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

BMC Biotechnology

J Hollis Rice³  Richard E Mundell¹  Reginald J Millwood³  Orlando D Chambers¹  C Neal Stewart³  H Maelor Davies² 

[1] Kentucky Tobacco Research & Development Center, University of Kentucky, Lexington, KY 40546, USA

[2] Department of Plant & Soil Sciences, University of Kentucky, Lexington, KY 40546, USA

[3] Department of Plant Sciences, University of Tennessee, Knoxville, TN 37996, USA

关键词: Plant-made-pharmaceuticals (PMPs);    Green fluorescent protein (GFP);    Nicotiana;    Bioconfinement;    Pharming;    Male-sterility;    Gene flow;   

Others  :  1121357
DOI  :  10.1186/1472-6750-13-63

 received in 2013-03-21, accepted in 2013-08-01,  发布年份 2013

【 摘 要 】

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.

【 授权许可】

   
2013 Rice et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Ma JK-C, Drake PMW, Christou P: The production of recombinant pharmaceutical proteins in plants. Nat Rev Genet 2003, 4:794-805.
• [2]He Y, Ning T, Xie T, Qiu Q, Zhang L, Sun Y, Jiang D, Fu K, Yin F, Zhang W: Large-scale production of functional human serum albumin from transgenic rice seeds. Proc Nat Acad Sci USA 2011, 108:19078-19083.
• [3]Ma JK-C, Chikwamba R, Sparrow P, Fischer R, Mahoney R, Twyman RM: Plant-derived pharmaceuticals–the road forward. Trends Plant Sci 2005, 10:580-585.
• [4]Lienard D, Sourrouille C, Gomord V, Faye L: Pharming and transgenic plants. Biotechnol Annual Rev 2007, 13:115-147.
• [5]Maxmen A: Drug-making plant blooms. Nature 2012, 485:160.
• [6]Stewart CN: Pharming in crop commodities. Nat Biotechnol 2008, 26:1222-1223.
• [7]Sparrow PAC, Irwin JA, Dale PJ, Twyman RM, Ma JK-C: Pharma-planta: road testing the developing regulatory guidelines for plant-made pharmaceuticals. Transgenic Res 2007, 16:147-161.
• [8]Hutchens TW: Tobacco seed. In Tobacco production, chemistry and technology. Edited by Davis DL, Nielsen MT. Oxford, UK: Blackwell Publishing Ltd; 1999:66-69.
• [9]Reichman JR, Watrud LS, Lee EH, Burdick CA, Bollman MA, Storm MJ, King GA, Mallory-Smith C: Establishment of transgenic herbicide-resistant creeping bentgrass (Agrostis stolonifera L.) in nonagronomic habitats. Mol Ecol 2006, 15:4243-4255.
• [10]Ramessar K, Sabalza M, Capell T, Christou P: Maize plants: an ideal production platform for effective and safe molecular pharming. Plant Sci 2008, 174:409-419.
• [11]Breyer D, Goossens M, Herman P, Sneyers M: Biosafety considerations associated with molecular farming in genetically modified plants. J Med Plants Res 2009, 3:825-838.
• [12]Trojak-Goluch A, Berbec A: Cytological investigations of the interspecific hybrids of Nicotiana tabacum L. × N. glauca Grah. J Applied Genetics 2003, 44:45-54.
• [13]Zaitlin D, Chambers OD, Li B, Mundell RE, Davies HM: Correspondence 1: drugs in crops. Nat Biotechnol 2004, 22:507-507.
• [14]Trojak-Goluch A, Berbeć A: Meiosis and fertility in interspecific hybrids of Nicotiana tabacum L. × N. glauca Grah. and their derivatives. Plant Breeding 2007, 126:201-206.
• [15]Al-Ahmad H, Galili S, Gressel J: Infertile interspecific hybrids between transgenically mitigated Nicotiana tabacum and Nicotiana sylvestris did not backcross to N. sylvestris. Plant Sci 2006, 170(5):953-961.
• [16]Haseloff J, Siemering KR, Prasher DC, Hodge S: Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Nat Acad Sci USA 1997, 94:2122-2127.
• [17]Morise H, Shimomura O, Johnson FH, Winant J: Intermolecular energy transfer in the bioluminescent system of Aequorea. Biochemistry 1974, 13:2656-2662.
• [18]Millwood R, Halfhill M, Harkins D, Russotti R, Stewart CN Jr: Instrumentation and methodology for quantifying GFP fluorescence in intact plant organs. Biotechniques 2003, 34:638-643.
