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Greenhouse and field cultivations of antigen-expressing potatoes focusing on the variability in plant constituents and antigen expression

  • Heike Mikschofsky
  • Elena Heilmann
  • Jörg Schmidtke
  • Kerstin Schmidt
  • Udo Meyer
  • Peter Leinweber
  • Inge Broer
Article

Abstract

The production of plant-derived pharmaceuticals essentially requires stable concentrations of plant constituents, especially recombinant proteins; nonetheless, soil and seasonal variations might drastically interfere with this stability. In addition, variability might depend on the plant organ used for production. Therefore, we investigated the variability in plant constituents and antigen expression in potato plants under greenhouse and field growth conditions and in leaves compared to tubers. Using potatoes expressing VP60, the only structural capsid protein of the rabbit haemorrhagic disease virus (RHDV), CTB, the non-toxic B subunit (CTB) of the cholera toxin (CTA-CTB5) and the marker protein NPTII (neomycinphosphotransferase) as a model, we compare greenhouse and field production of potato-derived antigens. The influence of the production organ turned out to be transgene specific. In general, yield, plant quality and transgene expression levels in the field were higher than or similar to those observed in the greenhouse. The variation (CV) of major plant constituents and the amount of transgene-encoded protein was not influenced by the higher variation of soil properties observed in the field. Amazingly, for specific events, the variability in the model protein concentrations was often lower under field than under greenhouse conditions. The changes in gene expression under environmental stress conditions in the field observed in another event do not reduce the positive influence on variability since events like these should excluded from production. Hence, it can be concluded that for specific applications, field production of transgenic plants producing pharmaceuticals is superior to greenhouse production, even concerning the stability of transgene expression over different years. On the basis of our results, we expect equal or even higher expression levels with lower variability of recombinant pharmaceuticals in the field compared to greenhouse production combined with approximately 10 times higher tuber yield in the field.

Keywords

VP60 CTB Molecular farming Field production Soil variability 

Notes

Acknowledgments

This work was supported by the Federal Ministry of Education and Research (BMBF) Germany, projects 03WKS07A and 03WKS02: Innovative Regionale Wachstumskerne: BioOK – Entwicklung von Zulassungs- und Überwachungsverfahren für transgene Nutzpflanzen.

Supplementary material

11103_2011_9774_MOESM1_ESM.doc (116 kb)
Supplementary material 1 (DOC 115 kb)

References

  1. Acciarri N, Restaino F, Vitelli G, Perrone D, Zottini M, Pandolfini T, Spena A, Rotino GL (2002) Genetically modified parthenocarpic eggplants: improved fruit productivity under both greenhouse and open field cultivation. BMC Biotechnol 2:7–13CrossRefGoogle Scholar
  2. Ahuja MR (2009) Transgene stability and dispersal in forest trees. Trees Struct Funct 23:1125–1135Google Scholar
  3. Arlen PA, Falconer R, Cherukumilli S, Cole A, Cole AM, Oishi KK, Daniell H (2007) Field production and functional evaluation of chloroplast-derived interferon-alpha 2b. Plant Biotechnol J 5:511–525PubMedCrossRefGoogle Scholar
  4. Basso B, Cammarano D, Chen D, Cafiero G, Amato M, Bitella G, Rossi R, Basso F (2009) Landscape position and precipitation effects on spatial variability of wheat yield and grain protein in Southern Italy. J Agronomy Crop Sci 195:301–312CrossRefGoogle Scholar
  5. Blume H-P, Stahr K, Leinweber P (2011) Bodenkundliches Praktikum, 3. Auflage, Spektrum Akademischer Verlag, Heidelberg, 260 SGoogle Scholar
  6. Bossdorf O, Pigliucci M (2009) Plasticity to wind is modular and genetically variable in Arabidopsis thaliana. Evol Ecol 23:669–685CrossRefGoogle Scholar
  7. Bradford MM (1976) Rapid and sensitive method for quantitation of microgram quantities of protein utilizing principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  8. Breyne P, Gheysen G, Jacobs A, Vanmontagu M, Depicker A (1992) Effect of T-DNA configuration on transgene expression. Mol Gen Genet 235:389–396PubMedCrossRefGoogle Scholar
  9. Broer I (1996) Stress inactivation of foreign genes in transgenic plants. Field Crops Res 45:19–25CrossRefGoogle Scholar
  10. Burlingame B, Mouille B, Charrondiere R (2009) Nutrients, bioactive non-nutrients and anti-nutrients in potatoes. J Food Comp Anal 22:494–502CrossRefGoogle Scholar
  11. Castanon S, Marin MS, Martin-Alonso JM, Boga JA, Casais R, Humara JM, Ordas RJ, Parra F (1999) Immunization with potato plants expressing VP60 protein protects against rabbit hemorrhagic disease virus. J Virol 73:4452–4455PubMedGoogle Scholar
  12. Castanon S, Martin-Alonso JM, Marin MS, Boga JA, Alonso P, Parra F, Ordas RJ (2002) The effect of the promoter on expression of VP60 gene from rabbit hemorrhagic disease virus in potato plants. Plant Sci 162:87–95CrossRefGoogle Scholar
  13. Castrignano A, Giugliarini L, Risaliti R, Martinelli N (2000) Study of spatial relationships among some soil physico-chemical properties of a field in central Italy using multivariate geostatistics. Geoderma 97(1-2):39–60Google Scholar
  14. Celis C, Scurrah M, Cowgill S, Chumbiauca S, Green J, Franco J, Main G, Kiezebrink D, Visser RGF, Atkinson HJ (2004) Environmental biosafety and transgenic potato in a centre of diversity for this crop. Nature 432:222–225PubMedCrossRefGoogle Scholar
  15. Chusainow J, Yang YS, Yeo YHM, Toh PC, Asvadi P, Wong NSC, Yap MGS (2009) A study of monoclonal antibody-producing CHO cell lines: what makes a stable high producer? Biotechnol Bioeng 102:1182–1196PubMedCrossRefGoogle Scholar
  16. De Bolle MFC, Butaye KMJ, Coucke WJW, Goderis IJWM, Wouters PFJ, van Boxel N, Broekaert WF, Cammue BPA (2003) Analysis of the influence of promoter elements and a matrix attachment region on the inter-individual variation of transgene expression in populations of Arabidopsis thaliana. Plant Sci 165:169–179CrossRefGoogle Scholar
  17. De Buck S, Windels P, De Loose M, Depicker A (2004) Single-copy T-DNAs integrated at different positions in the Arabidopsis genome display uniform and comparable beta-glucuronidase accumulation levels. Cell Mol Life Sci 61:2632–2645PubMedCrossRefGoogle Scholar
  18. Dong HZ, Li WJ (2007) Variability of endotoxin expression in Bt transgenic cotton. J Agronomy Crop Sci 193:21–29CrossRefGoogle Scholar
  19. Down RE, Ford L, Bedford SJ, Gatehouse LN, Newell C, Gatehouse JA, Gatehouse AMR (2001) Influence of plant development and environment on transgene expression in potato and consequences for insect resistance. Transgenic Res 10(3):223–236PubMedCrossRefGoogle Scholar
  20. Elomaa P, Helariutta Y, Griesbach RJ, Kotilainen M, Seppanen P, Teeri TH (1995) Transgene inactivation in Petunia-Hybrida is influenced by the properties of the foreign gene. Mol Gen Genet 248:649–656PubMedCrossRefGoogle Scholar
  21. Farre G, Ramessar K, Twyman RM, Capell T, Christou P (2010) The humanitarian impact of plant biotechnology: recent breakthroughs vs bottlenecks for adoption. Curr Opin Plant Biol 13:219–225PubMedCrossRefGoogle Scholar
  22. Fernandez-Fernandez MR, Mourino M, Rivera J, Rodriguez F, Plana-Duran J, Garcia JA (2001) Protection of rabbits against rabbit hemorrhagic disease virus by immunization with the VP60 protein expressed in plants with a potyvirus-based vector. Virology 280:283–291PubMedCrossRefGoogle Scholar
  23. Floris M, Mahgoub H, Lanet E, Robaglia C, Menand B (2009) Post-transcriptional regulation of gene expression in plants during abiotic stress. Int J Mol Sci 10:3168–3185PubMedCrossRefGoogle Scholar
  24. Gupta RK, Mostaghimi S, McClellan PW, Birch JB, Brann DE (1999) Modelling spatial variability of soil chemical parameters for site-specific farming using stochastic methods. Water Air Soil Pollut 110:17–34Google Scholar
  25. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vector strategy based on separation of vir-region and T-region of the agrobacterium-Tumefaciens Ti-plasmid. Nature 303:179–180CrossRefGoogle Scholar
  26. Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT (1985) A simple and general-method for transferring genes into plants. science 227:1229–1231CrossRefGoogle Scholar
  27. Huhns M, Neumann K, Hausmann T, Klemke F, Lockau W, Kahmann U, Kopertekh L, Staiger D, Pistorius EK, Reuther J, Waldvogel E, Wohlleben W, Effmert M, Junghans H, Neubauer K, Kragl U, Schmidt K, Schmidtke J, Broer I (2009) Tuber-specific cphA expression to enhance cyanophycin production in potatoes. Plant Biotechnol J 7:883–898PubMedCrossRefGoogle Scholar
  28. Iqbal J, Read JJ, Thomasson AJ, Jenkins JN (2005) Relationships between soil-landscape and dryland cotton lint yield. Soil Sci Soc Am J 69:872–882CrossRefGoogle Scholar
  29. Jimenez ME, Rossi AM, Samman NC (2009) Phenotypic, agronomic and nutritional characteristics of seven varieties of Andean potatoes. J Food Comp Anal 22:613–616CrossRefGoogle Scholar
  30. Karg SR, Kallio PT (2009) The production of biopharmaceuticals in plant systems. Biotechnol Adv 27:879–894PubMedCrossRefGoogle Scholar
  31. Knöchel N, Latzkow T, Nausch H, Kerbach S, Walter S, Broer I, Huckauf J (2011) Influence of UTR and terminator sequences on the expression of the herbicide resistance gene pat in Nicotiana tabacum. Submitted Plant Mol BiolGoogle Scholar
  32. Latzkow T, Knöchel N, Broer I, Huckauf J (2011) Influence of transgene sequence on heat-induced inactivation of the herbicide resistance gene pat in transgenic tobacco plants. Submitted Plant Mol BiolGoogle Scholar
  33. Lau OS, Sun SSM (2009) Plant seeds as bioreactors for recombinant protein production. Biotechnol Adv 27:1015–1022PubMedCrossRefGoogle Scholar
  34. Lheureux K, Menrad K (2004) A decade of European field trials with genetically modified plants. Environ Biosafety Res 3:99–107PubMedCrossRefGoogle Scholar
  35. Li JL, Meilan R, Ma C, Barish M, Strauss SH (2008) Stability of herbicide resistance over 8 years of coppice in field-grown, genetically engineered poplars. West J Appl For 23:89–93Google Scholar
  36. Logemann J, Schell J, Willmitzer L (1987) Improved method for the isolation of RNA from plant-tissues. Anal Biochem 163:16–20PubMedCrossRefGoogle Scholar
  37. Mallarino AP, Wittry DJ (2004) Efficacy of grid and zone soil sampling approaches for site-specific assessment of phosphorus, potassium, pH, and organic matter. Precis Agric 5:131–144Google Scholar
  38. Mason HS, Lam DMK, Arntzen CJ (1992) Expression of hepatitis-B surface-antigen in transgenic plants. Proc Natl Acad Sci USA 89:11745–11749PubMedCrossRefGoogle Scholar
  39. Meyers G, Wirblich C, Thiel HJ (1991) Rabbit hemorrhagic-disease virus—molecular-cloning and nucleotide sequencing of a Calicivirus genome. Virology 184:664–676PubMedCrossRefGoogle Scholar
  40. Mikschofsky H, Hammer M, Schmidtke J, Konig P, Keil G, Schirrmeier H, Schmidt K, Broer I (2009a) Optimization of growth performance of freshly induced carrot suspensions concerning PMP production. Vitro Cell Dev Biol Plant 45:740–749CrossRefGoogle Scholar
  41. Mikschofsky H, Konig P, Keil GM, Hammer M, Schirrmeier H, Broer I (2009b) Cholera toxin B (CTB) is functional as an adjuvant for cytoplasmatic proteins if directed to the endoplasmatic reticulum (ER), but not to the cytoplasm of plants. Plant Sci 177:35–42CrossRefGoogle Scholar
  42. Mikschofsky H, Mann G, Broer I (2009c) Soil adaptation of transgenic in vitro carrot plantlets. J Agric Sci 147:43–49CrossRefGoogle Scholar
  43. Mikschofsky H, Schirrmeier H, Keil GM, Lange B, Polowick PL, Keller W, Broer I (2009d) Pea-derived vaccines demonstrate high immunogenicity and protection in rabbits against rabbit haemorrhagic disease virus. Plant Biotechnol J 7:537–549PubMedCrossRefGoogle Scholar
  44. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–496CrossRefGoogle Scholar
  45. Murray MG, Thompson WF (1980) Rapid isolation of high molecular-weight plant DNA. Nucleic Acids Res 8:4321–4325PubMedCrossRefGoogle Scholar
  46. Negi PS, Nath N (2002) Effect of partial dehydration on quality of canned potatoes. Eur Food Res Technol 215:231–234CrossRefGoogle Scholar
  47. Neumann K, DrogeLaser W, Kohne S, Broer I (1997) Heat treatment results in a loss of transgene-encoded activities in several tobacco lines. Plant Physiol 115:939–947PubMedCrossRefGoogle Scholar
  48. Nikolopoulou D, Grigorakis K, Stasini M, Alexis MN, Iliadis K (2007) Differences in chemical composition of field pea (Pisum sativum) cultivars: effects of cultivation area and year. Food Chem 103:847–852CrossRefGoogle Scholar
  49. Ohlinger VF, Haas B, Meyers G, Weiland F, Thiel HJ (1990) Identification and characterization of the virus causing rabbit hemorrhagic-disease. J Virol 64:3331–3336PubMedGoogle Scholar
  50. Parra F, Prieto M (1990) Purification and characterization of a Calicivirus as the causative agent of a lethal hemorrhagic-disease in rabbits. J Virol 64:4013–4015PubMedGoogle Scholar
  51. Peach C, Velten J (1991) Transgene expression variability (position effect) of cat and Gus reporter genes driven by linked divergent T-DNA promoters. Plant Mol Biol 17:49–60PubMedCrossRefGoogle Scholar
  52. Po EA, Snapp SS, Kravchenko A (2009) Rotational and cover crop determinants of soil structural stability and carbon in a potato system. Agronomy J 101:175–183CrossRefGoogle Scholar
  53. Po EA, Snapp SS, Kravchenko A (2010) Potato yield variability across the landscape. Agronomy J 102:885–894CrossRefGoogle Scholar
  54. Qin HX, Dong YZ, von Arnim AG (2003) Epigenetic interactions between Arabidopsis transgenes: characterization in light of transgene integration sites. Plant Mol Biol 52:217–231PubMedCrossRefGoogle Scholar
  55. Quine TA, Zhang Y (2002) An investigation of spatial variation in soil erosion, soil properties, and crop production within an agricultural field in Devon, United Kingdom. J Soil Water Conserv 57:55–65Google Scholar
  56. Rask C, Fredriksson M, Lindblad M, Czerkinsky C, Holmgren J (2000) Mucosal and systemic antibody responses after peroral or intranasal immunization: effects of conjugation to enterotoxin B subunits and/or of co-administration with free toxin as adjuvant. APMIS 108:178–186PubMedCrossRefGoogle Scholar
  57. Redulla CA, Davenport JR, Evans RG, Hattendorf MJ, Alva AK, Boydston RA (2002) Relating potato yield and quality to field scale variability in soil characteristics. Am J Potato Res 79:317–323CrossRefGoogle Scholar
  58. Ritter E, Barandalla L, López R, Ruiz de Galarreta JI (2008) Exploitation of exotic cultivated solanum germplasm for breeding and commercial purposes. Potato Res 51:301–311CrossRefGoogle Scholar
  59. Schillberg S, Twyman RM, Fischer R (2005) Opportunities for recombinant antigen and antibody expression in transgenic plants–technology assessment. Vaccine 23:1764–1769PubMedCrossRefGoogle Scholar
  60. Schirrmeier H, Reimann I, Kollner B, Granzow H (1999) Pathogenic, antigenic and molecular properties of rabbit haemorrhagic disease virus (RHDV) isolated from vaccinated rabbits: detection and characterization of antigenic variants. Arch Virol 144:719–735PubMedCrossRefGoogle Scholar
  61. Sun B, Zhou S, Zhao Q (2003) Evaluation of spatial and temporal changes of soil quality based on geostatistical analysis in the hill region of subtropical China. Geoderma 115:85–99Google Scholar
  62. Walter C, Broer I, Hillemann D, Puhler A (1992) High-frequency, heat treatment-induced inactivation of the Phosphinothricin resistance gene in transgenic single cell-suspension cultures of Medicago-Sativa. Mol Gen Genet 235:189–196PubMedCrossRefGoogle Scholar
  63. Zhang P, Yang QB, Balkovetz DF, Lewis JP, Clements JD, Michalek SM, Katz J (2005) Effectiveness of the B subunit of cholera toxin in potentiating immune responses to the recombinant hemagglutinin/adhesin domain of the gingipain Kgp from Porphyromonas gingivalis. Vaccine 23:4734–4744PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Heike Mikschofsky
    • 1
  • Elena Heilmann
    • 2
  • Jörg Schmidtke
    • 3
  • Kerstin Schmidt
    • 3
  • Udo Meyer
    • 4
  • Peter Leinweber
    • 2
  • Inge Broer
    • 1
  1. 1.AgrobiotechnologieUniversität RostockRostockGermany
  2. 2.Universität RostockRostockGermany
  3. 3.BioMath GmbHGross LüsewitzGermany
  4. 4.Bioserv Analytik und Medizinprodukte GmbHRostockGermany

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