Protoplast-Independent Production of Transgenic Plants

  • Germán Spangenberg
  • Zeng-Yu Wang
  • Ingo Potrykus
Part of the Monographs on Theoretical and Applied Genetics book series (GENETICS, volume 23)


Problems encountered with protoplast-based methods for the generation of transgenic plants have prompted the development of alternative techniques for gene transfer in grasses. These problems relate mainly to plant regeneration from protoplasts and do not reflect specific barriers to the uptake of foreign DNA by isolated protoplasts. Examples of these difficulties are relatively low plating efficiencies and low plant regeneration frequencies from protoplasts, species and genotype dependence often observed in the regeneration process, and albinism and somaclonal variation revealed in protoplast-derived plants (Potrykus 1990). Plant regeneration from protoplasts is thus a delicate process depending upon parameters that are not under experimental control, such as wound response and genotype-dependent competence for regeneration (Vasil 1988; Potrykus 1990). Furthermore, transgenic plants recovered from protoplasts may show serious fertility constraints and undesired integration of multiple and rearranged transgene copies (Spangenberg et al. 1995a).


Transgenic Plant Tall Fescue Perennial Ryegrass Immature Zygotic Embryo Italian Ryegrass 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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  1. Asano Y, Otsuki Y, Ugaki M (1991) Electroporation-mediated and silicon carbide fiber-mediated DNA delivery in Agrostis alba L. (redtop). Plant Sci 79: 247–252CrossRefGoogle Scholar
  2. Batty NP, Evans JM (1992) Biological ballistics — no longer a shot in the dark. Transgenic Res 1: 107–113CrossRefGoogle Scholar
  3. Bilang R, Iida S, Peterhans A, Potrykus I, Paszkowski J (1991) The 3′ terminal region of the hygromycin-B-resistance gene is important for its activity in Escherichia coli and Nicotiana tabacum. Gene 100: 247–250PubMedCrossRefGoogle Scholar
  4. Casas AM, Kononowicz AK, Zehr UB, Tomes DT, Axtell JD, Butler LG, Bressan RA, Hasegawa PM (1993) Transgenic sorghum plants via microprojectile bombardment. Proc Natl Acad Sci USA 90: 11212–11216PubMedCrossRefGoogle Scholar
  5. Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM, Duncan DR, Conner TW, Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115: 971–980PubMedGoogle Scholar
  6. Christou P (1992) Genetic transformation of crop plants using microprojectile bombardment. Plant J 2: 275–281CrossRefGoogle Scholar
  7. Christou P (1993) Particle gun-mediated transformation. Curr Opin Biotechnol 4: 135–141CrossRefGoogle Scholar
  8. Christou (1996) Electric discharge particle acceleration (Accell®) technology for the creation of transgenic plants with altered characteristics. Field Crops Res 45: 143–151CrossRefGoogle Scholar
  9. Christou P, Ford T, Kofron M (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: 957–962CrossRefGoogle Scholar
  10. Conger BV, Hanning GE, Gray DJ, McDaniel JK (1983) Direct embryogenesis from mesophyll cells of orchardgrass. Science 221: 850–851PubMedCrossRefGoogle Scholar
  11. Dalton SJ, Bettany AJE, Timms E, Morris P (1998) Transgenic plants of Lolium multiflorum, Lolium perenne, Festuca arundinacea and Agrostis stolonifera by silicon carbide fibre-mediated transformation of cell suspension cultures. Plant Sci (in press)Google Scholar
  12. Denchev PD, Songstad DD, McDaniel JK, Conger BV (1997) Transgenic orchardgrass (Dactylis glomerata) plants by direct embryogenesis from microprojectile bombarded leaf cells. Plant Cell Rep 16: 813–819CrossRefGoogle Scholar
  13. Dunder E, Dawson J, Suttie J, Pace G (1995) Maize transformation by microprojectile bombardment of immature embryos. In: Potrykus I, Spangenberg G (eds) Gene transfer to plants. Springer, Berlin Heidelberg New York, pp 127–138Google Scholar
  14. Dunahay TG (1993) Transformation of Chlamydomonas reinhardtii with silicon carbide whiskers. BioTechniques 15: 452–460PubMedGoogle Scholar
  15. Finer JJ, Vain P, Jones MW, McMullen MD (1992) Development of the particle inflow gun for DNA delivery to plant cells. Plant Cell Rep 11: 323–328CrossRefGoogle Scholar
