of carnation by microproyectile bombardment. Scientia horticulturae 64: 177-185.
