Plant Biotechnology

Electronic Journal of Biotechnology ISSN: 0717-3458 Vol. 11 No. 5, Special Issue, 2008
© 2008 by Pontificia Universidad Católica de Valparaíso -- Chile  
DOI: 10.2225/vol11-issue5-fulltext-2
SHORT COMMUNICATION

Introduction of antifungal genes in sunflower via Agrobacterium

Laura Mabel Radonic
Instituto de Biotecnología
INTA Castelar
De los Reseros y Nicolás Repetto s/n (1712), Castelar
Buenos Aires, Argentina
Tel: 54 11 4621 1278/1447
Fax: 54 11 4621 0199
E-mail: lradonic@cnia.inta.gov.ar

Julián Marcos Zimmermann#
Instituto de Biotecnología
INTA Castelar
De los Reseros y Nicolás Repetto s/n (1712), Castelar
Buenos Aires, Argentina
Tel: 54 3878 494492
Fax: 54 3878 493190
E-mail: jzimmerman@cnia.inta.gov.ar

Diego Zavallo
Instituto de Biotecnología
INTA Castelar
De los Reseros y Nicolás Repetto s/n (1712), Castelar
Buenos Aires, Argentina
Tel: 54 11 4621 1278/1447
Fax: 54 11 4621 0199
E-mail: dzavallo@cnia.inta.gov.ar

Nilda López
Instituto de Biotecnología
INTA Castelar
De los Reseros y Nicolás Repetto s/n (1712), Castelar
Buenos Aires, Argentina
Tel: 54 11 4621 1278/1447
Fax: 54 11 4621 0199
E-mail: nelopez@cnia.inta.gov.ar

Marisa López Bilbao*
Instituto de Biotecnología
INTA Castelar
De los Reseros y Nicolás Repetto s/n (1712), Castelar
Buenos Aires, Argentina
Tel: 54 11 4621 1278/1447
Fax: 54 11 4621 0199
E-mail: mlopezbilbao@cnia.inta.gov.ar

*Corresponding author

Financial support: National Agency of Scientific and Technological Promotion (ANPCyT), PICTO Nº 08-13164 and 08-12925, Argentina.

Keywords: antifungal genes, double expression cassettes, polyprotein cassettes, sunflower, transgenic plants.

#Present Address: Chacra Experimental Agrícola, PO box nº 8 (A4531WAA), Colonia Santa Rosa, Salta, Argentina. E-mail: jzimmermann@chacraexperimental.org

Abbreviations:

CTAB: Cetyltrimethylammonium bromide
DNA: deoxyribonucleic acid
Km: kanamycin
PCR: polymerase chain reaction
Rip: ribosome inhibitor protein
TEV: tobacco etch virus
TMV: tobacco mosaic virus

Abstract   Full Text

There is evidence that overexpression of transgenes codifying antifungal proteins may confer protection to pathogen attack, and that this protection is increased due to the synergic effect of the expression of two or more genes. On the other hand it is well known that sunflower is a recalcitrant specie, highly difficult to be genetically transformed. In this context, the final aim of this project was to obtain sunflower plants expressing at least two antifungal genes, avoiding sequential transformation. The antifungal genes used encode for two enzymes that degrade the fungal wall (glucanase and chitinase), an osmotin and a ribosome inhibitor protein. Two types of transformation vectors were used: a more traditional system with a double cassette and a novel system producing a unique polyprotein with antifungal proteins released in equimolecular quantities. The polyprotein vector system generated hyperhydric shoots with necrotic areas and abnormal growth at the end of the tissue culture procedure, making impossible the use of this interesting vector in sunflower. Transformation assays carried out with the pHGC39 vector (including glucanase and chitinase genes) vector produced 0.83% efficiency, corresponding to 13 rooted shoots in kanamycin (Km) from a total of 1568 agroinfected shoots. T0 rooted shoots resulted positive by PCR analysis and were transferred to greenhouse to obtain their offspring. In addition, we corroborate the transformation protocol using Km as selective marker, previously described (Radonic et al. 2006) with a reporter gene, but in this opportunity with antifungal genes.

Supported by UNESCO / MIRCEN network