Anthocyanin expression in marker free transgenic wheat and triticale embryos

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 T 0 lines was performed by polymerase chain reaction (PCR), and further confirmation of PCR positives was done using realtime 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 T 1 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.

Analysis of T-DNA integration and generative segregation in transgenic winter triticale (x Triticosecale Wittmack)

“…A similar approach targeting haploid embryo-like structures resulted in only the transient expression of the transgene and no adult plants were regenerated [9]. A first applicative approach was published by Doshi et al (2007) who used the embryo-specific LTP1 promoter to show the effect of the C1 and Bperu maize genes on anthocyanin biosynthesis in triticale [10]. …”

Use of Quantitative Polymerase Chain Reaction for Determining Copy Numbers of Transgenes in <i>Lesquerella fendleri</i>

“…To date, qPCR technology has been applied to analyze copy number of transgenic plants, including soybean and peanut (Schmidt and Parrott, 2001), tomato (Mason et al, 2002), maize (Ingham et al, 2001;Shou et al, 2004;Song et al, 2002), rapeseed (Weng et al, 2004), wheat (Doshi et al, 2007;Li et al, 2004), rice (Jiang et al, 2009;Yang et al, 2005), tobacco (Subr et al, 2006), cotton (Yi et al, 2008), citrus (Omar et al, 2008), grape (Costa et al, 2009) and cassava (Beltran et al, 2009). To speed up the molecular analysis of transgenic plants, we describe here the development and application of a qPCR method that utilizes an endogenous calibrator and the threshold crossing point (Ct) calculated by the Applied Biosystem 7500 system for determination of transgene copy numbers in L. fendleri.…”

Evaluation of Clostridium ljungdahlii DSM 13528 reference genes in gene expression studies by qRT-PCR

“…Currently, qRT-PCR is one of the most widely used methods of gene expression analysis in a number of biological systems, such as bacteria (9,23) and plants (24,25). It is essential to select stable reference genes to ensure the accuracy of this technology.…”