Anthocyanin expression in marker free transgenic wheat and triticale embryos |
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Authors: | K M Doshi F Eudes A Laroche D Gaudet |
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Institution: | (1) Agriculture and Agri-Food Canada, Lethbridge Research Centre, P.O. Box 3000, Lethbridge, AB, Canada, T1J 4B1 |
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Abstract: | Wheat and triticale plants were transformed by bombardment of isolated scutella with a genetic construct consisting of the
two anthocyanin biosynthesis regulatory genes, C1 and Bperu, each under the control of the Ltp1 embryo-specific promoter. Transgenic plants were obtained in the absence of selective pressure and selectable marker gene
at a transformation frequency of 0.93% and 1.55% in triticale and wheat, respectively. Initial screening of T0 lines was performed by polymerase chain reaction (PCR), and further confirmation of PCR positives was done using real-time
PCR and by phenotypic observation. In this study, quantitative real-time PCR (qRT-PCR) was developed to determine the transgene
copy number in transgenic wheat and triticale. A conserved wheat housekeeping gene, puroindoline-b, was used as an internal control to calculate the transgene copy number in wheat and the SYBR green detection method with
a standard curve, constructed on the basis of serially diluted plasmid, was used to calculate the transgene copy in triticale.
Estimated transgene copies varied from 3 to 8 in wheat and 4 to 7 in triticale lines. The presence of anthocyanin regulatory
genes, promoter, and termination sequences was detected in six wheat lines and four triticale lines. However, anthocyanin-pigmented
embryos were only observed visually in mature T1 seeds of two transgenic wheat lines and a single triticale line. Multisite insertion and reorganization of transgenes was
likely the explanation for the failure of expression for the anthocyanin genes in the remaining wheat and triticale transgenic
lines. |
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Keywords: | Embryo-specific promoter Identity preservation qRT-PCR Stable transformation SYBR green method |
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