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Transformation and confirmation of GUS gene expression in Solanum melongena L. of PLR 1 cultivar

Vinod Kanna S., Jayabalan N.

Abstract


In the present study GUS gene transformation was carried out in eggplant using Agrobacterium strain with pBAL2 vector harboring gus gene and nptII as selection marker gene. The factors which are affecting (enhancing) the frequency of transient gus gene expression are different physical and biochemical variables has been carried out. It is observed that the 4 day precultured explants showed the minimum survival rate in the medium when compared with 2-day co cultivated medium. The explants which had undergone co-cultivation for 4 to 5 days showed GUS activity, the tissues were adversely affected due to the overgrowth of bacteria. The gene specific primers for nptII and gus gene were used for amplification and it has given 680bp and 1.9 kb amplified fragments respectively and recorded. The band was detected in the selected plants, but it was absent from the negative control (non-transformed) plant in the Southern hybridization. Our experiment showed 0.80-1.60 percentage of efficiency in transformation. With a total of 849 infected shoots were undergone confirmation tests which results 9 PCR positives (1.06% efficiency). The Transformant kept in the Environmental Growth Chamber and transferred to field condition subsequently.

Keywords


Agrobacterium tumifaciens, MS Salts, infection, pBAL2 and transformant

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References


Billings S, Jelenkovic G, Chin CK and Eberhadt J. The effect of growth regulationandantibiotics on eggplant transformation. Journal of American Society of Horticultural Science, 122 (1997): 158–162.

Binns AN and Thomashow MF. Cell biology of Agrobacterium infection andtransformation of plants. Annual Review of Microbiology, 42(1988): 575-606.

Cervera M, Juárez J, Navarro A, Pina JA, Durán-Vila N, Navarro Land Peña L. Genetic transformation and regeneration of mature tissues of woody fruitplantsbypassing the juvenile stage. Transgenic Research, 7(1998a):51–59.

Cervera M, Pina JA, Juárez J, Navarro A, Navarro L, and Peña L. Agrobacterium- mediated transformation of citrange: factors affectingtransformation and regeneration. Plant Cell Reports, 18(1998b): 271–278.

Cesarone CF, Bolongnesi C and Santi L. Improved microfluorometric DNA determination in biological material using 33258 Hoechst. Annals of Biochemistry, 100(1979):188 – 197.

Chakravarty and Pruski. Rapid regeneration of stable transformants in cultures of potato by improving factors influencing Agrobacterium-mediated transformation. Advances in Bioscience and Biotechnology, 1(2010):409-416.

Deblaere R, Bytebier B, De Greve H, Deboeck F, Schell J, Van Montagu M andLeemans J. Efficient octopineTi plasmid-derived vectors for Agrobacterium- mediated gene transfer to plants. Nucleic Acid Research, 13 (1985): 4777-4788.

Doyle JJ and Doyle JL. Isolation of plant DNA from fresh tissue. Focus (1990), 12: 13-15.

Edwards, K., Johnstone, C., and Thompson, C. (1991). A simple and rapid method for thepreparation ofgenomic plant DNA for PCR analysis. Nucleic Acids Research, 19(1990): 1349.

Franklin F, Sheeba CJ and Lakshmisita G. Regeneration of Eggplant (Solanummelongena L) from root explant. In Vitro Cellular and Developmental Biology – Plant, 40.2 (2004):188-191.

Guri A and Sink KC. Agrobacterium Transformation of Eggplant 1. Journal of Plant Physiology, 133.1 (1988): 52-55.

Guri A and Sink KC. Interspecific somatic hybrid plants between eggplant (Solanum melongena) and Solanum torvum. Theoretical and Applied Genetics, 76.4 (1988): 490-496.

Hamilton CM. A binary-BAC system for plant transformation with high-molecularweight DNA. Gene, 200(1997):107–116.

Hooykaas PJJ and Shilperoort RA. Agrobacterium and plant genetic engineering. Plant Molecular Biology, 19(1992):15-38.

Jefferson RA. Assaying chimeric genes in plants: The GUS gene fusion system. Plant Molecular Biology Report, 5(1987):387–405.

