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Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 134, Issue 2, pp 217–229 | Cite as

Optimization of Agrobacterium-mediated genetic transformation of Fraxinus nigra and development of black ash for possible emerald ash borer resistance

  • Jun Hyung Lee
  • Paula M. Pijut
Original Article

Abstract

Emerald ash borer (EAB; Agrilus planipennis Fairmaire) is the most devastating insect pest of North American ash species, including black ash (Fraxinus nigra Marsh.). As a first step in an effort to develop transgenic black ash plants resistant to EAB, we successfully established an efficient Agrobacterium-mediated transformation system for black ash hypocotyls. Kanamycin and timentin at 40 and 300 mg L−1, respectively, were most effective to select transformed explants and control excess Agrobacterium growth. Using a plant transformation vector harboring the enhanced green fluorescent protein (eGFP) gene, the effects of Agrobacterium strain, bacterial density, and the concentration of Silwet L-77 on transformation efficiency were evaluated. The best result was obtained when Agrobacterium strain EHA105 was used at a density of OD600 = 1.0. Silwet L-77 failed to promote transformation frequency and showed an adverse effect at higher concentrations (> 0.015%). Using this optimized transformation system, transgenic black ash shoots expressing a synthetic Bacillus thuringiensis toxin gene (cry8D2) were regenerated. Although no morphological abnormality was observed, transgenic shoots showed severe growth restriction. Three independent transgenic lines were selected for further assessment. All selected lines contained two copies of the cry8D2 gene, and the expression of the transgene was verified in transcript and protein levels. These transgenic shoots can be used for future bioassay to evaluate its efficacy against EAB.

Keywords

Black ash Emerald ash borer Fraxinus Genetic transformation 

Notes

Acknowledgements

This research was supported by partial funding from the USDA-APHIS-PPQ Center for Plant Health Science and Technology, the U.S. Endowment for Forestry and Communities, and members of the Indiana Hardwood Lumbermen’s Association. The authors gratefully acknowledge Dr. Dennis J. Gray, University of Florida, for the transformation vector pq35GR, and Dale Simpson, Natural Resources Canada, for the black ash seed. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that also may be suitable.

Author contributions

JHL and PMP conceived and designed the research. JHL conducted the experiments and analyzed the data. JHL and PMP wrote the manuscript. All authors read and approved the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. Abou-Alaiwi WA, Potlakayala SD, Goldman SL, Josekutty PC, Karelia DN, Rudrabhatla SV (2012) Agrobacterium-mediated transformation of the medicinal plant Centaurea montana. Plant Cell Tiss Organ Cult 109:1–8CrossRefGoogle Scholar
  2. Acharjee S, Sarmah BK, Kumar PA, Olsen K, Mahon R, Moar WJ, Moore A, Higgins TJV (2010) Transgenic chickpeas (Cicer arietinum L.) expressing a sequence-modified cry2Aa gene. Plant Sci 178:333–339CrossRefGoogle Scholar
  3. Alam P, Khan ZA, Abdin MZ, Khan JA, Ahmad P, Elkholy SF, Sharaf-Eldin MA (2017) Efficient regeneration and improved sonication-assisted Agrobacterium transformation (SAAT) method for Catharanthus roseus. 3 Biotech 7:26CrossRefPubMedPubMedCentralGoogle Scholar
  4. Asano S, Yamashita C, Iizuka T, Takeuchi K, Yamanaka S, Cerf D, Yamamoto T (2003) A strain of Bacillus thuringiensis subsp. galleriae containing a novel cry8 gene highly toxic to Anomala cuprea (Coleoptera: Scarabaeidae). Biol Control 28:191–196CrossRefGoogle Scholar
  5. Bai X, Rivera-Vega L, Mamidala P, Bonello P, Herms DA, Mittapalli O (2011) Transcriptomic signatures of ash (Fraxinus spp.) phloem. PLoS ONE 6:e16368CrossRefPubMedPubMedCentralGoogle Scholar
  6. Bauer LS, Londoño DK (2011) Effects of Bacillus thuringiensis SDS-502 on adult emerald ash borer. In: McManus KA, Gottschalk KW (Eds.) 2010 Proceedings of the 21st USDA Interagency Research Forum on Invasive Species, USDA Forest Services, Northern Research Station, Gen Tech Rep-NRS-P-75, pp 74–75Google Scholar
  7. Beasley RR, Pijut PM (2013) Regeneration of plants from Fraxinus nigra Marsh. hypocotyls. HortScience 48:887–890Google Scholar
  8. Beranová M, Rakouský S, Vávrová Z, Skalický T (2008) Sonication assisted Agrobacterium-mediated transformation enhances the transformation efficiency in flax (Linum usitatissimum L.). Plant Cell Tiss Organ Cult 94:253–259CrossRefGoogle Scholar
  9. Chakrabarti SK, Lutz KA, Lertwiriyawong B, Svab Z, Maliga P (2006) Expression of the cry9Aa2 B.t. gene in tobacco chloroplasts confers resistance to potato tuber moth. Transgenic Res 15:481–488CrossRefPubMedGoogle Scholar
  10. Chu M, Quiñonero C, Akdemir H, Alburquerque N, Pedreño M, Burgos L (2016) Agrobacterium-mediated transformation of Vitis Cv. Monastrell suspension-cultured cells: Determination of critical parameters. Biotechnol Prog 32:725–734CrossRefPubMedGoogle Scholar
  11. Cipollini D, Wang Q, Whitehill JGA, Powell JR, Bonello P, Herms DA (2011) Distinguishing defensive characteristics in the phloem of ash species resistant and susceptible to emerald ash borer. J Chem Ecol 37:450–459CrossRefPubMedGoogle Scholar
  12. Confalonieri M, Balestrazzi A, Bisoffi S (1994) Genetic transformation of Populus nigra by Agrobacterium tumefaciens. Plant Cell Rep 13:256–261CrossRefPubMedGoogle Scholar
  13. de Cosa B, Moar W, Lee S-B, Miller M, Daniell H (2001) Overexpression of the Bt cry2Aa2 operon in chloroplasts leads to formation of insecticidal crystals. Nat Biotechnol 19:71–74CrossRefPubMedPubMedCentralGoogle Scholar
  14. de Oliveira MLP, Febres VJ, Costa MGC, Moore GA, Otoni WC (2009) High-efficiency Agrobacterium-mediated transformation of citrus via sonication and vacuum infiltration. Plant Cell Rep 28:387–395CrossRefPubMedGoogle Scholar
  15. Diehn SH, De Rocher EJ, Green PJ (1996) Problems that can limit the expression of foreign genes in plants: lessons to be learned from B.t.-toxin genes. In: Setlow JK (ed) Genetic engineering: principles and methods. Springer, Boston, pp 83–99CrossRefGoogle Scholar
  16. Dong J, Kharb P, Teng W, Hall TC (2001) Characterization of rice transformed via an Agrobacterium-mediated inflorescence approach. Mol Breed 7:187–194CrossRefGoogle Scholar
  17. Du N, Pijut PM (2009) Agrobacterium-mediated transformation of Fraxinus pennsylvanica hypocotyls and plant regeneration. Plant Cell Rep 28:915–923CrossRefPubMedGoogle Scholar
  18. Ellison AM, Bank MS, Clinton BD, Colburn EA, Elliott K, Ford CR, Foster DR, Kloeppel BD, Knoepp JD, Lovett GM, Mohan J, Orwig DA, Rodenhouse NL, Sobczak WV, Stinson KA, Stone JK, Swan CM, Thompson J, Holle BV, Webster JR (2005) Loss of foundation species: consequences for the structure and dynamics of forested ecosystems. Front Ecol Environ 3:479–486CrossRefGoogle Scholar
  19. Fagard M, Vaucheret H (2000) (Trans)Gene silencing in plants: how many mechanisms? Annu Rev Plant Physiol Plant Mol Biol 51:167–194CrossRefPubMedGoogle Scholar
  20. Fan J, Liu X, Xu S-X, Xu Q, Guo W-W (2011) T-DNA direct repeat and 35S promoter methylation affect transgene expression but do not cause silencing in transgenic sweet orange. Plant Cell Tiss Organ Cult 107:225–232CrossRefGoogle Scholar
  21. Gahakwa D, Maqbool SB, Fu X, Sudhakar D, Christou P, Kohli A (2000) Transgenic rice as a system to study the stability of transgene expression: multiple heterologous transgenes show similar behaviour in diverse genetic backgrounds. Theor Appl Genet 101:388–399CrossRefGoogle Scholar
  22. Gambino G, Perrone I, Carra A, Chitarra W, Boccacci P, Marinoni DT, Barberis M, Maghuly F, Laimer M, Gribaudo I (2010) Transgene silencing in grapevines transformed with GFLV resistance genes: analysis of variable expression of transgene, siRNAs production and cytosine methylation. Transgenic Res 19:17–27CrossRefPubMedGoogle Scholar
  23. Génissel A, Leplé J-C, Millet N, Augustin S, Jouanin L, Pilate G (2003) High tolerance against Chrysomela tremulae of transgenic poplar plants expressing a synthetic cry3Aa gene from Bacillus thuringiensis ssp tenebrionis. Mol Breed 11:103–110CrossRefGoogle Scholar
  24. Han K-H, Meilan R, Ma C, Strauss SH (2000) An Agrobacterium tumefaciens transformation protocol effective on a variety of cottonwood hybrids (genus Populus). Plant Cell Rep 19:315–320CrossRefGoogle Scholar
  25. Harcourt RL, Kyozuka J, Floyd RB, Bateman KS, Tanaka H, Decroocq V, Llewellyn DJ, Zhu X, Peacock WJ, Dennis ES (2000) Insect- and herbicide-resistant transgenic eucalypts. Mol Breed 6:307–315CrossRefGoogle Scholar
  26. Herms DA, McCullough DG (2014) Emerald ash borer invasion of North America: history, biology, ecology, impacts, and management. Annu Rev Entomol 59:13–30CrossRefPubMedGoogle Scholar
  27. Khatodia S, Kharb P, Batra P, Chowdhury VK (2014) Development and characterization of transgenic chickpea (Cicer arietinum L.) plants with cry1Ac gene using tissue culture independent protocol. Int J Adv Res 2:323–331Google Scholar
  28. Ko T-S, Lee S, Krasnyanski S, Korban SS (2003) Two critical factors are required for efficient transformation of multiple soybean cultivars: Agrobacterium strain and orientation of immature cotyledonary explant. Theor Appl Genet 107:439–447CrossRefPubMedGoogle Scholar
  29. Kohli A, Twyman RM, Abranches R, Wegel E, Stoger E, Christou P (2003) Transgene integration, organization and interaction in plants. Plant Mol Biol 52:247–258CrossRefPubMedGoogle Scholar
  30. Lachance D, Hamel L-P, Pelletier F, Valéro J, Bernier-Cardou M, Chapman K, van Frankenhuyzen K, Séguin A (2007) Expression of a Bacillus thuringiensis cry1Ab gene in transgenic white spruce and its efficacy against the spruce budworm (Choristoneura fumiferana). Tree Genet Genomes 3:153–167CrossRefGoogle Scholar
  31. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  32. Larkin PJ, Scowcroft WR (1981) Somaclonal variation—a novel source of variability from cell cultures for plant improvement. Theor Appl Genet 60:197–214CrossRefPubMedGoogle Scholar
  33. Lee JH, Pijut PM (2017a) Adventitious shoot regeneration from in vitro leaf explants of Fraxinus nigra. Plant Cell Tiss Organ Cult 130:335–343CrossRefGoogle Scholar
  34. Lee JH, Pijut PM (2017b) Isolation and characterization of a floral homeotic gene in Fraxinus nigra causing earlier flowering and homeotic alterations in transgenic Arabidopsis. Plant Gene 10:17–25CrossRefGoogle Scholar
  35. Lefort F, Douglas GC (1999) An efficient micro-method of DNA isolation from mature leaves of four hardwood tree species Acer, Fraxinus, Prunus and Quercus. Ann For Sci 56:259–263CrossRefGoogle Scholar
  36. Li ZT, Jayasankar S, Gray DJ (2004) Bi-directional duplex promoters with duplicated enhancers significantly increase transgene expression in grape and tobacco. Transgenic Res 13:143–154CrossRefPubMedGoogle Scholar
  37. Li J-F, Park E, von Arnim AG, Nebenführ A (2009) The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species. Plant Methods 5:6CrossRefPubMedPubMedCentralGoogle Scholar
  38. Liu Z, Park B-J, Kanno A, Kameya T (2005) The novel use of a combination of sonication and vacuum infiltration in Agrobacterium-mediated transformation of kidney bean (Phaseolus vulgaris L.) with lea gene. Mol Breed 16:189–197CrossRefGoogle Scholar
  39. Liu S, Su L, Liu S, Zeng X, Zheng D, Hong L, Li L (2016) Agrobacterium rhizogenes-mediated transformation of Arachis hypogaea: an efficient tool for functional study of genes. Biotechnol Biotech Equip 30:869–878CrossRefGoogle Scholar
  40. Makarevitch I, Svitashev SK, Somers DA (2003) Complete sequence analysis of transgene loci from plants transformed via microprojectile bombardment. Plant Mol Biol 52:421–432CrossRefPubMedGoogle Scholar
  41. Matzke AJM, Neuhuber F, Park YD, Ambros PF, Matzke MA (1994) Homology-dependent gene silencing in transgenic plants: epistatic silencing loci contain multiple copies of methylated transgenes. Mol Gen Genet 244:219–229CrossRefPubMedGoogle Scholar
  42. Mercader RJ, Siegert NW, Liebhold AM, McCullough DG (2009) Dispersal of the emerald ash borer, Agrilus planipennis, in newly-colonized sites. Agric For Entomol 11:421–424CrossRefGoogle Scholar
  43. Mishiba K-I, Nishihara M, Nakatsuka T, Abe Y, Hirano H, Yokoi T, Kikuchi A, Yamamura S (2005) Consistent transcriptional silencing of 35S-driven transgenes in gentian. Plant J 44:541–556CrossRefPubMedGoogle Scholar
  44. Muirhead JR, Leung B, van Overdijk C, Kelly DW, Nandakumar K, Marchant KR, MacIsaac HJ (2006) Modelling local and long-distance dispersal of invasive emerald ash borer Agrilus planipennis (Coleoptera) in North America. Divers Distrib 12:71–79CrossRefGoogle Scholar
  45. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  46. Murray EE, Rocheleau T, Eberle M, Stock C, Sekar V, Adang M (1991) Analysis of unstable RNA transcripts of insecticidal crystal protein genes of Bacillus thuringiensis in transgenic plants and electroporated protoplasts. Plant Mol Biol 16:1035–1050CrossRefPubMedGoogle Scholar
  47. Okumura A, Shimada A, Yamasaki S, Horino T, Iwata Y, Koizumi N, Nishihara M, Mishiba K-I (2016) CaMV-35S promoter sequence-specific DNA methylation in lettuce. Plant Cell Rep 35:43–51CrossRefPubMedGoogle Scholar
  48. Palla KJ, Pijut PM (2015) Agrobacterium-mediated genetic transformation of Fraxinus americana hypocotyls. Plant Cell Tiss Organ Cult 120:631–641CrossRefGoogle Scholar
  49. Pawlowski WP, Somers DA (1998) Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA. PNAS 95:12106–12110CrossRefPubMedGoogle Scholar
  50. Poland TM, Ciaramitaro TM, McCullough DG (2016) Laboratory evaluation of the toxicity of systemic insecticides to emerald ash borer larvae. J Econ Entomol 109:705–716CrossRefPubMedGoogle Scholar
  51. Rawat P, Singh AK, Ray K, Chaudhary B, Kumar S, Gautam T, Kanoria S, Kaur G, Kumar P, Pental D, Burma PK (2011) Detrimental effect of expression of Bt endotoxin Cry1Ac on in vitro regeneration, in vivo growth and development of tobacco and cotton transgenics. J Biosci 36:363–376CrossRefPubMedGoogle Scholar
  52. Roome WJ (1992) Agrobacterium-mediated transformation of two forest tree species Prunus serotina and Fraxinus pennsylvanica. MS Thesis, College of Environmental Science and Forestry, State University of New YorkGoogle Scholar
  53. Sachs ES, Benedict JH, Stelly DM, Taylor JF, Altman DW, Berberich SA, Davis SK (1998) Expression and segregation of genes encoding CryIA insecticidal proteins in cotton. Crop Sci 38:1–11CrossRefGoogle Scholar
  54. SAS® Institute Inc. (2011) SAS® 9.3 software package. SAS® Institute Inc., CaryGoogle Scholar
  55. Shin D-I, Podila GK, Huang Y, Karnosky DF (1994) Transgenic larch expressing genes for herbicide and insect resistance. Can J For Res 24:2059–2067CrossRefGoogle Scholar
  56. Shu Q, Cui H, Ye G, Wu D, Xia Y, Gao M, Altosaar I (2002) Agronomic and morphological characterization of Agrobacterium-transformed Bt rice plants. Euphytica 127:345–352CrossRefGoogle Scholar
  57. Singh AK, Paritosh K, Kant U, Burma PK, Pental D (2016) High expression of Cry1Ac protein in cotton (Gossypium hirsutum) by combining independent transgenic events that target the protein to cytoplasm and plastids. PLoS ONE 11:e0158603CrossRefPubMedPubMedCentralGoogle Scholar
  58. