Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Direct gene transfer to protoplasts of two genotypes of Asparagus officinalis L. by electroporation

  • 36 Accesses

  • 12 Citations

Abstract

Callus-derived protoplasts of two genotypes of asparagus (Asparagus officinalis L.) were electroporated to introduce the β-glucuronidase gene (GUS). The level of GUS transient gene expression and the viability of the protoplasts were influenced by the voltage and duration of the electric pulse. The transient expression level was enhanced by increasing the plasmid DNA concentration and by the presence of polyethylene glycol (PEG) in the electroporation medium. A considerable increase in GUS activity was observed in presence of both PEG and upon heat-shock treatments compared to PEG treatment alone. An optimal level of GUS activity was obtained after electroporation with a capacitive discharge of 500 V/cm and 94 ms duration in both genotypes. The two genotypes differed in their responses to in vitro culture and also showed variable levels of transient expression. The present technique was found to be suitable to obtain transgenic plants as the histochemical GUS assay revealed GUS activity in the protoplast-derived micro-colonies as well as in callus tissues.

This is a preview of subscription content, log in to check access.

Abbreviations

CaMV:

cauliflower mosaic virus

CAT:

chloramphenicol acetyltransferase

CPW salts:

Cocking-Power-White salts

MES:

2-[N-Morpholino]ethanesulfonic acid

MU:

4-methyl umbelliferone

MUG:

4-methyl umbelliferyl glucuronide

μF:

microfarad

NAA:

α naphthaleneacetic acid

NOS:

nopaline synthase

NPT II:

neomycin phosphotransferase

X-Gluc:

5-bromo-4-chloro-3-indolyl-β-D-glucuronide

References

  1. Bytebier B, Deboeck F, De Greve H, Van Montagu M, Hemalsteens JP (1987) Proc Natl Acad Sci USA 84: 5345–5349

  2. Chibbar RN, Kartha KK, Datla RSS, Leung N, Caswell K, Mallard CS, Steinhauer L (1993) Plant Cell Rep 12: 506–509

  3. Delbreil B, Guerche P, Jullien M (1993) Plant Cell Rep 12: 129–132

  4. Doelling JH, Pikaard CS (1993) Plant Cell Rep 12: 241–244

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

  6. Galbraith DW (1981) Physiol Plant 53: 111–116

  7. Gilissen LJW, van Staveren MJ, Verhoeven HA, Sree Ramulu K (1992) Theor Appl Genet 84: 73–80

  8. Golovkin MV, Abraham M, Morocz S, Bottka S, Fehér A, Dudits D (1993) Plant Science 90: 41–52

  9. Higgs DC, Colbert JT (1993) Plant Cell Rep 12: 445–452

  10. Jefferson RA, Kavanagh TA, Bevan MW (1987) EMBO J 6: 3901–3907

  11. Lindsey K, Jones MGK (1987) Plant Mol Biol 10: 43–52

  12. Lindsey K, Jones MGK (1989) Plant Cell Rep 8: 71–74

  13. Manders G, dos Santos AVP, d'Utra Vaz FB, Davey MR, Power JB (1992) Plant Cell Tissue Organ Culture 30: 69–75

  14. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

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

  16. Negrutiu I, Dewulf J, Pietrzak M, Botterman M, Rietveld E, Wurzer-Figurelli EM, De Y, Jacobs M (1990) Physiol Plant 79: 197–205

  17. Paszkowski J, Saul MW, Potrykus I (1989) In: Schell J, Vasil IK (Eds) Cell culture and somatic cell genetics of plants, vol 6, Academic Press New York, pp 51–68

  18. Potrykus I (1991) Ann Rev Plant Physiol Plant Mol Biol 42: 205–225

  19. Puonti-Kaerlas J, Ottosson A, Eriksson T (1992) Plant Cell Tissue Organ Culture 30: 141–148

  20. Riggs CD, Bates GW (1986) Proc Natl Acad Sci USA 83: 5602–5606

  21. Rasmussen JO, Rasmussen OS (1993) Plant Science 89: 199–207

  22. Saunders JA, Bates GW (1992) In: Chang DC, Chassy BM, Saunders JA, Sowers AE (eds) Guide to electroporation and electrofusion, Academic Press New York, pp 471–483

  23. Schieder O, Gerdemann-Knorck M, Sacristan M-D (1991) Israel J Bot 40: 99–107.

  24. Séguin A, Lalonde M (1988) Plant Cell Rep 7: 367–370

  25. Shillito RD, Saul MW, Paszkowski J, Muller M, Potrykus I (1985) Bio/Technology 3: 1099–1103

  26. Songstad DD, Halaka FG, DeBoer DL, Armstrong CL, Hinchee MAW (1993) Plant Cell Tissue Organ Culture 33: 195–201

  27. Taylor MG, Vasil V, Vasil IK (1993) Plant Cell Rep 12: 491–495

  28. Widholm JM (1972) Stain Tech 47: 189–194

Download references

Author information

Additional information

Communicated by F. Constabel

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Mukhopadhyay, S., Desjardins, Y. Direct gene transfer to protoplasts of two genotypes of Asparagus officinalis L. by electroporation. Plant Cell Reports 13, 421–424 (1994). https://doi.org/10.1007/BF00231959

Download citation

Keywords

  • Transgenic Plant
  • Polyethylene Glycol
  • Transient Expression
  • Electric Pulse
  • Present Technique