An orange fluorescent protein tagging system for real-time pollen tracking
© Rice et al.; licensee BioMed Central Ltd. 2013
Received: 30 April 2013
Accepted: 24 September 2013
Published: 27 September 2013
Monitoring gene flow could be important for future transgenic crops, such as those producing plant-made-pharmaceuticals (PMPs) in open field production. A Nicotiana hybrid (Nicotiana. tabacum × Nicotiana glauca) shows limited male fertility and could be used as a bioconfined PMP platform. Effective assessment of gene flow from these plants is augmented with methods that utilize fluorescent proteins for transgenic pollen identification.
We report the generation of a pollen tagging system utilizing an orange fluorescent protein to monitor pollen flow and as a visual assessment of transgene zygosity of the parent plant. This system was created to generate a tagged Nicotiana hybrid that could be used for the incidence of gene flow. Nicotiana tabacum ‘TN 90’ and Nicotiana glauca were successfully transformed via Agrobacterium tumefaciens to express the orange fluorescent protein gene, tdTomato-ER, in pollen and a green fluorescent protein gene, mgfp5-er, was expressed in vegetative structures of the plant. Hybrids were created that utilized the fluorescent proteins as a research tool for monitoring pollen movement and gene flow. Manual greenhouse crosses were used to assess hybrid sexual compatibility with N. tabacum, resulting in seed formation from hybrid pollination in 2% of crosses, which yielded non-viable seed. Pollen transfer to the hybrid formed seed in 19% of crosses and 10 out of 12 viable progeny showed GFP expression.
The orange fluorescent protein is visible when expressed in the pollen of N. glauca, N. tabacum, and the Nicotiana hybrid, although hybrid pollen did not appear as bright as the parent lines. The hybrid plants, which show limited ability to outcross, could provide bioconfinement with the benefit of detectable pollen using this system. Fluorescent protein-tagging could be a valuable tool for breeding and in vivo ecological monitoring.
Increased use of transgenic crops has prompted the necessity of monitoring transgene flow in agroecological systems. Previous investigations have ascertained the utility of gene flow tracking with fluorescent proteins (FPs) [1–4]. These studies have shown that green fluorescent protein (GFP) is an effective tool for the purpose of gene flow tracking and can be targeted to various organs and tissues within plants, including pollen. This technology, in effect, could be used in an environmental monitoring system, one of the many uses of FPs in plants . One drawback of using native GFP as a marker in plants is the signal-to-noise ratio at GFP’s maximum excitation wavelength of 395 nm, often resulting in autofluorescence of plant tissue components . Fluorescent proteins emitting in the red/orange spectrum that require longer wavelengths for excitation have lower levels of autoflorescence in plant tissues compared to blue or UV light . One such widely used orange fluorescent protein (OFP), DsRed, is derived from Discosoma sp. its mutant variants have higher extinction coefficients and quantum yields . Coral-derived FPs should be useful for monitoring gene flow.
Nicotiana tabacum (tobacco) and Brassica napus (canola) plants have been transformed to synthesize GFP in pollen, using pollen-specific promoters [1, 4]. Long-range pollen tracking was conducted in canola species to assay pollen movement in real time (e.g. immediate detection of tagged pollen) using traps at various distances within field and greenhouse experiments. This method is quicker and less laborious for determining pollen flow than analyzing progeny from recipient plants (e.g. antibiotic screening, PCR, FP screening) . Drawing upon this previous body of work, it is logical to conceptualize a method to determine bioconfinement efficacy using FP tagging utilizing an improved fluorescent protein for plants.
The Nicotiana hybrid (Nicotiana tabacum × Nicotiana glauca), is highly sterile and prompted a further examination of bioconfinement through gene flow monitoring. Recently, we have shown that GFP tagging in vegetative plant tissues of this hybrid allows for gene tracking and assists with sterility assessments . Here we describe a modified system to tag pollen that is applicable to a real-time assay of pollen flow from FP-tagged plants. Our goal was to engineer each Nicotiana species for pollen-specific expression of an OFP gene that also had vegetative tissues that expressed a GFP gene. The transgenic plants could then be crossed to obtain interspecific hybrid Nicotiana s that had FP genes contributed from each parent. To achieve this goal, parent plants N. tabacum ‘TN 90’ and N. glauca were Agrobacterium-mediated transformed to synthesize the OFP tdTomato-ER in the pollen and bred to homozygosity, and then crossed to create the transgenic interspecific hybrid. Manual greenhouse crosses were performed to assess sexual compatibility and functionality of the system.
N. tabacum ‘TN 90’ used for transformation was from foundation seed lot # 86-02-K-4A, N. tabacum ‘MS TN 90’ from foundation seed lot # 86-03-KLC-15 is a male sterile variety of TN 90 that was used as a pollen recipient plant in crosses. N. tabacum ‘SN 2108’’, a morphologically distinct variety from the TN 90 cultivar used as a pollen donor in greenhouse crosses, is an experimental line developed into ‘KT D4’; all N. tabacum were obtained from the Kentucky Tobacco Seed Improvement Association, Inc. in Lexington, KY, USA. (38°8’N, 84°29’W). N. glauca used for transformation was from the US National Plant Germplasm System (plant introduction 307908, accession TW55 from Peru).
