AS  Vol.7 No.10 , October 2016
A Protocol for Endophyte-­Free Callus Tissue of the Grape Vitis aestivalis “Norton/Cynthiana” (Vitaceae)
Vitis aestivalis is used in commercial wine production. Vegetative propagation of V. aestivalis has shown a low success rate. Although plant tissue culture has been a successful method to propagate many species, V. aestivalis has not yet been reliably grown as pure callus culture due to a fungal endophyte that exists within the plant. This study reports a viable protocol for obtaining fungus-free tissue culture callus from V. aestivalis. Explant tissue was chosen from healthy, actively growing plants grown in a growth room and in a vineyard. Tissues were sterilized with a combination of isopropanol, bleach, and chlorine dioxide gas and plated onto media containing chlorothalonil. The results from this study suggest that in order to obtain endophyte-free callus tissue, vine explants are to be taken from plants grown in a growth chamber simulating springtime conditions, sterilized in a combination of alcohol, bleach, and chlorine dioxide, and plated on selection media containing an antifungal agent, such as chlorothalonil. This technique could potentially be used with plants that have associated endophytes or other contamination problems to establish callus tissue for research and/or commercial propagation efforts.
Cite this paper: Wilson, A. , Fuller, M. , Smith, S. , Johnston, T. and Du Bois, J. (2016) A Protocol for Endophyte-­Free Callus Tissue of the Grape Vitis aestivalis “Norton/Cynthiana” (Vitaceae). Agricultural Sciences, 7, 680-692. doi: 10.4236/as.2016.710064.

[1]   Ambers, R.K.R. and Ambers, C.P. (2004) Dr. Daniel Norborne Norton and the Origin of the Norton Grape. American Wine Society Journal, 36, 77-87.

[2]   Ambers, C.P. (2013) A Historical Hypothesis on the Origin of the Norton Grape. Journal of Wine Research, 24, 85-95.

[3]   Stover, E., Aradhya, M., Yang, J., Bautista, J. and Dangl, G.S. (2009) Investigations into the Origin of “Norton” Grape Using SSR Markers. Proceedings of the Florida State Horticultural Society, 122, 19-24.

[4]   Norton, M.A. and Skirvin, R.M. (2001) Micropropagation of “Norton” Winegrape. Hort Technology, 11, 206-208.

[5]   Parker, L.D., Bordallo, P.N. and Colova, V.M. (2009) Phylogenetics Analysis of North American Native “Cynthiana”/“Norton” Grape Cultivar Using DNA Microsatellite Markers. Acta Horticulturae, 2009, 225-228.

[6]   Krivanek, A.F. and Walker, M.A. (2005) Vitis Resistance to Pierce’s Disease Is Characterized by Differential Xylella fastidiosa Populations in Stems and Leaves. Phytopathology, 95, 44-52.

[7]   Polesani, M., Bortesi, L., Ferrarini, A., Zamboni, A., Fasoli, M., Zadra, C., Lovato, A., Pezzotti, M., Delledonne, M. and Polverari, A. (2010) General and Species-Specific Transcriptional Responses to Downy Mildew Infection in a Susceptible (Vitis vinifera) and a Resistant (V. riparia) Grapevine Species. BMC Genomics, 11, 117-132.

[8]   Espinoza, C., Vega, A., Medina, C., Schlauch, K., Cramer, G. and Arce-Johnson, P. (2007) Gene Expression Associated with Compatible Viral Diseases in Grapevine Cultivars. Functional & Integrative Genomics, 7, 95-110.

[9]   Fung, R.W.M., Qiu, W., Su, Y., Schachtman, D.P., Huppert, K., Fekete, C. and Kovacs, L.G. (2007) Gene Expression Variation in Grapevine Species Vitis vinifera L. and Vitis aestivalis Michx. Genetic Resources and Crop Evolution, 54, 1541-1553.

[10]   Hartmann, H.T., Keister, D.E. and Davies, F.T. (2002) Plant Propagation: Principles and Practices. Prentice Hall, Eaglewood Cliffs, New Jersey.

[11]   Qiu W., Fekete, S., Todd, T. and Kovacs, L. (2003) Facilitation of Microshoot Tip Propagation of Vitis aestivalis var. Norton by Combined Application of an Antioxidant and Cytokinins. American Journal of Enology and Viticulture, 55, 112-114.

[12]   Biggers, B. (2010) Micropropagation and Acclimatization of “Norton” Grapevine (Vitis aestivalis. University of Nebraska. Lincoln, Nebraska.

[13]   Murashige, T. (1974) Plant Propagation through Tissue Cultures. Annual Review of Plant Physiology, 25, 135-166.

[14]   García-Gonzáles, R., Quiroz, K., Carrasco, B. and Caligari, P. (2010) Plant Tissue Culture: Current Status, Opportunities and Challenges. Ciencia E Investigación Agraria, 37, 5-30.

[15]   Chugh, S., Guha, S. and Rao, I.U. (2009) Micropropagation of Orchids: A Review on the Potential of Different Explants. Scientia Horticulturae, 122, 507-520.

[16]   Akin-Idowu, P.E., Ibitoye, D.O. and Ademoyegun, O.T. (2009) Tissue Culture as a Plant Production Technique for Horticultural Crops. African Journal of Biotechnology, 8, 3782-3788.

[17]   Constabel, F. (2012) Callus Culture: Induction and Maintenance. In: Vasil, I., Ed., Laboratory Procedures and Their Applications, Academic Press, New York, 27-35.

[18]   Harris, R.E. and Stevenson, J.H. (1982) In Vitro Propagation of Vitis. Vitis, 21, 22-32.

[19]   Lloyd, G. and McGown, B. (1980) Commercially-Feasible Micropropagation of Mountain Laurel, Kalmia latifolia, by Use of Shoot-Tip Culture. Proceedings of the International Plant Propagators’ Society, 30, 421-427.

[20]   Preece, J.E. (2003) A Century of Progress with Vegetative Plant Propagation. Hortscience, 38, 1015-1025.

[21]   Leguillon, S., Charles, G. and Branchard, M. (2003) Plant Regeneration from Thin Cell Layers in Spinacia oleracea. Plant Cell, Tissue and Organ Culture, 74, 257-265.

[22]   Odnevall, A., Bjork, L. and Berglund, T. (1989) Rapid Establishment of Tissue Cultures from Seeds of Panax ginseng and Panax pseudoginseng. Biochemie und Physiologie der Pflanzen, 185, 131-134.

[23]   Bhawana, B., Miller, J.L. and Cahoon, A.B. (2014) Plant Cell Architecture of Arabidopsis thaliana (Brassicaceae) Using Focused Ion Beam-Scanning Electron Microscopy. Applications in Plant Sciences, 2, Article ID: 1300090.

[24]   Anderson, M.A. and Miller, B.T. (2011) Early Iron Deposition in Teeth of the Streamside Salamander, Ambystoma barbouri. Journal of Herpetology, 45, 336-338.