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Evaluation of potential impacts on biodiversity of the salt-tolerant transgenic Eucalyptus camaldulensis harboring an RNA chaperonic RNA-Binding-Protein gene derived from common ice plant


We recently reported that a genetic transformation of the RNA-Binding-Protein (McRBP), an RNA chaperone gene derived from common ice plant (Mesembryanthemum crystallinum), alleviated injury and loss of biomass production by salt stress in Eucalyptus camaldulensis in a semi-confined screen house trial. In this study, we assessed the potential environmental impact of the transgenic Eucalyptus in a manner complying with Japanese biosafety regulatory framework required for getting permission for experimental confined field trials. Two kinds of bioassays for the effects of allelopathic activity on the growth of other plants, i.e., the sandwich assay and the succeeding crop assay, were performed for three transgenic lines and three non-transgenic lines. No significant differences were observed between transgenic and non-transgenic plants. No significant difference in the numbers of cultivable microorganisms analyzed by the spread plate method were observed among the six transgenic and non-transgenic lines. These results suggested that there is no significant difference in the potential impact on biodiversity between the transgenic McRBP-E. camaldulensis lines and their non-transgenic comparators.

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  1. An G, Costa MA, Ha SB (1990) Nopaline synthase promoter is wound inducible and auxin inducible. Plant Cell 2:225–233.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. Asakawa Y, Fukumoto F, Hamaya E, Hasebe A, Ichikawa H, Matsuda I, Matsumura T, Okada M, Sato M, Shiyomi M, Ukai Y, Yokoyama K, Motoyoshi F, Ohashi Y, Ugaki M, Noguchi K (1992) Evaluation of the impact of the release of transgenic tomato plants with TMV resistance on the environment. Bull Natl Inst Agro-Environ Sci 8:1–51

    Google Scholar 

  3. Barker RF, Idler KB, Thompson DV, Kemp JD (1983) Nucleotide sequence of the T-DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Mol Biol 2:335–350.

    CAS  Article  PubMed  Google Scholar 

  4. Bevan M, Barnes WM, Chilton MD (1983) Structure and transcription of the nopaline synthase gene region of T-DNA. Nucleic Acids Res 11:369–385.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. Biosafety-Clearing-House (2015) Commercial Release of genetically modified eucalyptus—Event H421. Technical Opinion no. 4408/2015. FuturaGene Brasil Tecnologia Ltda, São Paulo, Brazil.

  6. Boland DJ, Brooker MIH, Chippendale G, Hall N, Hyland B, Johnston R, Kleinig D, McDonald M, Turner J (2006) Forest trees of Australia. CSIRO publishing, Australia

    Book  Google Scholar 

  7. CAB-International (2000) Eucalyptus camaldulensis. In: Forestry compendium global module. CAB International, Wallingford, UK

  8. CAB-International (2018) Eucalyptus camaldulensis (red gum). In: Invasive Species Compendium. CAB International, Wallingford, UK.

  9. CBD-COP-MOP8 (2016) Guidance on Risk Assessment of Living Modified Organisms and Monitoring in the Context of Risk Assessment (vol UNEP/CBD/BS/COP-MOP/8/8/Add.1). Cancun, Mexico

  10. Cremer K (1977) Distance of seed dispersal in eucalypts estimated from seed weights. Aust For Res 7:225–228

    Google Scholar 

  11. Depicker A, Stachel S, Dhaese P, Zambryski P, Goodman H (1982) Nopaline synthase: transcript mapping and DNA sequence. J Mol Appl Genet 1:561–573

    CAS  PubMed  Google Scholar 

  12. Doran JC, Brophy JJ (1990) Tropical red gums—a source of 1,8-cineole-rich Eucalyptus oil. New Forest 4:157–178.

    Article  Google Scholar 

  13. EFSA (2007) Statement on the safe use of the nptII antibiotic resistance marker gene in genetically modified plants by the Scientific Panel on genetically modified organisms (GMO). EFSA J 5:742–748.

    Article  Google Scholar 

  14. Eldridge K, Davidson J, Harwood C, Gv Wyk (1994) Eucalypt domestication and breeding. Clarendon Press, Gloucestershire

    Google Scholar 

  15. FAO (2010) Forests and genetically modified trees. Rome, Italy.

  16. FAO (2015) Global Forest Resources Assessment 2015: How have the world’s forests changed? Rome, Italy.

  17. Fuchs RL, Ream JE, Hammond BG, Naylor MW, Leimgruber RM, Berberich SA (1993) Safety assessment of the neomycin phosphotransferase II (NPTII) Protein. Bio-Technology 11:1543–1547.

