Summary
Transient expression of the β-glucuronidase (GUS) gene has been studied in leaf-derived embryogenic callus of sweetpotatoIpomoea batatas L. (Lam.) by electroporation. The influence of several factors including electric field strength, buffer composition, time course of transientGUS gene expression, DNA concentration, enzyme, and polyethylene glycol (PEG) treatment was examined onGUS gene expression (number of blue spots). MaximumGUS gene expression (an average of 90 blue spots/fifty mg fresh weight callus tissue) was observed after 48 h when callus pieces were preincubated with electroporation (EPR) buffer for 1 h, followed by electroporation with a single electric pulse of 500 V/cm discharged from a 960-μF capacitor in the presence of 20 μg DNA/ml and 8.3 μl NaCl (3M). Changing the electroporation buffer conductivity (by varying the buffer composition with low-high salt concentrations), had only slight effect on the number of blue spots. Similarly, the time course study ofGUS gene expression revealed that GUS activity could be detected 12 h after electroporation with a maximum activity after 72 h (112 blue spots). Increasing the amount of DNA from 5 to 50 μg/ml in the EPR buffer had a slight effect on the expression frequency (from 20–110 blue spots, and 112 blue spots with 20 μg/ml). The number of blue spots was increased by enzymatic wounding of callus pieces for 10 min and by addition of 200 μl PEG 4000 (15%) before electroporation. These results suggest that intact cell electroporation can be used for producing transgenic sweetpotato tissue.
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References
Anonymous. Program and Budget. International Potato Center (CIP), Lima Peru. 16–18; 1995.
Akella, V.; Lurquin, P. F. Expression in cowpea seedlings of chimeric transgenes after electroporation into seed-derived embryos. Plant Cell Rep. 12:110–117; 1993.
Al-Juboory, K. H.; Skirvin, R. M..In vitro regeneration ofAgrobacterium-transformed sweetpotato (Ipomoea batatas L.). Plant Growth Regul. Soc. Amer. Q. 19:82–89; 1991.
Arencibia, A.; Molina, P. R.; Riva, G., et al. Production of transgenic sugarcane (Saccharum officinarum L.) plants by intact cell electroporation. Plant Cell Rep. 14:305–309; 1995.
Chee, R. P.; Schultheis, J. R.; Cantliffe, D. J. Plant recovery from sweetpotato somatic embryos. HortScience 25:795–797; 1990.
Chowrira, G. M.; Akella, V.; Lurquin, P. F.. Electroporated-mediated gene transfer into intact nodal meristemsin planta. Mol. Biotech. 3:17–23; 1995.
Christou, P.. Particle bombardment for genetic engineering of plants (eds). Texas: Landes RG Co. 1–42; 1996.
Dekeyser, R. A.; Claes, B.; DeRycke, R. M. U., et al. Transient gene expression in intact and organized rice tissues. Plant Cell 2:591–602; 1990.
D’Halluin, K.; Bonne, E.; Bossut, M., et al. Transgenic maize plants by tissue electroporation. Plant Cell 4:1495–1505; 1992.
Dhir, S. K.; Dhir, S.; Hepburn, A., et al. Factors affecting transient gene expression in electroporatedGlycine max protoplasts. Plant Cell Rep. 10:106–110; 1991.
Dillen, W.; Engler, G.; Montagu, M. V. Electroporation-mediated DNA delivery to seedling tissues ofPhaseolus vulgaris L. (common bean). Plant Cell Rep. 115:119–124; 1995.
Fromm, M.; Taylor, L. P.; Walbot, V. Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc. Natl. Acad. Sci. USA 82:5824–5828; 1985.
Gama, I. C. S.; Leite, R. P., Jr.; Cordeiro, A. R., et al. Transgenic sweetpotato plants obtained byAgrobacterium tumefaciens-mediated transformation. Plant Cell Tissue Organ Cult. 46:237–244; 1996.
Gustafson, V. D.; Baenziger, S. P.; Mitra, A.. Electroporation of wheat anther culture-derived embryoids. Cereal Res. Commun. 23:207–231; 1995.
Hansch, R.; Koprek, T.; Heydemann, H., et al. Electroporation-mediated transient gene expression in isolates scutella ofHordeum vulgare. Physiol. Plant. 98:20–27; 1996.
Hooykaas, P. J. J.Agrobacterium tumefaciens, a natural vector system. In: Potrykus, I.; Spangenberg, G., ed. Gene transfer to plants. New York: Springer-Verlag, 1–3; 1995.
Jardinaud, M. F.; Sourve, A.; Beckert, M., et al. Optimisation of DNA transfer and transient β-glucuronidase expression in electroporated maize (Zea mays L.) microspores. Plant Cell Rep. 15:55–58; 1995.
Jefferson, R. A.; Kananagh, T. A.; Bevan, M. W.. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J. 6:3901–3907; 1987.
Kloti, A.; Iglesias, V. A.; Wunn, J., et al. Gene transfer by electroporation into intact scutellum cells of wheat embryos. Plant Cell Rep. 12:671–675; 1993.
