Green fluorescent protein as a marker for expression of a second gene in transgenic plants

Abstract

The use of transgenic crops has generated concerns about transgene movement to unintended hosts and the associated ecological consequences. Moreover, the in-field monitoring of transgene expression is of practical concern (e.g., the underexpression of an herbicide tolerance gene in crop plants that are due to be sprayed with herbicide). A solution to these potential problems is to monitor the presence and expression of an agronomically important gene by linking it to a marker gene, such as GFP. Here we show that GFP fluorescence can indicate expression of the Bacillus thuringiensus cry1Ac gene when co-introduced into tobacco and oilseed rape, as demonstrated by insect bioassays and western blot analysis. Furthermore we conducted two seasons of field experiments to characterize the performance of three different GFP genes in transgenic tobacco. The best gene tested was mGFP5er, a mutagenized GFP gene that is targeted to the endoplasmic reticulum. We also demonstrated that host plants synthesizing GFP in the field suffered no fitness costs.

An orange fluorescent protein tagging system for real-time pollen tracking

“…GFP was visible in leaves, stems, and roots (data not shown) and OFP was visible in pollen under a microscope (Figure 5) with the aforementioned filter set. GFP, regulated by the CaMV 35S promoter, was not visible in pollen in accordance with previous findings [19,20]. Highly fluorescent individual plants from the most fluorescent N. glauca TD-GFP-K lines were crossed with highly fluorescent TN 90 TD-GFP-H lines to ensure hybrids had both antibiotic resistance genes and would fluoresce brightly, thereby facilitating detection.…”

An orange fluorescent protein tagging system for real-time pollen tracking

“…GFP was visible in leaves, stems, and roots (data not shown) and OFP was visible in pollen under a microscope (Figure 5) with the aforementioned filter set. GFP, regulated by the CaMV 35S promoter, was not visible in pollen in accordance with previous findings [19,20]. Highly fluorescent individual plants from the most fluorescent N. glauca TD-GFP-K lines were crossed with highly fluorescent TN 90 TD-GFP-H lines to ensure hybrids had both antibiotic resistance genes and would fluoresce brightly, thereby facilitating detection.…”

Bt-transgenic oilseed rape hybridization with its weedy relative,Brassica rapa

“…These results reinforce the evidence that gene flow readily occurs from transgenic oilseed rape to its weedy relative, B. rapa (Harper et al, 1999;Halfhill et al, 2001;Metz et al, 1997;Snow et al, 1999). Furthermore, the resultant hybrids synthesize the Bt Cry1Ac protein at similar levels to the parental oilseed rape lines.…”

Spectroscopic investigations on plant responses to growth environment

“…Our results compared very well with previous reports that the RD21 gene in A. thaliana was induced by dehydration and high salt environmental stress conditions, whereby the cognate mRNA transcripts were detected after 1.5 h and accumulated steadily up to 24 h (Yamaguchi- Shinozaki, Koizumi et al 1992). This pattern of promoter activity in a heterologous environment was not surprising, as stress responses in plants are thought to be mediated via universal signaling pathways (Harper, Mabon et al 1999;Hu and Cheng 1995;Naylor 1999).…”

An orange fluorescent protein tagging system for real-time pollen tracking

“…GFP was visible in leaves, stems, and roots (data not shown) and OFP was visible in pollen under a microscope (Figure 5) with the aforementioned filter set. GFP, regulated by the CaMV 35S promoter, was not visible in pollen in accordance with previous findings [19,20]. Highly fluorescent individual plants from the most fluorescent N. glauca TD-GFP-K lines were crossed with highly fluorescent TN 90 TD-GFP-H lines to ensure hybrids had both antibiotic resistance genes and would fluoresce brightly, thereby facilitating detection.…”

Bt-transgenic oilseed rape hybridization with its weedy relative,Brassica rapa

“…These results reinforce the evidence that gene flow readily occurs from transgenic oilseed rape to its weedy relative, B. rapa (Harper et al, 1999;Halfhill et al, 2001;Metz et al, 1997;Snow et al, 1999). Furthermore, the resultant hybrids synthesize the Bt Cry1Ac protein at similar levels to the parental oilseed rape lines.…”

Spectroscopic investigations on plant responses to growth environment

“…Our results compared very well with previous reports that the RD21 gene in A. thaliana was induced by dehydration and high salt environmental stress conditions, whereby the cognate mRNA transcripts were detected after 1.5 h and accumulated steadily up to 24 h (Yamaguchi- Shinozaki, Koizumi et al 1992). This pattern of promoter activity in a heterologous environment was not surprising, as stress responses in plants are thought to be mediated via universal signaling pathways (Harper, Mabon et al 1999;Hu and Cheng 1995;Naylor 1999).…”

An orange fluorescent protein tagging system for real-time pollen tracking

“…GFP was visible in leaves, stems, and roots (data not shown) and OFP was visible in pollen under a microscope (Figure 5) with the aforementioned filter set. GFP, regulated by the CaMV 35S promoter, was not visible in pollen in accordance with previous findings [19,20]. Highly fluorescent individual plants from the most fluorescent N. glauca TD-GFP-K lines were crossed with highly fluorescent TN 90 TD-GFP-H lines to ensure hybrids had both antibiotic resistance genes and would fluoresce brightly, thereby facilitating detection.…”

Bt-transgenic oilseed rape hybridization with its weedy relative,Brassica rapa

“…These results reinforce the evidence that gene flow readily occurs from transgenic oilseed rape to its weedy relative, B. rapa (Harper et al, 1999;Halfhill et al, 2001;Metz et al, 1997;Snow et al, 1999). Furthermore, the resultant hybrids synthesize the Bt Cry1Ac protein at similar levels to the parental oilseed rape lines.…”

Spectroscopic investigations on plant responses to growth environment

“…Our results compared very well with previous reports that the RD21 gene in A. thaliana was induced by dehydration and high salt environmental stress conditions, whereby the cognate mRNA transcripts were detected after 1.5 h and accumulated steadily up to 24 h (Yamaguchi- Shinozaki, Koizumi et al 1992). This pattern of promoter activity in a heterologous environment was not surprising, as stress responses in plants are thought to be mediated via universal signaling pathways (Harper, Mabon et al 1999;Hu and Cheng 1995;Naylor 1999).…”