Abstract
Transgenic, fertile barley (Hordeum vulgare L.) from the Finnish elite cultivar Kymppi was obtained by particle bombardment of immature embryos. Immature embryos were bombarded to the embryonic axis side and grown to plants without selection. Neomycin phosphotransferase II (NPTII) activity was screened in small plantlets. One out of a total of 227 plants expressed the transferred nptII gene. This plant has until now produced 98 fertile spikes (T0), and four of the 90 T0 spikes analyzed to date contained the nptII gene. These shoots were further analyzed and they expressed the transferred gene. From green grains, embryos were isolated and grown to plantlets (T1). The four transgenic shoots of Toivo (the T0 plant) produced 25 plantlets as T1 progeny. Altogether fifteen of these T1 plants carried the transferred nptII gene as detected with the PCR technique, fourteen of which expressed the nptII gene. The integration and inheritance of the transferred nptII gene was confirmed by Southern blot hybridization. Although present as several copies, the transferred gene was inherited as a single Mendelian locus into the T2 progeny.
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References
Birnboim HC, Doly J: A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7: 1513–1523 (1979).
Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 76: 248–254 (1976).
Christou P, Ford TL, Kofron M: 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: (1991).
Dellaporta SL, Wood J, Hicks JB: A plant DNA mini-preparation: Version II. Plant Mol Biol Rep 1: 19–21 (1983).
Foroughi-Wehr B, Mix G, Gaul H, Wilson HM: Plant production from cultured anthers of Hordeum vulgare L. Z Pflanzenzücht 77: 198–204 (1976).
Gordon-Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start WG, O'Brien JV, Chambers SA, AdamsJr WR, Willetts NG, Rice TB, Mackey CJ, Krueger RW, Kausch AP, Lemaux PG: Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2: 603–618 (1990).
Hunter CP: Plant generation method. European patent application 0 245 898 A2: 1–8 (1987).
International Rules for Seed Testing. International Seed Testing Association, Seed Sci. & Technol. 21, Supplement (1993).
Jefferson RA: Assaying chimeric genes in plants. The GUS gene fusion system. Plant Mol Biol Rep 5: 387–405 (1987).
Kartha KK, Chibbar RN, Georges F, Leung N, Caswell K, Kendall E, Qureshi J: Transient expression of chloramphenicol acetyltransferase (CAT) gene in barley cell cultures and immature embryos through microprojectile bombardment. Plant Cell Rep 8: 429–432 (1989).
Klein TM, Wolf ED, Wu R, Sanford JC: High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327: 70–73 (1987).
Lazzeri PA, Brettschneider R, Lührs R, Lörz H: Stable transformation of barley via PEG-induced direct DNA uptake into protoplasts. Theor Appl Genet 81: 437–444 (1991).
McDonnell RE, Clark RD, Smith WA, Hinchee MA: A simplified method for the detection of neomycin phosphotransferase II activity in transformed plant tissues. Plant Mol Biol Rep 5: 380–386 (1987).
Mendel G: Versuche über Pflanzenhybriden. Verhandlungen des naturforschenden Vereines in Brünn 4: 3–47 (1865).
Mendel RR, Müller B, Schulze J, Kolesnikov V, Zelenin A: Delivery of foreign genes to intact barley cells by high-velocity microprojectiles. Theor Appl Genet 78: 31–34 (1989).
Murashige T, Skoog F: A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15: 473–497 (1962).
Reiss B, Sprengel R, Will H, Schaller H: A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts. Gene 30: 211–218 (1984).
Ritala A, Mannonen L, Aspegren K, Salmenkallio-Marttila M, Kurtén U, Hannus R, Mendez Lozano J, Teeri TH, Kauppinen V: Stable transformation of barley tissue culture by particle bombardment. Plant Cell Rep 12: 435–440 (1993).
Rogers SW, Rogers JC: The importance of DNA methylation for stability of foreign DNA in barley. Plant Mol Biol 18: 945–961 (1992).
Salmenkallio M, Hannus R, Teeri TH, Kauppinen V: Regulation of α-amylase promoter by gibberellic acid and abscisic acid in barley protoplasts transformed by electroporation. Plant Cell Rep 9: 352–355 (1990).
Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).
Sanford JC, Klein TM, Wolf ED, Allen N: Delivery of substances into cells and tissues using a particle bombardment process. Particulate Sci Technol 5: 27–37 (1987).
Somers DA, Rines HW, Gu W, Kaeppler HF, Bushnell WR: Fertile, transgenic oat plants. Bio/Technology 10: 1589–1594 (1992).
Southern EM: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98: 503–517 (1975).
Torres JT, Block A, Hahlbrock K, Somssich IE: Influence of bacterial strain genotype on transient expression of plasmid DNA in plant protoplasts. Plant J 4: 587–592 (1993).
Van denBroeck G, Timko MP, Kausch AP, Cashmore AR, VanMontagu M, Herrera-Estrella L: 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 (1985).
Vasil V, Castillo AM, Fromm ME, Vasil IK: Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Bio/Technology 10: 667–674 (1992).
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Ritala, A., Aspegren, K., Kurtén, U. et al. Fertile transgenic barley by particle bombardment of immature embryos. Plant Mol Biol 24, 317–325 (1994). https://doi.org/10.1007/BF00020170
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DOI: https://doi.org/10.1007/BF00020170