Abstract
We have developed a system for the biolistic transformation of barley using freshly-isolated microspores as the target tissue. Independent transformation events led, on average, to the recovery of one plant per 1×107 bombarded microspores. Putative transformants have been regenerated using phosphinothricin as a selective agent. R0 plants have been transferred to soil approximately 2 months after bombardment. Integration of the marker genes bar and uidA has been confirmed by Southern analysis. The marker genes are inherited in all progeny plants confirming the expected homozygous nature of the R0 plants.
Similar content being viewed by others
References
Barcelo P, Hagel C, Becker D, Martin A, Lörz H (1994) Transgenic cereal (Tritordeum) plants obtained at high efficiency by microprojectile bombardment of inflorescence tissue. The Plant J 5(4): 583–592
Becker D, Brettschneider R, Lörz H (1994) Fertile transgenic wheat from microprojectile bombardment of scutellar tissue. The Plant J 5(2):299–307
Birch RG, Franks T (1991) Development and optimisation of microprojectile systems for plant genetic transformation. Aust J Plant Physiol 18:453–469
Bolik M, Koop HU (1991) Identification of embryogenic microspores of barley (Hordeum vulgare L.) by individual selection and culture, and their potential for transformation by microinjection. Protoplasma 162:61–68
Bower R, Birch RG (1992) Transgenic sugarcane plants via microprojectile bombardment. The Plant J 2:409–416
Cao J, Duan X, McElroy D, Wu R (1992) Regeneration of herbicideresistant transgenic rice plants following microprojectile-mediated transformation of suspension culture cells. Plant Cell Rep 11:586–591
Christou P, Ford TL, Kofron M (1991) Production of transgenic rice (Oryza sativa L.) plants from agronomically-important indica and japonica varieties via electrical discharge particle-acceleration of exogenous DNA into immature zygotic embryos. Bio/Technol 9: 957–962
Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 4:19–21
D'Halluin K, Bonne E, Bossut M, De Beuckeleer M, Leemans J (1993) Transgenic maize plants by tissue electroporation. Plant Cell 4:1495–1505
Fennell A, Hauptmann R (1992) Electroporation and PEG delivery of DNA into maize microspores. Plant Cell Rp. 11:567–570
Funatsuki H, Lörz H, Lazzeri PA (1992) Use of feeder cells to improve barley protoplast culture and regeneration. Plant Sci 85:179–187
Golds TJ, Babczinsky J, Mordhorst AP, Koop HU (1994) Protoplast preparation without centrifugation: plant regeneration from barley (Hordeum vulgare L.). Plant Cell Rep 13:188–192
Gordon Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start WG, O`Brian JV, Chambers SA, Adams JWR, Willetts NG, Rice TB, Mackey CJ, Krueger W, Kausch AP, Lemaux PG (1990) Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2:603–618
Hoekstra S, van Zijderveld MH, Louwerse JD, Heidekamp F, van der Mark F (1992) Anther and microspore culture of Hordeum vulgare L. cv Igri. Plant Sci 86:89–96
Hoekstra S, van Zijderveld MH, Heidekamp F, van der Mark F (1993) Microspore culture of Hordeum vulgare L.: the influence of density and osmolality. Plant Cell Rep 12:661–665
Huang C, Yan H, Yan Q, Zhu M, Yuan M, Xu A (1993) Establishment and characterization of embryogenic cell-suspension cultures from immature and mature embryos of barley (Hordeum vulgare L.). Plant Cell Tissue Org Cult 32:19–25
Jähne A, Lazzeri PA, Jäger-Gussen M (1991a) Plant regeneration from embryogenic cell suspensions derived from anther cultures of barley (Hordeum vulgare L.). Theor Appl Genet 82:74–80
Jähne A, Lazzeri PA, Lörz H (1991b) Regeneration of fertile plants from protoplasts derived from embryogenic cell suspensions of barley (Hordeum vulgare L.). Plant Cell Rep 10:1–6
Jefferson RA (1987) Assaying chimeric genes in plants by the GUS fusion system. Plant Mol Biol Rep 5:387–405
