Keywords
Protoplasts
Beta vulgaris
Electrofusion
Sugar Beet
How to Cite
Abstract
Somatic hybridization between different plants through protoplast fusion represent an efficient experimental approach to produce genetically transformed plant species. Electrofution of mesophyll protoplasts in sugar beet was occurred to overcome the barriers faced breeding program of this economically industrial crop Protoplasts were successfully isolated from leave's mesophyll of two varieties of sugar beet (Beta vulgaris L.). Various enzyme solutions were assessed for the cell wall degrading ability. They express different efficiency in isolation of mesophyll protoplasts of var. Baraka. The protoplasts yield was 18 × 104 cell ml-1 using the mixture consisting of 0.5% Cellulase RS, 1.0% Hemicellulase and 0.1% Pectolyase Y-23 with 13% mannitol. A total of 16 hrs. for cell wall digestion, and protoplast viability approached 93%. Protoplasts were isolated from leaf mesophyll of var. Carola using the same enzymatic mixtures. High protoplasts yield 20 × 104 cell ml-1 was obtained, requiring the same period 16 hrs. to approach viability 96%. The protoplasts were spherical in shape, varied in chloroplast distribution, having size ranged 12 – 52 µm. The present study succeeded in electrofusion between Baraka × Carola mesophyll protoplasts, producing somatic hybrid cells under conditions of 1MHz, 1000 Vcm-1, 2 pulses, 1.5 msec./pulse with fusion percent of 73%.
References
Al-Nema, Q.S., Al-Mallah, M.K. (2013). Protoplast isolation from leaf mesophyll of sugar beet Beta vulgaris L. axenic seedlings. J. Biotech. Res. Cent., 7, 36-42.
Arnon, D.I., Hoagland, D.R. (1944). The investigation of plant nutrition by artificial culture methods. Boil. Rev., 19, 55-67.
Badr-Elden, A., Ahmed, N., Nasr, M., Ibrahim, A. (2010). Isolation and fusion of protoplasts in sugar beet (Beta vulgaris L.). Sugar Tech., 12, 53-58.
Bhojwani, S.S., Razdan, M.K. (1996). Plant Tissue Culture: Theory and Practice. A revised edition. Elsevier. India.
Davey, M.R. and Anthony, P. (2010). Plant Cell Culture Essential Methods. Wiley-Blackwell, UK.
Davey, M.R., Anthony, P., Patel, D., Power, J.B. (2010). Plant protoplasts: isolation, culture and plant regeneration. In: Davey, M.R. and P. Anthony, Plant Cell Culture Essential Methods. Wiley-Blackwell, UK. pp. 153-155.
Davey, M.R., Anthony, P., Power, J.B., Lowe, K.C. (2005). Plant protoplasts: status and biotechnological perspectives. Biotech. Adv., 23, 131-171.
Frearson, E.M., Power, J.B., Cocking, E.C. (1973). The isolation, culture and regeneration of Petunia leaf protoplasts. Dev. Biol., 33, 130-137.
Galbraith, D.W. (1981). The use of Calcofluor White to visualize cell walls. Bio/Technol., 3: 1104-1106. (C. F. Davey et al., 2010).
Grzebelus, E., Szklarczyk, M., Gren, J., Sniegowska, K., Jopek, M., Kacinska, I., Mrozek, K. (2012). Phytosulfokine stimulates cell division in sugar beet (Beta vulgaris L.) mesophyll protoplast cultures. Plant Growth Regul., 67, 93-100.
Guo, W.W., Deng, X.X., Yi, H.L. (2000). Somatic hybrids between novel orange (Citrus sinensis) and grapefruit (C. paradisi) for seedless triploid breeding. Euphytica, 116, 281-285.
Jones, B., lynch, P.T., Malaure, R.S., Handley, G.J., Blackhall, N.W., Hammatt, N., et al., (1994). Low cost equipment for the efficient large-scale electromanipulation of plant protoplasts. BioTechniques, 16, 312-321.
Kao, K.N. (1975). A Nuclear Staining Method for Plant Protoplasts. In: Gamborg, O.L. and Wetter, L.R. (eds). Plant Tissue Culture Methods. 60-61. National Research Council of Canada.
Majewska-Sawka, A., Munster, A. (2003). Cell wall antigens in mesophyll cells and mesophyll-derived protoplasts of sugar beet: possible implication in protoplast recalcitrance? Plant Cell Repts., 21, 946-954.
Mi Jeon, j., Ahn, N.Y., Son, B.H., Kim, C.Y., Han, C., Kim, G., et al., (2007). Efficient transient expression and transformation of PEG-mediated gene uptake into mesophyll protoplasts of pepper (Capsicum annuum L.). Plant Cell, Tiss. Org. Cult., 88, 225-232.
Murashige, T., Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol. Plant., 15, 473-497.
Niedz, R.P., Mckendree, W.L., Shatters, R.G. (2003). Electroporation of embryogenic protoplasts of sweet orange (Citrus sinensis L.) and regeneration of transformed plants. In vitro Cell. Dev. Biol. Plant., 39, 586-594.
Power, J.B., Cummins, S.E., Cocking, E.C. (1970). Fusion of isolated plant protoplasts. Nature, 225, 1016-1018.
Power, J.B., Davey, M.R., McLellan, M., Wilson, D. (1989). Laboratory Manual: Plant Tissue Culture. Plant Genetic Manipulation Group, University of Nottingham, UK.
Ritchie, G.A., Short, K.C., Davey, M.R. (1989). In vitro shoot regeneration from callus leaf axils and petioles of sugar beet (Beta vulgaris L.). J. Exp. Bot., 40, 277-283.
Rathore, K.S., Chowdhury, V.K., Hodges, T.K. (1993). Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts. Plant Mol. Biol., 21, 871-884.
Senda, M. Takeda, J. Abe, S., Nakamura, T. (1979). Induction of cell fusion of plant protoplasts by electrical stimulation. Plant Cell Physiol., 20, 1441-1443.
Szczerbakowa, A., Maciejewska, U., Zimnoch-Guzowska, E., Pawlowski, P., Skierski, S., Wielgat, B. (2001). Electrofusion of protoplasts from Solanum tuberosum, S. nigrum and S. bulbocastanum. Acta Physiol. Plant., 23, 169-179.
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