Summary
Transgenic barley plants (Hordeum vulgare L. cv. Kymppi) were obtained by particle bombardment of various tissues. Immature embryos and microspore-derived cultures were bombarded with gold particles coated with plasmid DNA carrying the gene coding for neomycin phosphotransferase II (NPTII), together with plasmid DNA containing the gene for β-glucuronidase (GUS).
Bombarded immature embryos were grown to plants without selection and NPTII activity was screened in small plantlets. One plant proved to be transgenic (T0). This chimeric plant passed the transferred nptII gene to its T1 progeny. The presence of the nptII gene was demonstrated by the PCR technique and enzyme activity was analyzed by an NPTII gel assay. Four T0 spikes and 15 T1 offspring were transgenic. The integration and inheritance was confirmed by Southern blot hybridization. Transgenic T2 and T3 plants were produced by isolating embryos from green grains of transgenic T1 and T2 plants, respectively and growing them to plants. After selfing, the ratio of transgenic to non-transgenic T2 offspring was shown to follow the rule of Mendelian inheritance. The general performance of transgenic plants was normal and no reduction in fertility was observed.
Microspore-derived cultures were bombarded one and four weeks after microspore isolation. After bombardment, cultures were grown either with or without antibiotic selection (geneticinR or kanamycin). When cultures were grown without selection and regenerated plants were transferred to kanamycin selection in rooting phase, one out of a total of about 1500 plants survived. This plant both carried and expressed the transferred nptII gene. The integration was confirmed by Southern blot hybridization. This plant was not fertile.
Similar content being viewed by others
References
Birnboim H.C. & J. Doly, 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acid Res. 7: 1513–1523.
Christou P., T.L. Ford & M. Kofron, 1991. Production of transgenic rice (Oryza sativa L.) plants from agronomically important indica and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos. Bio/Technology 9: 957–962.
Chu C.C., C.C. Wang, C.S. Sun, C. Hsũ, K.C. Yin, C.Y. Chu & F.Y. Bi, 1975. Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Sci. Sin. 18: 659–668.
Dellaporta S.L., J. Wood & J.B. Hicks, 1983. A plant DNA minipreparation: Version II. Plant Mol. Biol. Rep. 1: 19–21.
Foroughi-Wehr B., G. Mix, H. Gaul & H.M. Wilson, 1976. Plant production from cultured anthers of Hordeum vulgare L.. Z. Pflanzenzüchtg. 77: 198–204.
Gordon-Kamm W.J., T.M. Spencer, M.L. Mangano, T.R. Adams, R.J. Daines, W.G. Start, J.V. O'Brien, S.A. Chambers, W.R. AdamsJr., N.G. Willets, T.B. Rice, C.J. Mackey, R.W. Krueger, A.P. Kausch & P.G. Lemaux, 1990. Transformation of maize cells and regenration of fertile transgenic plants. Plant Cell. 2: 603–618.
Hunter, C.P., 1987. Plant generation method. European patent application 0 245 898 A2: 1–8.
International Rules for Seed Testing, 1993. International Seed Testing Association. Seed Sci. & Technol. 21, Supplement.
Jefferson R.A., 1987. Assaying chimeric genes in plants: The GUS fusion system. Plant Mol. Biol. Rep. 5: 387–405.
Klein T.M., E.D. Wolf, R. Wu & J.C. Sanford, 1987. High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327: 70–73.
Lazzeri P.A., R. Brettschneider, R. Lührs & H Lörz, 1991. Stable transformation of barley via PEG-induced direct DNA uptake into protoplasts. Theor. Appl. Genet. 81: 437–444.
McDonnell R.E., R.D. Clark, W.A. Smith & M.A. Hinchee, 1987. A simplified method for the detection of neomycin phosphotransferase II activity in transformed plant tissues. Plant Mol. Biol. Rep. 5: 380–386.
Murashige T. & F. Skoog, 1962. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant 15: 473–497.
Olsen F.L., 1991. Isolation and cultivation of embryogenic microspores from barley (Hordeum vulgare L.). Hereditas 115: 255–266.
Reiss B., R. Sprengel, H. Will & H. Schaller, 1984. A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts. Gene 30: 211–218.
Ritala A., L. Mannonen, K. Aspegren, M. Salmenkallio-Marttila, U. Kurtén, R. Hannus, J. Mendez Lozano, T.H. Teeri & V. Kauppinen, 1993. Stable transformation of barley tissue culture by particle bombardment. Plant Cell Rep. 12: 435–440.
Ritala A., K. Aspegren, U. Kurtén, M. Salmenkallio-Marttila, L. Mannonen, R. Hannus, V. Kauppinen, T.H. Teeri & T.M. Enari, 1994. Fertile transgenic barley by particle bombardment of immature embryos. Plant Mol. Biol. 24: 317–325.
Sambrook J., E.F. Fritsch & T. Maniatis, 1989. Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Sanford J.C., T.M. Klein, E.D. Wolf & N. Allen, 1987. Delivery of substances into cells and tissues using a particle bombardment process. Particulate Sci. Technol. 5: 27–37.
Somers D.A., H.W. Rines, W. Gu, H.F. Kaeppler & W.R. Bushnell, 1992. Fertile, transgenic oat plants. Bio/Technology 10: 1589–1594.
Southern E.M., 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503–517.
Van den Broeck G., M.P. Timko, A.P. Kausch, A.R. Cashmore, M.Van Montagu & L. Herrera-Estrella, 1985. Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose 1,5-bisphosphate carboxylase. Nature 313: 358–363.
Vasi V., A.M. Castillo, M.E. Fromm & I.K. Vasil, 1992. Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Bio/Technology 10: 667–674.
Wan Y. & P.G. Lemaux, 1994. Generation of large numbers of independently transformed fertile barley plants. Plant Physiol. 104: 37–48.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Ritala, A., Aikasalo, R., Aspegren, K. et al. Transgenic barley by particle bombardment. Inheritance of the transferred gene and characteristics of transgenic barley plants. Euphytica 85, 81–88 (1995). https://doi.org/10.1007/BF00023933
Issue Date:
DOI: https://doi.org/10.1007/BF00023933