Kott LS, Kasha KJ (1984) Initiation and morphological development of somatic embryoids from barley cell cultures. Can J Bot 62:1245–1249
Koziel GM, Beland GL, Bowman C, Carozzi NB, Crenshaw R, Crossland L, Dawson J, Desai N, Hill M, Kadwell S, Launis K, Lewis K, Maddox D, McPherson K, Meghji MR, Merlin E, Rhodes R, Warren GW, Wright M, Evola SV (1993) Field performance of elite transgenic maize plants expressing an insecticidial protein derived from Bacillus thuringiensis. Bio/Technol 11:194–200
Kuhlmann U, Foroughi-Wehr B (1989) Production of doubled haploid lines in frequencies sufficient for barley breeding programs. Plant Cell Rep 8:78–81
Kuhlmann U, Foroughi-Wehr B, Graner A, Wenzel G (1991) Improved culture system for microspores of barley to become a target for DNA uptake. Plant Breed 107:165–168
Lazzeri PA, Brettschneider R, Lührs R, Lörz H (1991) Stable transformation of barley via PEG-induced direct DNA uptake into protoplasts. Theor Appl Genet 81:437–444
McCabe DE, Swain WF, Martinell BJ, Christou P (1988) Stable transformation of soybean (Glycine max) by particle acceleration. Bio/Technol 6:923–926
Mordhorst AP, Lörz H (1992) Electrostimulated regeneration of plantlets from protoplasts derived from cell suspensions of barley (Hordeum vulgare L.). Physiol Plant 85:289–294
Mordhorst AP, Lörz H (1993) Embryogenesis and development of isolated barley (Hordeum vulgare L.). Microspores are influenced by the amount and composition of nitrogen sources in the culture media. J Plant Physiol 142:485–492
Neuhaus-Url G, Neuhaus G (1993) The use of the nonradioactive digoxigenin chemiluminescent technology for plant genomic Southern-blot hybridization: a comparison with radioactivity. Transgenic Res 2:115–120
Olsen FL (1987) Induction of microspore embryogenesis in cultured anthers of Hordeum vulgare. The effects of ammonium nitrate, glutamine and asparagine as nitrogen sources. Carlsberg Res Commun 5:393–404
Olsen FL (1991) Isolation and cultivation of embryogenic microspores from barley (Hordeum vulgare L.). Hereditas 115:255–266
Sanford J C (1990) Biolistic plant transformation. Physiol Plant 79:206–209
Simonson RL, Baenziger P S (1992) The effect of gelling agents on wheat anther and immature embryo culture. Plant Breed 109:211–217
Somers DA, Rines HW, Gu W, Kaeppler HF, Bushnell WR (1992) Fertile transgenic oat plants. Bio/Technol 10:1589–1594
Sorvari S (1986) Comparison of anther cultures of barley cultivars in barley-starch and agar-gelatinized media. Ann Agric Fenn 25:249–254
Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517
Vasil IK, Vasil V (1992) Advances in cereal protoplast research. Physiol Plant 85:279–283
Vasil V, Castillo AM, Fromm ME, Vasil IK (1992) Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Bio/Technol 10:667–674
Wan Y, Lemaux PG (1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiol 104:37–48
Weeks JT, Anderson OD, Blechl AE (1993) Rapid production of multiple independent lines of fertile transgenic wheat (Triticum aestivum). Plant Physiol 102:1077–1084
Widholm JM (1972) The use of fluorescein diacetate and phenosafranine for determining the viability of cultured plant cells. Stain Technol 47:189–194
Ziauddin A, Marsolais A, Simion D, Kasha KJ (1992) Improved plant regeneration from wheat and barley microspore culture using phenylacetic acid (PAA). Plant Cell Rep 11:489–498
Author information
Authors and Affiliations
Additional information
Communicated by G.Wenzel
Rights and permissions
About this article
Cite this article
Jähne, A., Becker, D., Brettschneider, R. et al. Regeneration of transgenic, microspore-derived, fertile barley. Theoret. Appl. Genetics 89, 525–533 (1994). https://doi.org/10.1007/BF00225390
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00225390