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Assessing the bioconfinement potential of a Nicotiana hybrid platform for use in plant molecular farming applications.

Rice JH, Mundell RE, Millwood RJ, Chambers OD, Stewart CN, Davies HM - BMC Biotechnol. (2013)

Bottom Line: The introduction of pharmaceutical traits in tobacco for commercial production could benefit from the utilization of a transgene bioconfinement system.GFP was used as a useful proxy for pharmaceutical transgenes.In two field experiments, one each in Tennessee and Kentucky, we detected outcrossing at only one location (Tennessee) at 1.4%.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plant Sciences, University of Tennessee, Knoxville, TN 37996, USA.

ABSTRACT

Background: The introduction of pharmaceutical traits in tobacco for commercial production could benefit from the utilization of a transgene bioconfinement system. It has been observed that interspecific F1Nicotiana hybrids (Nicotiana tabacum × Nicotiana glauca) are sterile and thus proposed that hybrids could be suitable bioconfined hosts for biomanufacturing. We genetically tagged hybrids with green fluorescent protein (GFP), which was used as a visual marker to enable gene flow tracking and quantification for field and greenhouse studies. GFP was used as a useful proxy for pharmaceutical transgenes.

Results: Analysis of DNA content revealed significant genomic downsizing of the hybrid relative to that of N. tabacum. Hybrid pollen was capable of germination in vitro, albeit with a very low frequency and with significant differences between plants. In two field experiments, one each in Tennessee and Kentucky, we detected outcrossing at only one location (Tennessee) at 1.4%. Additionally, from 50 hybrid plants at each field site, formation of 84 and 16 seed was observed, respectively. Similar conclusions about hybrid fertility were drawn from greenhouse crosses. In terms of above-ground biomass, the hybrid yield was not significantly different than that of N. tabacum in the field.

Conclusion: N. tabacum × N. glauca hybrids show potential to contribute to a bioconfinement- and biomanufacturing host system. Hybrids exhibit extremely low fertility with no difference of green biomass yields relative to N. tabacum. In addition, hybrids are morphologically distinguishable from tobacco allowing for identity preservation. This hybrid system for biomanufacturing would optimally be used where N. glauca is not present and in physical isolation of N. tabacum production to provide total bioconfinement.

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Design of field gene flow experiment. A modified Nelder wheel design was used evaluate the gene flow of hybrid GFP plants. Three plant types were used in the experiment: male sterile N. tabacum ‘MS TN 90’ were pollen ‘receptor’ plants, hybrid GFP was fluorescently tagged to enable gene flow tracking, and N. tabacum type SN 2108 was used as a pollen donor to assure pollen flow was occurring in the field by seed set on MS TN 90 and to test for female sterility of the hybrid GFP plants. A center pollen source patch contained 50 alternating hybrid GFP and fertile SN 2108 plants, spaced approximately 1 m apart. Sixteen 1 m2 blocks of male sterile MS TN 90 pollen receptor plants were placed at 9, 23, 38, and 54 m distances from the center and were used to detect pollen via seed formation. Each MS TN 90 plot was 22.5° relative to the adjacent plot as viewed from the center. A honeybee hive was placed at the center of the field site to vector pollen.
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Figure 1: Design of field gene flow experiment. A modified Nelder wheel design was used evaluate the gene flow of hybrid GFP plants. Three plant types were used in the experiment: male sterile N. tabacum ‘MS TN 90’ were pollen ‘receptor’ plants, hybrid GFP was fluorescently tagged to enable gene flow tracking, and N. tabacum type SN 2108 was used as a pollen donor to assure pollen flow was occurring in the field by seed set on MS TN 90 and to test for female sterility of the hybrid GFP plants. A center pollen source patch contained 50 alternating hybrid GFP and fertile SN 2108 plants, spaced approximately 1 m apart. Sixteen 1 m2 blocks of male sterile MS TN 90 pollen receptor plants were placed at 9, 23, 38, and 54 m distances from the center and were used to detect pollen via seed formation. Each MS TN 90 plot was 22.5° relative to the adjacent plot as viewed from the center. A honeybee hive was placed at the center of the field site to vector pollen.

Mentions: Natural outcrossing rates of fluorescently tagged hybrids were estimated in two field experiments conducted at Versailles, Kentucky, USA (38.075784, -84.740575) (KY) and Knoxville, Tennessee, USA (35.891769, -83.959786) (TN). A modified Nelder wheel design (Figure 1) [29] covered approximately 0.931 hectares and contained three plant types. GFP-tagged hybrids and non-transgenic SN 2108 plants were used as pollen donors in the center of the plot, and located along the spokes of the Nelder wheel MS TN 90 plants were used as pollen recipients (Figure 1). The pollen source plot measured approximately 15 m in diameter and contained 3 concentric circles consisting of 50 plants each of alternating hybrid GFP and SN 2108 spaced approximately 1 m apart; a honeybee hive was located at the center of the experiment to ensure pollinator presence. Honeybees were observed on plot flowers. Surrounding this central plot were sixteen blocks of five MS TN 90 plants, each used to detect outcrossing at 9, 23, 38, and 54 m from the center plot in each cardinal direction. Each block of MS TN 90 plants were 22.5° relative to the adjacent block with respect to the center plot in contrast to Nelder’s design where the outer plots are arranged in a linear fashion. This modification was made to take advantage of honeybee flight patterns [30], for flight to and from the center patch. Blocks of MS TN 90 plants and hybrid GFP flowers were monitored throughout the season for formation of pods, which were promptly collected at maturity. SN 2108 plants were used solely as a pollen source and seed set was not of interest because of high self-fertilization rates.