• [19]Horsch R, Fry J, Hoffmann N, Eichholtz D, Rogers SG, Fraley R: A simple and general method for transferring genes into plants. Science 1985, 227:1229-1231.
• [20]McCormick S, Niedermeyer J, Fry J, Barnason A, Horsch R, Fraley R: Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens. Plant Cell Rep 1986, 5:81-84.
• [21]Stewart CN Jr, Via LE: A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications. Biotechniques 1993, 14:748-750.
• [22]Hudson LC, Chamberlain D, Stewart CN Jr: GFP-tagged pollen to monitor pollen flow of transgenic plants. Mol Ecol Notes 2001, 1:321-324.
• [23]Halfhill MD, Millwood RJ, Weissinger AK, Warwick SI, Stewart CN Jr: Additive transgene expression and genetic introgression in multiple green-fluorescent protein transgenic crop × weed hybrid generations. Theoretical Applied Genetics 2003, 107:1533-1540.
• [24]Galbraith DW: Simultaneous flow cytometric quantification of plant nuclear DNA contents over the full range of described angiosperm 2C values. Cytometry Part A 2009, 75:692-698.
• [25]Doležel J, Sgorbati S, Lucretti S: Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiol Plant 1992, 85:625-631.
• [26]Galbraith DW, Harkins KR, Maddox JM, Ayers NM, Sharma DP, Firoozabady E: Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 1983, 220:1049-1051.
• [27]Hudson LC, Stewart CN Jr: Effects of pollen-synthesized green fluorescent protein on pollen grain fitness. Sexual Plant Reprod 2004, 17:49-53.
• [28]Brewbaker JL, Kwack BH: The essential role of calcium ion in pollen germination and pollen tube growth. Am J Botany 1963, 50:859-865.
• [29]Nelder JA: New kinds of systematic designs for spacing experiments. Biometrics 1962, 18:283-307.
• [30]Ginsberg H: Honey bee orientation behaviour and the influence of flower distribution on foraging movements. Ecol Entomol 1986, 11:173-179.
• [31]Association IST: International rules for seed testing. Seed Sci Technol 1999, 27(Suppl):333.
• [32]Shapiro SS, Wilk MB: An analysis of variance test for normality (complete samples). Biometrika 1965, 52:591-611.
• [33]Levene H: Robust tests for equality of variances. In Contributions to probability and statistics: essays in honor of Harold Hotelling. vol. 2 edition. Edited by Olkin I. Palto Alto, CA: Stanford University Press; 1960:278-291.
• [34]Tremblay R, Wang D, Jevnikar AM, Ma S: Tobacco, a highly efficient green bioreactor for production of therapeutic proteins. Biotechnol Adv 2010, 28:214-221.
• [35]Daniell H, Streatfield SJ, Wycoff K: Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants. Trends Plant Sci 2001, 6:219-226.
• [36]Ling HY, Edwards AM, Gantier MP, DeBoer KD, Neale AD, Hamill JD, Walmsley AM: An interspecific Nicotiana hybrid as a useful and cost-effective platform for production of animal vaccines. PloS ONE 2012, 7:e35688.
• [37]Hiatt A, Caffferkey R, Bowdish K: Production of antibodies in transgenic plants. Nature 1989, 342:76-78.
• [38]Wang DJ, Brandsma M, Yin Z, Wang A, Jevnikar AM, Ma S: A novel platform for biologically active recombinant human interleukin-13 production. Plant Biotechnol J 2008, 6:504-515.
• [39]Arumuganathan K, Earle E: Nuclear DNA content of some important plant species. Plant Mol Biol Reporter 1991, 9:208-218.
• [40]Narayan R: Nuclear DNA changes, genome differentiation and evolution in Nicotiana (Solanaceae). Plant System Evol 1987, 157:161-180.
• [41]Leitch I, Bennett M: Genome downsizing in polyploid plants. Biol J Linnean Soc 2004, 82:651-663.
• [42]Laughnan JR, Gabay-Laughnan S: Cytoplasmic male sterility in maize. Annu Rev Genet 1983, 17:27-48.
• [43]Janska H, Sarria R, Woloszynska M, Arrieta-Montiel M, Mackenzie SA: Stoichiometric shifts in the common bean mitochondrial genome leading to male sterility and spontaneous reversion to fertility. Plant Cell 1998, 10:1163-1180.
• [44]USDA: The PLANTS database. Greensboro, NC USA: National Plant Data Team; 2012.