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Efficient regeneration of Brassica oleracea hypocotyl protoplasts and high frequency genetic transformation by direct DNA uptake

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Summary

Efficient regeneration (80%) and high frequency genetic transformation (10–33%) were achieved by culturing protoplasts isolated from hypocotyl tissues of six day old Brassica oleracea seedlings and by subjecting these protoplasts to PEG mediated direct plasmid uptake. Three different plasmid vectors carrying marker genes for resistance to methotrexate (dhfr), hygromycin (hpt) and phosphinotricin (bar) were constructed and used for transformation. Large number of normal, fertile transformants were obtained with vectors carrying hpt and bar genes. No transformants could be regenerated for resistance to methotrexate as it severely suppressed shoot differentiation.

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Abbreviations

bar/PAT:

bialaphos resistance gene/phosphinotricin acetyltransferase

2,4-D:

2,4-di-chlorophenoxyacetic acid

dhfr/DHPR:

dihydrofolate reductase gene/enzyme

gus/GUS:

β-glucuronidase gene/enzyme

hpt/HPT:

hygromycin phosphotransferase gene/enzyme

Kn:

kinetin

PEG:

polyethylene glycol

RH:

relative humidity

References

  • Boulter ME, Croy E, Simpson P, Shields R, Croy RRD, Shirsat AH (1990) Plant Sci 70: 91–99

    Google Scholar 

  • Damm B, Schmidt R, Willmitzer L (1989) Mol Gen Genet 217: 6–12

    Google Scholar 

  • Datta SK, Peterhans A, Datta K, Potrykus I (1990) Bio/Technology 8: 736–740

    Google Scholar 

  • De Block M, De Brouwer D, Tenning P (1989) Plant Physiol 91: 694–701

    Google Scholar 

  • Dellaporta SL, Wood J, Hicks JB (1983) Plant Mol Biol Reporter 1: 19–21

    Google Scholar 

  • Frearson EM, Power JB, Cocking EC (1973) Dev Biol 33: 130–137

    Google Scholar 

  • Glimelius K (1984) Physiol Plant 61: 38–44

    Google Scholar 

  • Golz C, Köhler F, Schieder O. (1990) Plant Mol Biol 15: 475–483

    Google Scholar 

  • Guerche P, Charbonnier M, Jouanin L, Tourneur C, Paszkowski J, Pelletier G (1987) Plant Sci 52: 111–116

    Google Scholar 

  • Guerche P, De Almeida ERP, Schwarztein MA, Gander E, Krebbers E, Pelletier G (1990) Mol Gen Genet 221: 306–314

    Google Scholar 

  • Hain R, Stabel P, Czernilofski AP, Steinbiβ HH, Herrera-Estrella L, Schell J (1985) Mol Gen Genet 199: 161–168

    Google Scholar 

  • Jefferson RA (1987) Plant Mol Biol Reporter 5: 387–405

    Google Scholar 

  • Jourdan PS, Earle ED, Mutschler MA (1990) Plant Cell, Tissue and Organ Culture 21: 227–236

    Google Scholar 

  • Jourdan PS, Earle ED (1989) J Amer Soc Hort Sci 114: 343–349

    Google Scholar 

  • Kao HM, Keller WA, Gleddie S, Brown GC (1990) Plant Cell Reports 9: 311–315

    Google Scholar 

  • Lillo C, Olsen JE (1989) Plant Cell, Tissue and Organ Culture 17: 91–100

    Google Scholar 

  • Menczel L, Nagy I, Kizz ZR, Maliga P (1981) Theor Appl Genet 59: 191–195

    Google Scholar 

  • Murashige T, Skoog F (1962) Physiol Plant 15: 473–497

    CAS  Google Scholar 

  • Nagy JJ, Maliga P (1976) Z Pflanzenphysiol 78: 453–455

    Google Scholar 

  • Negrutiu I, Shillito RD, Potrykus I, Basini G, Sala F (1987) Plant Mol Biol 8: 363–373

    Google Scholar 

  • Perbal B (1988) A practical Guide to Molecular Cloning (2nd ed) John Wiely & Sons, Inc., Chichester, UK

    Google Scholar 

  • Potrykus I, Paszkowski J, Saul MW, Petruska J, Shillito RD (1985) Mol Gen Genet 199: 169–177

    Google Scholar 

  • Pua EC, Mehra-Palta A, Nagy F, Chua NH (1987) Bio/Technology 5: 815–817

    Google Scholar 

  • Rhodes CA, Pierce DA, Mettler IJ, Mascarenhas D, Detmer JJ (1988) Science 240: 204–207

    Google Scholar 

  • Robertson D, Earle ED (1986) Plant Cell Reports 5: 61–64

    Google Scholar 

  • Saul MW, Shillito RD, Negrutiu I (1988) Plant Mol Biol, Manual AI, Kluwer Academic Publishers, Belgium, pp. 1–16

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning — A Laboratory Manual (2nd ed) Cold Spring Harbor Laboratory Press

  • Shimamoto K, Terada R, Izawa T, Fujimoto H (1989) Nature 338: 274–276

    Google Scholar 

  • Simonsen CC, Levinson AD (1983) Proc Natl Acad Sci 80: 2495–2499

    Google Scholar 

  • Thomzik JE, Hain R (1990) Plant Cell Reports 9: 233–236

    Google Scholar 

  • Töpfer R, Matzeit V, Gronenborn B, Schell J, Steinbiβ HH (1987) Nucl Acids Res 15: 5890

    Google Scholar 

  • Töpfer R, Pröls M, Schell J, Steinbiβ HH (1988b) Plant Cell Reports 7: 225–228

    Google Scholar 

  • Töpfer R, Schell J, Steinbiβ HH (1988a) Nucl Acids Res 16: 8725

    Google Scholar 

  • Xu ZH, Davey MR, Cocking EC (1982) Plant Sci Lett 24: 117–121

    Google Scholar 

  • Yamashita Y, Shimamoto K (1989) In: Bajaj YPS (ed) Biotechnology in Agriculture and Forestry 8, Springer, Berlin, Heidelberg, New York, pp. 193–205

    Google Scholar 

  • Yang H, Zhang HM, Davey MR, Mulligan BJ, Cocking EC (1988) Plant Cell Reports 7: 421–425

    Google Scholar 

  • Zhang HM, Yang H, Rech EL, Golds TJ, Davis AS, Mulligan BJ, Cocking EC, Davey MR (1988) Plant Cell Reports 7: 379–384

    Google Scholar 

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Authors and Affiliations

  1. Tata Energy Research Institute, 90 Jor Bagh, 110 003, New Delhi, India

    Arundhati Mukhopadhyay, Akshay K. Pradhan, Yaspal S. Sodhi & Deepak Pental

  2. Max-Planck-Institut für Züchtungsforschung, Carl-von-Linné-Weg 10, W-5000, Köln, Germany

    Reinhard Töpfer, Hans-Henning Steinbiß & Jeff Schell

Authors
  1. Arundhati Mukhopadhyay
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  2. Reinhard Töpfer
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  3. Akshay K. Pradhan
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  4. Yaspal S. Sodhi
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  5. Hans-Henning Steinbiß
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  6. Jeff Schell
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  7. Deepak Pental
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Communicated by H. Lörz

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Mukhopadhyay, A., Töpfer, R., Pradhan, A.K. et al. Efficient regeneration of Brassica oleracea hypocotyl protoplasts and high frequency genetic transformation by direct DNA uptake. Plant Cell Reports 10, 375–379 (1991). https://doi.org/10.1007/BF00232604

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  • DOI: https://doi.org/10.1007/BF00232604

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