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3 October 2007 Anthocyanin expression in marker free transgenic wheat and triticale embryos
K. M. Doshi, F. Eudes, A. Laroche, D. Gaudet
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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.

K. M. Doshi, F. Eudes, A. Laroche, and D. Gaudet "Anthocyanin expression in marker free transgenic wheat and triticale embryos," In Vitro Cellular and Developmental Biology - Plant 43(5), 429-435, (3 October 2007). https://doi.org/10.1007/s11627-007-9089-7
Received: 15 August 2007; Accepted: 23 August 2007; Published: 3 October 2007
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KEYWORDS
Embryo-specific promoter
Identity preservation
qRT-PCR
Stable transformation
SYBR green method
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