Articles
Production of marker-free cisgenic apple plants using inducible site-specific recombinase and a bifunctional selectable gene
Article number
1261_24
Pages
149 – 156
Language
English
Abstract
The presence of marker genes, especially for antibiotic resistance, in genetically modified plants is of concern to our society due to fears associated with risks for the environment and human health.
The creation of transgenic plants that do not contain foreign genetic material, especially of bacterial and viral origin, largely alleviates this tension.
In our research we used the pMF system containing the Zygosaccharomyces rouxii recombinaseR and a CodA-nptII bifunctional selectable gene for producing marker-free transgenic apple plants carrying the super sweet thaumatin II gene from the tropical plant Thaumatococcus daniellii under control of E8 gene fruit specific promoter and rbsS3A terminator.
We have obtained three independent transgenic lines that have been thoroughly analysed by PCR for the presence of T-DNA sequences.
Two of them contained a partial sequence of the T-DNA, recombination site (RS) near the left border was missed.
We then used a delayed strategy for the selection of marker-free plants with one checked line.
After induction of recombinase activity in leaf explants we have obtained on selective media with 5-fluorocytosine more than 30 sublines, most of them lost their resistance to kanamycin.
PCR analysis revealed that all undesirable genes and sequences between RS sites were removed by the recombination process while genes of interest with regulatory elements were present in all obtained plants.
So, using a vector based on an pMF system we developed an acceptable protocol for the production of marker-free cisgenic apple plants.
The creation of transgenic plants that do not contain foreign genetic material, especially of bacterial and viral origin, largely alleviates this tension.
In our research we used the pMF system containing the Zygosaccharomyces rouxii recombinaseR and a CodA-nptII bifunctional selectable gene for producing marker-free transgenic apple plants carrying the super sweet thaumatin II gene from the tropical plant Thaumatococcus daniellii under control of E8 gene fruit specific promoter and rbsS3A terminator.
We have obtained three independent transgenic lines that have been thoroughly analysed by PCR for the presence of T-DNA sequences.
Two of them contained a partial sequence of the T-DNA, recombination site (RS) near the left border was missed.
We then used a delayed strategy for the selection of marker-free plants with one checked line.
After induction of recombinase activity in leaf explants we have obtained on selective media with 5-fluorocytosine more than 30 sublines, most of them lost their resistance to kanamycin.
PCR analysis revealed that all undesirable genes and sequences between RS sites were removed by the recombination process while genes of interest with regulatory elements were present in all obtained plants.
So, using a vector based on an pMF system we developed an acceptable protocol for the production of marker-free cisgenic apple plants.
Publication
Authors
V.R. Timerbaev, T.Y. Mitiouchkina, S.V. Dolgov
Keywords
marker-free plants, apple, Malus × domestica, thaumatin II, CodA, R/RS recombination system
Groups involved
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