  16. Frame BR, Drayton PR, Bagnall SV, Lewnau CJ, Bullock WP, Wilson HM, Dunwell JM, Thompson JA, Wang K (1994) Production of fertile transgenic maize plants by silicon carbide whisker-mediated transformation. Plant J 6: 941–948CrossRefGoogle Scholar
  17. Fuchs RL, Ream JE, Hammond BG, Naylor MW, Leimgruber RM, Berberich SA (1993) Case study in safety assessment (II): safety assessment of the neomycin phosphotransferase II (nptII) protein. Bio/Technology 11: 1543–1547PubMedCrossRefGoogle Scholar
  18. Hartman CL, Lee L, Day PR, Turner NE (1994) Herbicide resistant turfgrass (Agrostis palustris Huds.) by biolistic transformation. Bio/Technology 12: 919–923CrossRefGoogle Scholar
  19. Hensgens LAM, de Bakker EPHM, van Os-Ruygrok EP, Rueb S, van de Mark F, van der Maas HM, van der Veen S, Kooman-Gersmann M, Hart L, Schilperoort RA (1993) Transient and stable expression of gusA fusions with rice genes in rice, barley and perennial ryegrass. Plant Mol Biol 22: 1101–1127PubMedCrossRefGoogle Scholar
  20. Hiei J, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6: 271–282PubMedCrossRefGoogle Scholar
  21. Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Bio/Technology 14: 745–750CrossRefGoogle Scholar
  22. Kaeppler HF, Gu W, Somers DA, Rines HW, Cockburn AF (1990) Silicon carbide fiber-mediated DNA delivery into plant cells. Plant Cell Rep 9: 415–418CrossRefGoogle Scholar
  23. Kaeppler HF, Somers DA, Rines HW, Cockburn AF (1992) Silicon carbide fiber-mediated stable transformation of plant cells. Theor Appl Genet 84: 560–566CrossRefGoogle Scholar
  24. Klein TM (1995) The biolistic transformation system. In: Potrykus I, Spangenberg G (eds) Gene transfer to plants. Springer, Berlin Heidelberg New York, pp 115–117Google Scholar
  25. Klein TM, Fitzpatrick-McElligott S (1993) Particle bombardment — a universal approach for gene transfer to cells and tissues. Curr Opin Biotechnol 4: 583–590PubMedCrossRefGoogle Scholar
  26. Klein TM, Wolf ED, Wu R, Sanford JC (1987) High velocity microprojectiles for delivering nucleic acids into living cells. Nature 327: 70–73CrossRefGoogle Scholar
  27. Lee L (1996) Turfgrass biotechnology. Plant Sci 115: 1–8CrossRefGoogle Scholar
  28. McCabe DE, Christou P (1993) Direct DNA transfer using electric discharge particle acceleration (Accell®) technology. Plant Cell Tissue Organ Culture 33: 227–236CrossRefGoogle Scholar
  29. Pérez-Vicente R, Wen XD, Wang ZY, Leduc N, Sautter C, Wehrli E, Potrykus I, Spangenberg G (1993) Culture of vegetative and floral meristems in ryegrasses: potential targets for microballistic transformation. J Plant Physiol 142: 610–617Google Scholar
  30. Potrykus I (1990) Gene transfer to cereals: an assessment. Bio/Technology 8: 535–542CrossRefGoogle Scholar
  31. Rashid H, Yokoi S, Toriyama K, Hinata K (1996) Transgenic plant production mediated by Agrobacterium in Indica rice. Plant Cell Rep 15: 727–730CrossRefGoogle Scholar
  32. Sanford JC (1988) The biolistic process. TIBTECH 6: 299–302CrossRefGoogle Scholar
  33. Sanford JC, Klein TM, Wolf ED, Allen NJ (1987) Delivery of substances into cells and tissues using a particle bombardment process. J Particulate Sci Technol 6: 559–563Google Scholar
  34. Sanford JC, Smith FD, Russell JA (1993) Optimizing the biolistic process for different biological applications. Methods Enzymol 217: 483–509PubMedCrossRefGoogle Scholar
  35. Sautter C, Waldner H, Neuhaus-Url G, Galli A, Neuhaus G, Potrykus I (1991) Micro-targeting: high efficiency gene transfer using a novel approach for the acceleration of microprojectiles. Bio/Technology 9: 1080–1085PubMedCrossRefGoogle Scholar
  36. Sautter C (1993) Development of a microtargeting device for particle bombardment of plant meristems. Plant Cell Tissue Organ Cult 33: 251–257CrossRefGoogle Scholar
  37. Spangenberg G, Wang ZY, Nagel J, Potrykus I (1994) Protoplast culture and generation of transgenic plants in red fescue (Festuca rubra L.). Plant Sci 97: 83–94CrossRefGoogle Scholar
  38. Spangenberg G, Wang ZY, Wu XL, Nagel J, Iglesias VA, Potrykus I (1995a) Transgenic tall fescue (Festuca arundinacea) and red fescue (F. rubra) plants from microprojectile bombardment of embryogenic suspension cells. J Plant Physiol 145: 693–701Google Scholar