Larkin, P. J.; Taylor, B. H.; Gersmann, M., et al. Direct gene transfer to protoplasts. Aust. J. Plant Physiol. 17:291–302; 1990.
Laursen, C. M.; Krzyzek, R. A.; Flick, C. E., et al. Production of fertile transgenic maize by electroporation of suspension culture cells. Plant Mol. Biol. 24:51–61; 1994.
Lee, N.; Wang, Y.; Yang, J. Efficient transformation and regeneration of rice small cell groups. Proc. Natl. Acad. Sci. USA 88:6389–6393; 1991.
Lindsey, K.; Jones, M. G. K., Transient gene expresion in electroporated protoplasts and intact cells of sugar beet. Plant Mol. Biol. 10:43–52; 1987.
Luong, H. T.; Shewry, P. R.; Lazzeri, P. A. Transient gene expression in cassava somatic embryos by tissue electroporation. Plant Sci. 107:105–115; 1995.
Lowe, J. M.; Hamilton, W. D. O.; Newell, C. A. Genetic transformation inIpomoea batatas (L.) Lam. (sweetpotato) In: Bajaj, Y. P. S., ed. Biotechnology in agriculture and forestry., Vol. 29. Plant protoplasts and genetic engineering V. New York: Springer-Verlag; 1994:308–320.
Mukhopadhyay, S.; Desjardins, Y. Direct gene transfer to protoplasts of two genotypes ofAsparagus officinalis L. by electroporation. Plant Cell Rep. 13:421–424; 1994.
Nagata, T. Cell biological aspects of gene delivery into plant protoplasts by electroporation. Int. Rev. Cytol. 116:229–255; 1989.
Newell, C. A.; Lowe, J. M.; Merryweather, A., et al. Transformation of sweetpotato [Ipomoea batatas (L.) Lam] withAgrobacterium tumefaciens and regeneration of plants expressing cowpea trypsin inhibitor and snowdrop lectin. Plant Sci. 107:215–227; 1995.
Okada, K.; Takebe, I.; Nagata, T. Expression and integration of genes introduced into highly synchronized protoplasts. Mol. Gen. Genet. 205:398–403; 1986.
Otani, M.; Mii, M.; Handa, T., et al. Transformation of sweetpotato [Ipomoea batatas (L.) Lam.] plants byAgrobacterium rhizogenes. Plant Sci. 94:151–159; 1993.
Penza, R.; Akella, V.; Lurquin, P. F. Transient expression and histological localization of agus chimeric gene after direct transfer to mature cowpea embryos. BioTechniques 13:576–578; 1992.
Pescitelli, S. M.; Sukhapinda, K. Stable transformation via electroporation into maize type II callus and regeneration of fertile transgenic plants. Plant Cell Rep. 14:712–716; 1995.
Potrykus, I. Direct gene transfer to protoplasts. In: Potrykus, I.; Spangenberg, G., ed. Gene transfer to plants. New York: Springer-Verlag 195–201; 1995.
Prakash, C. S.; Varadarajan, U. Genetic transformation of sweetpotato by particle bombardment. Plant Cell. Rep. 11:53–57; 1992.
Prols, M.; Topfer, R.; Schell J., et al. Transient gene expression in tobacco protoplasts. I. Time course of CAT appearance. Plant Cell Rep. 7:221–224; 1988.
Rao, K. V. Transient gene expression in electroporated immature embryos of rice (Oryza sativa L.) J. Plant Physiol. 147:71–74; 1995.
Songstad, D. D.; Halaka, F. G.; DeBoer, D. L., et al. Transient gene expression of GUS and anthocyanin constructs in intact maize immature embryos following electroporation. Plant Cell Tissue Organ Cult. 33:195–201; 1993.
Songstad, D. D.; Somers, D. A.; Griesbach, R. J. Advances in alternative DNA delivery techniques. Plant Cell Tissue Organ Cult. 40:1–15; 1995.
Tada, Y.; Sakamoto, M.; Fujimura, T. Efficient gene introduction into rice by electroporation and analysis of transgenic plants: use of electroporation buffer lacking chloride ions. Theor. Appl. Genet. 80:475–480; 1990.
Xiayi, K.; Xiuwen, Z.; Heping, S., et al. Electroporation of immature maize zygotic embryos and regeneration of transgenic plants. Transgenic Res. 5:219–221; 1996.
Xu, X.; Li, B.. Fertile transgenic indica rice plants obtained by electroporation of the seed embryo, cells. Plant Cell Rep. 13:237–242; 1994.
Zheng, Q.; Dessai, A. P.; Prakash, C. S. Rapid and repetitive plant regeneration in sweetpotato via somatic embryogenesis. Plant Cell Rep. 15:381–385; 1996.
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Mitchell, T.D., Bhagsari, A.S., Ozias-Akins, P. et al. Electroporation-mediated transient gene expression in intact cells of sweetpotato. In Vitro Cell.Dev.Biol.-Plant 34, 319–324 (1998). https://doi.org/10.1007/BF02822741
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DOI: https://doi.org/10.1007/BF02822741