    CAS  Article  PubMed  Google Scholar 

  18. Fujii Y, Parvez SS, Parvez MM, Ohmae Y, Iida O (2003) Screening of 239 medicinal plant species for allelopathic activity using the sandwich method. Weed Biol Manag 3:233–241.

    Article  Google Scholar 

  19. Government of Japan (2003) Act on the conservation and sustainable use of biological diversity through regulations on the use of living modified organisms.

  20. Griffin A (1980) Floral Phenology of a Stand of Mountain Ash (Eucalyptus regnans F. Muell.) In Gippsland, Victoria. Aust J Bot 28:393–404.

    Article  Google Scholar 

  21. Häggman H, Raybould A, Borem A, Fox T, Handley L, Hertzberg M, Lu M-Z, Macdonald P, Oguchi T, Pasquali G, Pearson L, Peter G, Quemada H, Séguin A, Tattersall K, Ulian E, Walter C, McLean M (2013) Genetically engineered trees for plantation forests: key considerations for environmental risk assessment. Plant Biotechnol J 11:785–798.

    Article  PubMed  PubMed Central  Google Scholar 

  22. ISAAA (2016) Global Status of Commercialized Biotech/GM Crops: 2016. ISAAA Brief 52, Metro Manila, Philippines.

  23. ISAAA (2017) Pocket K No. 5: Documented Benefits of GM Crops. International Service for the Acquisition of Agri-biotech Applications. Metro Manila, Philippines.

  24. ISAAA (2019) GM Approval Database. Accessed 12 October 2019

  25. Itani T, Nakahata Y, Kato-Noguchi H (2013) Allelopathic activity of some herb plant species. Int J Agric Biol 15:1359–1362

    Google Scholar 

  26. Kikuchi A, Kawaoka A, Shimazaki T, Yu X, Ebinuma H, Watanabe KN (2006) Trait stability and environmental biosafety assessments on three transgenic Eucalyptus lines (Eucalyptus camaldulensis Dehnh. codA 12-5B, codA 12-5C, codA 20-C) conferring salt tolerance (in Japanese with English summary). Breed Res 8:17–26

    Google Scholar 

  27. Kikuchi A, Yu X, Shimazaki T, Kawaoka A, Ebinuma H, Watanabe KN (2009) Allelopathy assessments for the environmental biosafety of the salt-tolerant transgenic Eucalyptus camaldulensis, genotypes codA12-5B, codA 12-5C, and codA 20C. J Wood Sci 55:149–153.

    CAS  Article  Google Scholar 

  28. Ko S-S, Liu Y-C, Chung M-C, Shih M-C, Mohammadmehdi H, Oguchi T, Watanabe KN, Yeh K-W (2019) Environmental biosafety assessment on transgenic Oncidium orchid modified by RNA interference of Phytoene Synthase genes. Plant Biotechnol 36:181–185.

    CAS  Article  Google Scholar 

  29. MAFF (2013) Concerning the aplication for approval of type 1 use regulations with regard to the genetically modified plants, the production or circulation of which falls within the jurisdiction of the Minister of Agriculture, Forestry and Fisheries. Notification no.8999, Food Safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry, and Fishery, Japan

  30. Mardani H, Sekine T, Azizi M, Mishyna M, Fujii Y (2015) Identification of safranal as the main allelochemical from saffron (Crocus sativus). Nat Prod Commun 10:775–777.

    Article  PubMed  Google Scholar 

  31. MEXT, MoE (2004) The Ministerial Ordinance providing containment measures to be taken in type 2 use of living modified organisms for research and development (2004-1-29), Ministory of Education, Culture, Sports, Science and Technology and Ministory of Environment of Japan. Accessed 08 Sept 2020

  32. MoE, MAFF (2007) Notification No. 8999. Concerning the Application for Approval of Type 1 Use Regulations with regard to the genetically modified plants, the production or circulation of which falls within the jurisdiction of the Minister of Agriculture, Forestry and Fisheries. (2007-12-10), Wildlife Division, Nature Conservation Bureau, Ministry of the Environment of Japan and Consumer Safety Bureau, Ministry of Agriculture and Fisheries of Japan Accessed 08 Sept 2020

  33. Morikawa CIO, Miyaura R, Tapia Y, Figueroa MDL, RengifoSalgado EL, Fujii Y (2012) Screening of 170 Peruvian plant species for allelopathic activity by using the Sandwich Method. Weed Biol Manag 12:1–11.