Generation of transgenic plants
Hybrid Nicotiana production
Plants were bred to obtain lines that had both constructs for a complete tracking of pollen. To ensure multiple transgene copies were stacked into the hybrid, our goal was to produce hybrids containing one TD-GFP-K and one TD-GFP-H construct, using dual antibiotic screening to ensure to select hybrids that were transgenic for each construct.
Fluoresence measurements and observations
Transgenic line selection
With the assumption of a single insertion event, transgene zygosity was estimated using epifluorescent microscopy. Plants with 100% fluorescent pollen (deemed homozygous) were bagged and self-fertilized as previously described. In addition to the FP pollen assay, we used progeny assays to assure that we selected homozygosity of each T2 line. Germinated seed were screened with a handheld UV light to determine zygosity of each T2 line (using ratios of GFP to non-GFP plants). Seeds of each T2 line were also screened for inheritance of antibiotic resistance genes by germination on MS media  containing kanamycin (200 mg/L) or hygromycin (50 mg/L) wherein observation of plant health allowed for determination of segregation; 100% survival indicated a homozygous line for selection. The selected transgenic T2 lines, TN 90 TD-GFP-H and N. glauca TD-GFP-K, were crossed (TN 90 TD-GFP-H × N. glauca TD-GFP-K). Hybrid lines derived from parents lines transformed with the TD-GFP-K/TD-GFP-H constructs were named ‘Hybrid OFP’ plants. These hybrid seeds were germinated on MSO media containing both kanamycin (200 mg/L) and hygromycin (50 mg/L) to ensure both constructs were integrated into the hybrid genome.
Fertility assessment in hybrids
Greenhouse crosses performed with hybrid OFP plants
Crosses forming seed
Total seed count
Detectable gene flow
(MS TN 90 × hybrid OFP)
(hybrid OFP × SN 2108)
All analysis of variance (ANOVA) routines were performed using SAS (Version 9.3 SAS Institute Inc, Cary, NC, USA) using the MIXED procedure with a significance level of p < 0.05. When ANOVA results were found to be statistically significant, the least significant differences were used for mean separations.
Results and discussion
Transformation of N. tabacum ’TN 90’ and N. glauca were successful except for N. glauca TD-GFP-H where multiple attempts failed to produce hygromycin-resistant plants. 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. Since we selected plants on the basis of green-fluorescence shoots, it is not surprising that pollen orange fluorescence was also bright in these lines. Hybrid OFP lines were 100% resistant to kanamycin and hygromycin when screened on MSO media containing both antibiotics (data not shown), indicating inclusion of both cassettes into the F1 hybrids.
Manual plant crosses revealed that the hybrids were able to backcross to a non-transgenic male sterile N. tabacum ‘MS TN 90’ (Table 1), forming entirely non-viable seed in 2% of the crosses (98% of the crosses produced no seed), thus restricting detectable transgene transmission rates in progeny to 0%. This result was in contrast to our previous findings where few viable seeds (2 out of 445 seeds from 96 crosses) were generated from a similar (MS TN 90 × hybrid) cross that employed a different construct using mgfp5-er. In addition, we have determined that male fertility varies among hybrid lines from 0 to 3% . When the fertile line, SN 2108, was used to pollinate hybrid OFP plants, limited seed set (19% of crosses) was observed. Only 10 germinated seedlings out of 12 expressed GFP, (83% detectable transgene transmission), indicating that transgenes might be segregating out of some hybrid OFP × SN 2108 progeny.
It was unknown if tdTomato-ER would be visible in the pollen of the Nicotiana hybrid as the plant largely produces immature pollen where many pollen mother cells cease to develop past the tetrad stage . Many of the immature pollen grains apparently did not synthesize sufficient tdTomato-ER for visual detection. The FP was only obvious in larger, more mature hybrid OFP pollen and did not appear to fluoresce as brightly as TN 90 TD-GFP-K and N. glauca TD-GFP-H. The pollen-specific promoter LAT52, regulates gene expression during microspore mitosis, allowing transcription until anthesis [14, 22]. Our observation of few mature fluorescent pollen grains produced in the hybrids demonstrates that the interspecific hybrid system could be a viable candidate for transgene bioconfinement.
A bright orange fluorescent protein, tdTomato-ER, can be synthesized in pollen when its gene is under the control of the LAT52 pollen promoter. Fluorescently-tagged pollen is highly distinguishable from non-tagged pollen, and shows low autofluorescence. The plants produced in this study further increase the number of tools available for gene flow studies. Crossing studies demonstrated that hybrid OFP plants had low fertility and provided bioconfinement by limiting successful crosses made to the maternal line, N. tabacum. As pollen tracking is possible with this fluorescently tagged hybrid, more research is needed to determine the efficacy of pollen detection with this system and how it relates to bioconfinement in a field setting.
The authors wish to thank Charleson Poovaiah and Christy Rose for assistance in plant transformation experiments and Justin McDuffie for assistance with data collection. This study was funded by the NIFA Biotechnology Risk Assessment Grant Program and all research was conducted in compliance of USDA-APHIS-BRS regulations.
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