Transient expression assay for optimization of direct gene transfer into cucumber meristem protoplasts by electroporation

• Autorzy: Burza W , Wochniak P. , Wroblewski T. , Malepszy S.
• Języki publikacji: EN

Abstrakty

EN

The paper presents a new way of obtaining viable and very homogeneous cucumber protoplasts. Protoplasts from cells formed in the shoot tip meristem culture were isolated from suspension. Plasmid pBI121 was introduced using impulse electric field. Effectiveness of transformation process was determined by the transient expression of ß-glucuronidase (GUS) gene, controlled by promotor 35S. The activity of ß-glucuronidase enzyme as a product of GUS reporter gene was estimated by fluorimetric method (JEFFERSON 1987). Parameters of electroporation process were optimized. The transient expression of GUS gene was measured 24 h after electroporation. The highest effectiveness of transformation process was achieved using three electric impulses at the initial voltage of 250-350 V at 10-sec. intervals as a result of discharging a 140 µF capacitor and 50-70 µg × cm⁻³ plasmid DNA in the presence of 50 µg × cm⁻³ carrier DNA. The system presented is an effective method of exogenic DNA transfer, which is indicated by a high transient expression of the reporter gene. In comparison to Agrobacterium tumefaciens and A. rhizogenes, this alternative method of gene transfer can be used for obtaining transgenic cucumber plants.

Słowa kluczowe

EN

electroporation; gene transfer; protoplast; transient expression; cucumber; Cucumis sativus

• Wydawca: -
• Czasopismo: Journal of Applied Genetics
• Rocznik: 1995
• Tom: 36
• Numer: 1
• Opis fizyczny: p.1-10,fig.

Twórcy

autor

Burza W

• Warsaw Agricultural University, ul.Nowoursynowska 166, 02-766 Warszawa, Poland

autor

Wochniak P.

autor

Wroblewski T.

autor

Malepszy S.

Bibliografia

• CHEE P.P. (1990). Transformation of cucumber via Agrobacterium tumefaciens and the regeneration of transformed plants. Plant Cell Rep. 9: 245-248.
• CHUPEAU M.Ch., BELLINI C.,GUERCHE P.,MAISONNENVE B., VASTRA G., CHUPEAU Y. (1989). Transgenic plants of lettuce (Lactuca saliva) obtained through electroporation of protoplasts. Biotechnology 7: 503-508.
• FROMM M., TAYLOR L.P., WALBOT V. (1985). Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc. Natl. Acad. Sci. USA 82: 5824-5828.
• FROMM M., TAYLOR L.P., WALBOT V. (1986). Stable transformation of maize after transfer by electroporation. Nature 319: 791-793.
• GUERCHE P., BELLINI C., Le MOULLEC J.M., CABOCHE M. (1987a). Use of transient expression assay for the optimization of direct gene transfer into tobacco mesophyll protoplasts by electroporation. Biochemie 69: 621-628.
• GUERCHE P., CHARBONNIER M., JOUANIN L., TOURNEUR C., PASZKOWSKI J., PELLETIER G. (1987b). Direct gene transfer by electroporation in Brassica napus. Plant Science 52: 111-116.
• JEFFERSON R.A. (1987). Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5: 387-405.
• JONES H., OOMS G., JONES M.G.K. (1989).Transient gene expression in electroporated Solanum protoplasts. Plant Mol. Biol. 13: 503-511.
• KRENS F.A., MOLENDIJK L., WULLEMS G.J., SCHILPEROOT R.A. (1982). In vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296: 72-74.
• LARKIN P.J., TAYLOR B.H., GERSMANN M., BRETTEL R.I.S. (1990). Direct gene transfer to protoplasts. Aust. J. Plant Physiol. 17: 291-302.
• LINDSEY K., JONES M.G.K. (1987). Transient gene expression in electroporated protoplasts and intact cells of sugar beet. Plant Mol. Biol. 10: 43-52.
• ŁYŻNIK L.A., RYAN R.D., RITCHIE S.W., HADGES T.K. (1989). Stable cotransformation of maize protoplasts with gus A and neo genes. Plant Mol. Biol. 13: 151-161.
• MALEPSZY S. (1988). Cucumber (Cucumis sativus L.). Biotechnology in Agriculture and Forestry. Vol. 6, Crops II (Y.P.S. Bajaj, ed.). Springer-Verlag. Berlin-Heidelberg: 227-293.
• MURASHIGE T., SKOOG F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15: 473-497.
• NAGATA T., TAKEBE J. (1971). Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts. Planta 92: 301-308.
• ORCZYK W., MALEPSZY S. (1985). In vitro culture of Cucumis sativus L. V. Stabilizing effect of glycine on leaf protoplasts. Plant Cell Rep. 4: 269-273.
• PASZKOWSKI J., SHILLITO R.D., SAUL M.W., MANDAK V., HOHN T., HOHN B., POTRYKUS J. (1984). Direct gene transfer to plants. EMBO J. 3: 27i7-2722.
• RIGGS C.D., BATES G.W. (1986). Stable transformation of tobacco by electroporation. Proc. Natl. Academ. Sci. USA 83: 5602-5606.
• SAUL M.W., SHILLITO R.D., NAGRUTIU I. (1988). Direct DNA transfer to protoplasts with and without electroporation. Plant Molecular Biology Manual A1: 1-16. Kluwer Academic Publishers, Dordrecht-Printed in Belgium.
• SHILLITO R.D., SAUL M.W., PASZKOWSKI J., MULLER M., POTRYKUS J. (1985). High efficiency direct gene transfer to plants. Biotechnology 3: 1099-1103.
• TAUTORUS T.E., BEKKAONI F., PILON M., DALTA R.S.S., CROSBY W.L., FOWKE L.C., DUNSTAN D.J. (1989). Factors affecting transient expression in electroporated black spruce (Picea mariana) and jack pine (Pinus bankstona) protoplasts. Theor. App. Genet. 78: 531-536.
• TRULSON A.J., SIMPSON R.B., SHAHIN E.A. (1986). Transformation of cucumber (Cucumis salivus L.) plants with Agrobacterium rhizogenes. Theor. Appl. Genet. 73: 11-15.
• WRÓBLEWSKI T., MALEPSZY S. (1992). In vitro culture of Cucumis sativus L. XVI. Established long-term culture. Proceedings - Fifth EUCARPIA Cucurbitaceae Symposium. July 27-31, Skierniewice, Poland: 104-107.