Assessing the bioconfinement potential of a Nicotiana hybrid platform for use in plant molecular farming applications.

Rice JH, Mundell RE, Millwood RJ, Chambers OD, Stewart CN, Davies HM - BMC Biotechnol. (2013)

Design of field gene flow experiment. A modified Nelder wheel design was used evaluate the gene flow of hybrid GFP plants. Three plant types were used in the experiment: male sterile N. tabacum ‘MS TN 90’ were pollen ‘receptor’ plants, hybrid GFP was fluorescently tagged to enable gene flow tracking, and N. tabacum type SN 2108 was used as a pollen donor to assure pollen flow was occurring in the field by seed set on MS TN 90 and to test for female sterility of the hybrid GFP plants. A center pollen source patch contained 50 alternating hybrid GFP and fertile SN 2108 plants, spaced approximately 1 m apart. Sixteen 1 m2 blocks of male sterile MS TN 90 pollen receptor plants were placed at 9, 23, 38, and 54 m distances from the center and were used to detect pollen via seed formation. Each MS TN 90 plot was 22.5° relative to the adjacent plot as viewed from the center. A honeybee hive was placed at the center of the field site to vector pollen.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750662&req=5

Figure 1: Design of field gene flow experiment. A modified Nelder wheel design was used evaluate the gene flow of hybrid GFP plants. Three plant types were used in the experiment: male sterile N. tabacum ‘MS TN 90’ were pollen ‘receptor’ plants, hybrid GFP was fluorescently tagged to enable gene flow tracking, and N. tabacum type SN 2108 was used as a pollen donor to assure pollen flow was occurring in the field by seed set on MS TN 90 and to test for female sterility of the hybrid GFP plants. A center pollen source patch contained 50 alternating hybrid GFP and fertile SN 2108 plants, spaced approximately 1 m apart. Sixteen 1 m2 blocks of male sterile MS TN 90 pollen receptor plants were placed at 9, 23, 38, and 54 m distances from the center and were used to detect pollen via seed formation. Each MS TN 90 plot was 22.5° relative to the adjacent plot as viewed from the center. A honeybee hive was placed at the center of the field site to vector pollen.
Mentions: Natural outcrossing rates of fluorescently tagged hybrids were estimated in two field experiments conducted at Versailles, Kentucky, USA (38.075784, -84.740575) (KY) and Knoxville, Tennessee, USA (35.891769, -83.959786) (TN). A modified Nelder wheel design (Figure 1) [29] covered approximately 0.931 hectares and contained three plant types. GFP-tagged hybrids and non-transgenic SN 2108 plants were used as pollen donors in the center of the plot, and located along the spokes of the Nelder wheel MS TN 90 plants were used as pollen recipients (Figure 1). The pollen source plot measured approximately 15 m in diameter and contained 3 concentric circles consisting of 50 plants each of alternating hybrid GFP and SN 2108 spaced approximately 1 m apart; a honeybee hive was located at the center of the experiment to ensure pollinator presence. Honeybees were observed on plot flowers. Surrounding this central plot were sixteen blocks of five MS TN 90 plants, each used to detect outcrossing at 9, 23, 38, and 54 m from the center plot in each cardinal direction. Each block of MS TN 90 plants were 22.5° relative to the adjacent block with respect to the center plot in contrast to Nelder’s design where the outer plots are arranged in a linear fashion. This modification was made to take advantage of honeybee flight patterns [30], for flight to and from the center patch. Blocks of MS TN 90 plants and hybrid GFP flowers were monitored throughout the season for formation of pods, which were promptly collected at maturity. SN 2108 plants were used solely as a pollen source and seed set was not of interest because of high self-fertilization rates.

Bottom Line: The introduction of pharmaceutical traits in tobacco for commercial production could benefit from the utilization of a transgene bioconfinement system.GFP was used as a useful proxy for pharmaceutical transgenes.In two field experiments, one each in Tennessee and Kentucky, we detected outcrossing at only one location (Tennessee) at 1.4%.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plant Sciences, University of Tennessee, Knoxville, TN 37996, USA.

ABSTRACT

Background: The introduction of pharmaceutical traits in tobacco for commercial production could benefit from the utilization of a transgene bioconfinement system. It has been observed that interspecific F1Nicotiana hybrids (Nicotiana tabacum × Nicotiana glauca) are sterile and thus proposed that hybrids could be suitable bioconfined hosts for biomanufacturing. We genetically tagged hybrids with green fluorescent protein (GFP), which was used as a visual marker to enable gene flow tracking and quantification for field and greenhouse studies. GFP was used as a useful proxy for pharmaceutical transgenes.

Results: Analysis of DNA content revealed significant genomic downsizing of the hybrid relative to that of N. tabacum. Hybrid pollen was capable of germination in vitro, albeit with a very low frequency and with significant differences between plants. In two field experiments, one each in Tennessee and Kentucky, we detected outcrossing at only one location (Tennessee) at 1.4%. Additionally, from 50 hybrid plants at each field site, formation of 84 and 16 seed was observed, respectively. Similar conclusions about hybrid fertility were drawn from greenhouse crosses. In terms of above-ground biomass, the hybrid yield was not significantly different than that of N. tabacum in the field.

Conclusion: N. tabacum × N. glauca hybrids show potential to contribute to a bioconfinement- and biomanufacturing host system. Hybrids exhibit extremely low fertility with no difference of green biomass yields relative to N. tabacum. In addition, hybrids are morphologically distinguishable from tobacco allowing for identity preservation. This hybrid system for biomanufacturing would optimally be used where N. glauca is not present and in physical isolation of N. tabacum production to provide total bioconfinement.

Show MeSH
Related in: MedlinePlus