Electroporation-mediated transient gene expression in intact cells of sweetpotato

  • Tonya D. Mitchell
  • Ajmer S. Bhagsari
  • Peggy Ozias-Akins
  • Sarwan K. Dhir
Genetic Transformation/Somatic Cell Genetics


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.

Key words

sweetpotato Ipomoea batatas L. (Lam.) electroporation transient gene expression intact cells biotechnology 


  1. Anonymous. Program and Budget. International Potato Center (CIP), Lima Peru. 16–18; 1995.Google Scholar
  2. 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.CrossRefGoogle Scholar
  3. 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.Google Scholar
  4. 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.CrossRefGoogle Scholar
  5. Chee, R. P.; Schultheis, J. R.; Cantliffe, D. J. Plant recovery from sweetpotato somatic embryos. HortScience 25:795–797; 1990.Google Scholar
  6. Chowrira, G. M.; Akella, V.; Lurquin, P. F.. Electroporated-mediated gene transfer into intact nodal meristemsin planta. Mol. Biotech. 3:17–23; 1995.CrossRefGoogle Scholar
  7. Christou, P.. Particle bombardment for genetic engineering of plants (eds). Texas: Landes RG Co. 1–42; 1996.Google Scholar
  8. 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.PubMedCrossRefGoogle Scholar
  9. D’Halluin, K.; Bonne, E.; Bossut, M., et al. Transgenic maize plants by tissue electroporation. Plant Cell 4:1495–1505; 1992.PubMedCrossRefGoogle Scholar
  10. 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.Google Scholar
  11. 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.CrossRefGoogle Scholar
  12. 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.PubMedCrossRefGoogle Scholar
  13. 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.CrossRefGoogle Scholar
  14. Gustafson, V. D.; Baenziger, S. P.; Mitra, A.. Electroporation of wheat anther culture-derived embryoids. Cereal Res. Commun. 23:207–231; 1995.Google Scholar
  15. Hansch, R.; Koprek, T.; Heydemann, H., et al. Electroporation-mediated transient gene expression in isolates scutella ofHordeum vulgare. Physiol. Plant. 98:20–27; 1996.CrossRefGoogle Scholar
  16. 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.Google Scholar
  17. 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.CrossRefGoogle Scholar
  18. 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.PubMedGoogle Scholar
  19. 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.CrossRefGoogle Scholar
  20. Larkin, P. J.; Taylor, B. H.; Gersmann, M., et al. Direct gene transfer to protoplasts. Aust. J. Plant Physiol. 17:291–302; 1990.CrossRefGoogle Scholar
  21. 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.PubMedCrossRefGoogle Scholar
  22. Lee, N.; Wang, Y.; Yang, J. Efficient transformation and regeneration of rice small cell groups. Proc. Natl. Acad. Sci. USA 88:6389–6393; 1991.PubMedCrossRefGoogle Scholar
  23. 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.CrossRefGoogle Scholar
  24. 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.CrossRefGoogle Scholar
  25. 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.Google Scholar
  26. Mukhopadhyay, S.; Desjardins, Y. Direct gene transfer to protoplasts of two genotypes ofAsparagus officinalis L. by electroporation. Plant Cell Rep. 13:421–424; 1994.CrossRefGoogle Scholar
  27. Nagata, T. Cell biological aspects of gene delivery into plant protoplasts by electroporation. Int. Rev. Cytol. 116:229–255; 1989.CrossRefGoogle Scholar
  28. 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.CrossRefGoogle Scholar
  29. Okada, K.; Takebe, I.; Nagata, T. Expression and integration of genes introduced into highly synchronized protoplasts. Mol. Gen. Genet. 205:398–403; 1986.CrossRefGoogle Scholar
  30. Otani, M.; Mii, M.; Handa, T., et al. Transformation of sweetpotato [Ipomoea batatas (L.) Lam.] plants byAgrobacterium rhizogenes. Plant Sci. 94:151–159; 1993.CrossRefGoogle Scholar
  31. 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.PubMedGoogle Scholar
  32. 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.CrossRefGoogle Scholar
  33. Potrykus, I. Direct gene transfer to protoplasts. In: Potrykus, I.; Spangenberg, G., ed. Gene transfer to plants. New York: Springer-Verlag 195–201; 1995.Google Scholar
  34. Prakash, C. S.; Varadarajan, U. Genetic transformation of sweetpotato by particle bombardment. Plant Cell. Rep. 11:53–57; 1992.CrossRefGoogle Scholar
  35. 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.CrossRefGoogle Scholar
  36. Rao, K. V. Transient gene expression in electroporated immature embryos of rice (Oryza sativa L.) J. Plant Physiol. 147:71–74; 1995.Google Scholar
  37. 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.CrossRefGoogle Scholar
  38. Songstad, D. D.; Somers, D. A.; Griesbach, R. J. Advances in alternative DNA delivery techniques. Plant Cell Tissue Organ Cult. 40:1–15; 1995.CrossRefGoogle Scholar
  39. 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.CrossRefGoogle Scholar
  40. 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.CrossRefGoogle Scholar
  41. Xu, X.; Li, B.. Fertile transgenic indica rice plants obtained by electroporation of the seed embryo, cells. Plant Cell Rep. 13:237–242; 1994.CrossRefGoogle Scholar
  42. 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.CrossRefGoogle Scholar

Copyright information

© Society for In Vitro Biology 1998

Authors and Affiliations

  • Tonya D. Mitchell
    • 1
  • Ajmer S. Bhagsari
    • 1
  • Peggy Ozias-Akins
    • 2
  • Sarwan K. Dhir
    • 1
  1. 1.Plant Science, Agricultural Research StationFort Valley State UniversityFort Valley
  2. 2.Department of Horticulture, Coastal Plain Experiment StationUniversity of GeorgiaTifton

Personalised recommendations