Breeding

Electronic Journal of Biotechnology ISSN: 0717-3458 Vol. 9 No. 3, Issue of April 15, 2006
© 2006 by Pontificia Universidad Católica de Valparaíso -- Chile  
DOI: 10.2225/vol9-issue3-fulltext-19  
RESEARCH ARTICLE

Rooting in Km selective media as efficient in vitro selection method for sunflower genetic transformation

Laura Mabel Radonic
Instituto de Biotecnología
INTA Castelar
De los Reseros y Las Cabañas s/n (1712), Castelar
Buenos Aires, Argentina
E-mail: lradonic@cicv.inta.gov.ar

Julián Marcos Zimmermann
Instituto de Biotecnología
INTA Castelar
De los Reseros y Las Cabañas s/n (1712), Castelar
Buenos Aires, Argentina
E-mail: jzimmerman@cicv.inta.gov.ar

Diego Zavallo
Instituto de Biotecnología
INTA Castelar
De los Reseros y Las Cabañas s/n (1712), Castelar
Buenos Aires, Argentina
E-mail: dzavallo@cicv.inta.gov.ar

 Nilda López
Instituto de Biotecnología
INTA Castelar
De los Reseros y Las Cabañas s/n (1712), Castelar
Buenos Aires, Argentina
E-mail: nlopez@cicv.inta.gov.ar

 Marisa López Bilbao*
Instituto de Biotecnología
INTA Castelar
De los Reseros y Las Cabañas s/n (1712), Castelar
Buenos Aires, Argentina
E-mail: mlopezbilbao@cicv.inta.gov.ar

*Corresponding author

Financial support: This project was supported by the Agencia Nacional de Promoción Científica y Tecnológica (PICTO Nº 08-13164 and 08-12925), Argentina.

Keywords: genetic transformation, kanamycin, selection, sunflower.

Abbreviations:

A: agroinfected
AIM: Agrobacterium inducing media
BAP: benzyl amino purine
C: controls
CC: co culture
CTAB: Cetyltrimethylammonium bromide
Km: kanamycin
MS: Murashige-Skoog media
NAA: naphthalene acetic acid
OD: optical density
PCR: polymerase chain reaction
Re: Regenerating media
Tmt: timentine


Abstract   Reprint (PDF)

Despite of numerous publications in sunflower genetic transformation, there is no efficient or reproducible protocol with low number of escapes. The latter would indicate that the selection method is not effective. In this work we used Km as selective agent, Agrobacterium tumefaciens EHA105 strain and a vector with the nptII gene under the nos promoter and uidA gene under 35S promoter. The response of agroinfected (A) and control (C) explants during the in vitro culture was studied and in both cases in presence or absence of Km in order to assign a differential morphologic response between transformed and non-transformed plants. The characteristics analyzed were: height, colour/aspect of the plantlets, in vitro rooting and in vitro bud-flower development. Selection was applied from the third regeneration media. Among the A plantlets two were capable of rooting, being positive by PCR, whereas the C were unable to root in presence of Km. One of them gave 6 seeds and in these plants, it was determined the presence of the transgene by PCR and GUS staining. This work shows that in Km selection, colour/aspect of shoots is not useful as selection criteria whereas rooting is an effective selection method in which no escapes were obtained. 

 
Supported by UNESCO / MIRCEN network 
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