Li D, Zhao K, Xie B, Zhang B and Luok. Establishment of highly efficient transformation system for pepper (Capsicum annuum L.). Plant Cell Reports, 21(2003): 785-788.

Magioli C, Rocha APM, Oliveira DE and Mansur E. Efficient shootorganogenesisofeggplant (Solanum melongena L.) induced by thidiazuron. Plant Cell Reports, 17(1998): 661-663.

Magioli C, Rocha APM, Tarré E, Santiago-Fernandes LD, Oliveira DE, Krul WR andMansurE. Effect of morphological factors, antibiotics and Agrobacteriumco-cultivation in theefficiency of somatic embryogenesis of eggplant (SolanummelongenaL.). Journal of Plant Biotechnology, 3.1 (2001): 19-25.

Maniatis T, Fritsch EF and Sambrook J. Molecular Cloning. A Laboratory Manual, published by Cold Spring Harbor Laboratory Press, NY (1982).

McHughen A, Jordan M and Fiest G. A preculture period prior to Agrobacteriuminoculatedincrease production to transgenic plants. Journal of Plant Physiology, 135(1989):245-248.

Mourgues F, Chevreau E, Lambert C and de Bondt A. Efficient Agrobacterium mediated transformation and recovery of transgenic plants from pear (Pyruscommunis L.). Plant Cell Reports, 16 (1996):245–249.

Nauerby B, Billing K and Wyndaele R. Influence of the antibiotic timentin on plantregeneration compared to car benicillin and cefotaxime in concentrations suitable forelimination of Agrobacterium tumefaciens. Plant Science, 123(1997): 169-177.

Nester EW, Gordon MP, Amasino RM, and Yanofsky MF. Crown gall: A molecular and physiological analysis. Annual Review of Plant Physiology, 35(1984): 387–413.

Pelayo Pérez-Piñeiro1, Jorge Gago1, Mariana Landín and Pedro P. Gallego. Agrobacterium-Mediated Transformation of Wheat: General Overview and New Approaches to Model and Identify the Key Factors Involved. Transgenic Plants - Advances and Limitations. INTECH, UK. 2012.

Prabhavathi V, Yadav JS, Kumar PA and Rajam MV. Abiotic stress tolerance intransgenic eggplant (Solanum melongena L.) by introduction of bacterial mannitolphosphodehydrogenase gene. Molecular Breeding, 9.2 (2002): 137-147.

Sheikholeslam SN and Weeks DP. Acetosyringone promotes high efficiencytransformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Molecular Biology, 8.4 (1987):291-298.

Smith EF and Townsend CO. A plant tumour of bacterial origin. Science, 25(1907): 671-673.

Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology, 98(1975): 503-517.

Sujatha M and Sailaja M. Stable genetic transformation of castor (Ricinus communisL.) via Agrobacterium tumefaciens-mediated gene transfer using embryo axesfrommatureseeds. Plant Cell Reports, 23(2005):803-810.

Torisky RS, Kovacs L, Avdiushko S, Newman JD, Hunt AG and Collins GB. Development of a binary vector system for plant transformation based on supervirulentAgrobacterium tumefaciens strain Chry5. Plant Cell Reports, 17(1997):102-108.

Vinod Kanna S and Jayabalan N. Influence of N6-(2-isopentenyl) adenine on in vitro shoot proliferation in Solanum melongena L. International Journal of Academic Research. 2(2010): 98-100.

Vinod kanna S and N. Jayabalan. Regeneration via Direct Organogenesis from Leaf Segments of Eggplant (Solanum melongena L.). Journal of Plant Sciences, 10.3 (2015): 90-98.

Wallroth M, Gerats AGM, Rogers SG, Fraley RT and Horsch RB. Chromosolmal Localization in Petunia hybrid. Molecular Genetics and Genomics, 202(1986): 6- 15.

Zupan JR, and Zambryski PC. Transfer of TDNA from Agrobacterium to the plantcell. Plant Physiology, 107(1995):1041.1047.




DOI: https://doi.org/10.21746/aps.2018.7.4.19



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