Isopentenyl transferase gene expression offers the positive selection of marker-free transgenic plant of Kalanchoe blossfeldiana

Abstract

The technologies allowing the production of transgenic plants without selectable marker genes, is of great interest in public and environmental safety. For generating such marker-free transgenic plants, possibility has been offered by Multi-Auto-Transformation [MAT] vector system, which combines positive selection, using the isopentenyl transferase (ipt) gene, with a site-specific recombination that generates marker-free plants. In this study Agrobacterium tumefaciens strain EHA105 harboring an ipt-type MAT vector, pMAT21, containing lacZ, gus genes and the removable cassette in the T-DNA region was used to produce marker-free transgenic Kalanchoe blossfeldiana Poelln., employing ipt gene as the selectable marker gene. Co-cultivated explants were cultured on hormone-and selective agent-free MS medium, and 85% of the regenerated shoots showed ipt-shooty phenotype with GUS expression. Forty-one morphologically normal shoots were produced during the subculture. More than ninety percent of the normal shoots were ipt -, gus -but lacZ ? as determined by PCR analyses. These results indicate that the ipt phenotype was clearly distinguishable from non-transgenic as well as transgenic marker-free shoots. This study opens interesting perspective for the generation of marker-free transgenic K. blossfeldiana with objective useful transgene.

Increased resistance to fusarium wilt in transgenic tobacco lines co‐expressing chitinase and wasabi defensin genes

“…The PCR product was expected to be 0·8 kb for the ipt gene, 0·7 kb for the npt II gene, a 0·9‐kb fragment for the ChiC and 0·5 kb for wasabi gene (Khan et al. , 2006b, 2008; Thirukkumaran et al. , 2009).…”

“…PCR amplification was performed using genomic DNA of each plant as a target and oligonucleotide primers for ipt, ChiC, nptII and WD. The PCR product was expected to be 0AE8 kb for the ipt gene, 0AE7 kb for the nptII gene, a 0AE9-kb fragment for the ChiC and 0AE5 kb for wasabi gene (Khan et al, 2006b(Khan et al, , 2008Thirukkumaran et al, 2009).…”

Generation of phenotypically normal marker-free transgenic plants of Kalanchoe blossfeldiana through hairy root induction

“…This may be due to the silencing of the rol gene. In our previous study on the production of marker-free transgenic K. blossfeldiana plants by using ipt-type MAT vector, we observed some non-transgenic normal shoots from ipt-shooty line (Thirukkumaran et al 2009). This may indicate that the non-transgenic shoots were generated from the cells, which obtained excessive cytokinin from the adjacent transgenic cells.…”

“…Therefore, Kalanchoe species can be considered as model plants for conducting biotechnological studies particularly for generation of transgenic plants in ornamental species. In our previous study (Thirukkumaran et al 2009) we used ipt-type MAT vector, pMAT21, for the production of phenotypically normal marker-free transgenic Kalanchoe blossfeldiana Poelln.. However, a number of escapes have been found using ipt gene as the selection marker.…”

Rol (root loci) gene as a positive selection marker to produce marker-free Petunia hybrida

“…This system does not need any antibiotic or herbicide resistance genes as selection markers. Ipt-type MAT vector system has been evaluated in tobacco (Ebinuma et al 1997a, b;Sugita et al 2000), hybrid aspens (Matsunaga et al 2002), rice (Endo et al 2002), Nierembergia (Khan et al 2006), white poplar (Zelasco et al 2007), Citrus (Ballester et al 2007), Cassava (Saelim et al 2009), Medicago truncatula (Scaramelli et al 2009) and Kalanchoe blossfeldiana (Thirukkumaran et al 2009). However, rol-type MAT vector has been tried only in a few plant species; Antirrhinum majus (Cui et al 2000(Cui et al , 2001, tobacco and white poplar (Zelasco et al 2007), and needs to be evaluated in other plant species.…”