  39. Spangenberg G, Wang ZY, Wu XL, Nagel J, Potrykus I (1995b) Transgenic perennial ryegrass (Lolium perenne) plants from microprojectile bombardment of embryogenic suspension cells. Plant Sci 108: 209–217CrossRefGoogle Scholar
  40. Spangenberg G, Wang ZY, Potrykus I (1998) Biotechnology in forage and turf grass improvement. In: Frankel R, Grossman M, Linskens HF, Maliga P, Riley R (eds) Monographs on theoretical and applied genetics, this volume. Springer, Berlin Heidelberg New YorkGoogle Scholar
  41. Svab Z, Maliga P (1993) High-frequency plastid transformation in tobacco by selection for a chimeric aadA gene. Proc Natl Acad Sci USA 90: 913–917PubMedCrossRefGoogle Scholar
  42. Svab Z, Hajdukiewicz P, Maliga P (1990) Stable transformation of plastids in higher plants. Proc Natl Acad Sci USA 87: 8526–8530PubMedCrossRefGoogle Scholar
  43. Takamizo T, Suginobu KI, Ohsugi R (1990) Plant regeneration from suspension culture derived protoplasts of tall fescue (Festuca arundinacea Schreb.) of a single genotype. Plant Sci 72: 125–131CrossRefGoogle Scholar
  44. Taylor MG, Vasil IK (1991) Histology of, and physical factors affecting transient GUS expression in pearl millet (Pennisetum glaucum (L.) R. Br.) embryos following microprojectile bombardment. Plant Cell Rep 10: 120–125CrossRefGoogle Scholar
  45. Tingay S, McElroy D, Kalla R, Fieg S, Wang M, Thornton S, Brettell R (1997) Agrobacterium tumejaciens-mediated barley transformation. Plant J 11: 1369–1376CrossRefGoogle Scholar
  46. Trigiano RN, Gray DJ, Conger BV, McDaniel JK (1989) Origin of direct somatic embryos from cultured leaf segments of Dactylis glomerata. Bot Gaz 150: 72–77CrossRefGoogle Scholar
  47. Vain P, Keen N, Murillo J, Rathus C, Nemes C, Finer JJ (1993) Development of the particle inflow gun. Plant Cell Tissue Organ Cult 33: 237–246CrossRefGoogle Scholar
  48. Van der Maas HM, de Jong ER, Rueb S, Hensgens LAM, Krens FA (1994) Stable transformation and long-term expression of the gusA reporter gene in callus lines of perennial ryegrass (Lolium perenne L.). Plant Mol Biol 24: 401–405PubMedCrossRefGoogle Scholar
  49. Vasil IK (1988) Progress in the regeneration and genetic manipulation of cereal crops. Bio/Technology 6: 397–402CrossRefGoogle Scholar
  50. Vasil V, Srivastava V, Castillo AM, Fromm ME, Vasil IK (1993) Rapid production of transgenic wheat plants by direct bombardment of cultured immature embryos. Bio/Technology 11: 1553–1558CrossRefGoogle Scholar
  51. Wan Y, Lemaux PG (1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiol 104: 37–48PubMedGoogle Scholar
  52. Wang ZY, Takamizo T, Iglesias VA, Osusky M, Nagel J, Potrykus I, Spangenberg G (1992) Transgenic plants of tall fescue (Festuca arundinacea Schreb.) obtained by direct gene transfer to protoplasts. Bio/Technology 10: 691–696.PubMedCrossRefGoogle Scholar
  53. Wang K, Frame BR, Drayton PR, Thompson JA (1995) Silicon carbide whisker-mediated transformation: regeneration of transgenic maize plants. In: Potrykus I, Spangenberg G (eds) Gene transfer to plants. Springer, Berlin Heidelberg New York, pp 186–192Google Scholar
  54. Xiao L, Ha SB (1997) Efficient selection and regeneration of creeping bentgrass transformants following particle bombardment. Plant Cell Rep 16: 874–878CrossRefGoogle Scholar
  55. Ye XD (1997) Gene transfer to ryegrasses (Lolium spp.): modification of fructan metabolism in transgenic plants. PhD Diss, Swiss Federal Institute of Technology, ZürichGoogle Scholar
  56. Ye XD, Wang ZY, Wu XL, Potrykus I, Spangenberg G (1997) Transgenic Italian ryegrass (Lolium multiflorum) plants from microprojectile bombardment of embryogenic suspension cells. Plant Cell Rep 16: 379–384Google Scholar
  57. Zhong H, Bolyard MG, Srinivasan C, Stickelen M (1993) Transgenic plants of turfgrass (Agrostis palustris Huds.) from microprojectile bombardment of embryogenic callus. Plant Cell Rep 13: 1–6CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1998

Authors and Affiliations

  • Germán Spangenberg
    • 1
  • Zeng-Yu Wang
    • 1
  • Ingo Potrykus
    • 2
  1. 1.Plant Sciences and Biotechnology, Agriculture Victoria, Department of Natural Resources and Environment and CRC for Molecular Plant BreedingLa Trobe UniversityBundooraAustralia
  2. 2.Institute of Plant SciencesSwiss Federal Institute of TechnologyZürichSwitzerland

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