CREATING MARKER-FREE PLANTS BY COMBINING SITE-SPECIFIC RECOMBINATION AND THE DAO1 GENE THAT ALLOWS BOTH POSITIVE AND NEGATIVE SELECTION

R.C. García-Almodóvar, C. Petri, I.M.G. Padilla , L. Burgos
The gene dao1 from the yeast Rhodotorula gracilis encodes a D-amino acid oxidase (DAAO) that catalyses stereospecifically the oxidative deamination of D-amino acids (Alonso et al., 1998). It has been successfully used to transform Arabidopsis plants by using D-amino acids as selective agents (Erikson et al., 2004). Site specific recombination has allowed the production of marker-free transgenic plants. The Cre-loxP system in the plasmid pX6-GFP (Zuo et al., 2001) uses the recombinase Cre, under the control of an inducible promoter tightly regulated by β-estradiol, to remove marker genes between the loxP sites. We have replaced the nptII gene, for antibiotic resistance, with the dao1 gene within the pX6-GFP vector. Transgenic tobacco plants were efficiently produced, by positive selection of dao1 gene, including 6 mM D-alanine in the regeneration medium. Plants with different number of transgene insertions were selected and T1 and T2 generations produced by self-pollination. Excision of marker genes was induced both in leaves from transgenic lines and in homozygous T2 seeds in the presence of β-estradiol, by negative selection of dao1 gene, in selective medium containing 6 mM or 35 mM D-valine, respectively. Selection of marker-free transgenic plants using the pX6-GFP vector relied on gfp expression, after excision occurred, and may be difficult in plants where chlorophyll autofluorescence masks marker expression. The dao1 gene helps to overcome this problem allowing the selection of the marker-free plants more efficiently and independently of marker genes expression.
García-Almodóvar, R.C., Petri, C., Padilla , I.M.G. and Burgos, L. (2012). CREATING MARKER-FREE PLANTS BY COMBINING SITE-SPECIFIC RECOMBINATION AND THE DAO1 GENE THAT ALLOWS BOTH POSITIVE AND NEGATIVE SELECTION. Acta Hortic. 929, 429-435
DOI: 10.17660/ActaHortic.2012.929.62
https://doi.org/10.17660/ActaHortic.2012.929.62
D-amino acids, Cre-loxP, marker excision
English

Acta Horticulturae