    Article  Google Scholar 

  34. Nagaya S, Kawamura K, Shinmyo A, Kato K (2010) The HSP terminator of Arabidopsis thaliana increases gene expression in plant cells. Plant Cell Physiol 51:328–332.

    CAS  Article  PubMed  Google Scholar 

  35. Nakamura R, Teshima R, Takagi K, Sawada J (2005) Development of Allergen Database for Food Safety (ADFS): an integrated database to search allergens and predict allergenicity. Bull Nat Inst Health Sci 123:32–36

    CAS  Google Scholar 

  36. Nakamura R, Nakamura R, Adachi R, Hachisuka A, Yamada A, Ozeki Y, Teshima R (2014) Differential analysis of protein expression in RNA-binding-protein transgenic and parental rice seeds cultivated under salt stress. J Proteome Res 13:489–495.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. National Academies of Sciences, Engineering, and Medicine (2016) Genetically Engineered Crops: Experiences and Prospects. The National Academies Press, Washington, DC

    Google Scholar 

  38. Nishimura H (1987) Eucalyptus as biochemical resources in the future. Uchida Rokakuho, Tokyo

    Google Scholar 

  39. Nonaka S, Someya T, Zhou S, Takayama M, Nakamura K, Ezura H (2017) An Agrobacterium tumefaciens strain with gamma-aminobutyric acid transaminase activity shows an enhanced genetic transformation ability in plants. Sci Rep 7:1–11.

    CAS  Article  Google Scholar 

  40. OECD (2016) safety assessment of transgenic organisms in the environment, volume 6: OECD consensus documents. In: Harmonisation of regulatory oversight in biotechnology. OECD Publishing, Paris

  41. OGTR (2017) Risk Assessment reference: marker genes in GM plants. Methods of plant genetic modification. Australian Government Department of Health, Canberra, Australia.

  42. Oguchi T, Kashimura Y, Mimura M, Yu X, Matsunaga E, Nanto K, Shimada T, Kikuchi A, Watanabe KN (2014) A multi-year assessment of the environmental impact of transgenic Eucalyptus trees harboring a bacterial choline oxidase gene on biomass, precinct vegetation and the microbial community. Transgenic Res 23:767–777.

    CAS  Article  PubMed  Google Scholar 

  43. Osakabe Y, Kajita S, Osakabe K (2011) Genetic engineering of woody plants: current and future targets in a stressful environment. Physiol Planta 142:105–117.

    CAS  Article  Google Scholar 

  44. Parisi C, Tillie P, Rodríguez-Cerezo E (2016) The global pipeline of GM crops out to 2020. Nat Biotechnol 34:31–36.

    CAS  Article  PubMed  Google Scholar 

  45. Percival SL, Williams DW (2014) Escherichia coli. In: Percival SL, Yates MV, Williams DW, Chalmers RM, Gray NF (eds) Microbiology of waterborne diseases, 2nd edn. Academic Press, London, pp 89–117

    Chapter  Google Scholar 

  46. Perry JN, ter Braak CJF, Dixon PM, Duan JJ, Hails RS, Huesken A, Lavielle M, Marvier M, Scardi M, Schmidt K, Tothmeresz B, Schaarschmidt F, van der Voet H (2009) Statistical aspects of environmental risk assessment of GM plants for effects on non-target organisms. Environ Biosaf Res 8:65–78.

    Article  Google Scholar 

  47. Rothstein SJ, Jorgensen R, Yin J-P, Yong-Di Z, Johnson R, Reznikoff W (1981) Genetic organization of Tn5. Cold Spring Harbor Symp Quant Biol 45:99–105.

    CAS  Article  PubMed  Google Scholar 

  48. Ruthrof KX, Loneragan WA, Yates CJ (2003) Comparative population dynamics of Eucalyptus cladocalyx in its native habitat and as an invasive species in an urban bushland in south-western Australia. Divers Distrib 9:469–483.

    Article  Google Scholar 

  49. Shaw CH, Carter GH, Watson MD, Shaw CH (1984) A functional map of the nopaline synthase promoter. Nucleic Acids Res 12:7831–7846.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. Shirasawa-Seo N, Sano Y, Nakamura S, Murakami T, Gotoh Y, Naito Y, Hsia CN, Seo S, Mitsuhara I, Kosugi S, Ohashi Y (2005) The promoter of Milk vetch dwarf virus component 8 confers effective gene expression in both dicot and monocot plants. Plant Cell Rep 24:155–163.

    CAS  Article  PubMed  Google Scholar 

  51. Sugio T, Satoh J, Matsuura H, Shinmyo A, Kato K (2008) The 5′-untranslated region of the Oryza sativa alcohol dehydrogenase gene functions as a translational enhancer in monocotyledonous plant cells. J Biosci and Bioeng 105:300–302.

    CAS  Article  Google Scholar 

  52. Tran N-HT, Oguchi T, Matsunaga E, Kawaoka A, Watanabe KN, Kikuchi A (2018a) Environmental risk assessment of impacts of transgenic Eucalyptus camaldulensis events highly expressing bacterial Choline Oxidase A gene. Plant Biotechnol 35:393–397.

    CAS  Article  Google Scholar 

  53. Tran N-HT, Oguchi T, Matsunaga E, Kawaoka A, Watanabe KN, Kikuchi A (2018b) Transcriptional enhancement of a bacterial Choline Oxidase A gene by an HSP terminator improves the glycine betaine production and salinity stress tolerance of Eucalyptus camaldulensis trees. Plant Biotechnol 35:215–224.

    CAS  Article  Google Scholar 

  54. Tran N-HT, Oguchi T, Akatsuka N, Matsunaga E, Kawaoka A, Yamada A, Ozeki Y, Watanabe KN, Kikuchi A (2019) Development and evaluation of novel salt-tolerant Eucalyptus trees by molecular breeding using an RNA-Binding-Protein gene derived from common ice plant (Mesembryanthemum crystallinum L.). Plant Biotechnol J 17:801–811.

    CAS  Article  PubMed  Google Scholar 

  55. UNDP (2016) United Nation Development Programme support to the Implementation of Sustainable Development Goal. New York, NY, 10017 USA. Accessed 05 April 2018

  56. USDA (2019) Petitions for Determination of Nonregulated Status. Animal and Plant Health Inspection Service, United States Department of Agriculture. Accessed 05 October 2019

  57. Watanabe KN, Teab M, Okusu H (2004) Japanese controversies over transgenic crop regulation. Scinece 305:1572.

    CAS  Article  Google Scholar 

  58. Yu X, Kikuchi A, Matsunaga E, Morishita Y, Nanto K, Sakurai N, Suzuki H, Shibata D, Shimada T, Watanabe KN (2009) Establishment of the evaluation system of salt tolerance on transgenic woody plants in the special netted-house. Plant Biotechnol 26:135–141.

    CAS  Article  Google Scholar 

  59. Yu X, Kikuchi A, Matsunaga E, Shimada T, Watanabe KN (2013a) Environmental biosafety assessment on transgenic Eucalyptus globulus harboring the choline oxidase (codA) gene in semi-confined condition. Plant Biotechnol 30:73–76.

    CAS  Article  Google Scholar 

  60. Yu X, Kikuchi A, Shimazaki T, Yamada A, Ozeki Y, Matsunaga E, Ebinuma H, Watanabe KN (2013b) Assessment of the salt tolerance and environmental biosafety of Eucalyptus camaldulensis harboring a mangrin transgene. J Plant Res 126:141–150.

    CAS  Article  PubMed  Google Scholar 

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This research was supported in part by the New Energy and Industrial Technology Development Organization (NEDO) of Japan (P07015), and by a grant from the Plant Transgenic Design Initiative (PTraD), Gene Research Center, Tsukuba Plant Innovation Research Center, University of Tsukuba, Japan (Grant Nos.1220,1322, 1423, 1520, 1630, and 1725).

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Correspondence to Taichi Oguchi.

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Tran, NH.T., Oguchi, T., Matsunaga, E. et al. Evaluation of potential impacts on biodiversity of the salt-tolerant transgenic Eucalyptus camaldulensis harboring an RNA chaperonic RNA-Binding-Protein gene derived from common ice plant. Transgenic Res 30, 23–34 (2021).

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  • RNA-binding protein
  • Mesembryanthemum crystallinum
  • Transgenic trees
  • Environmental risk assessment (ERA)
  • Biosafety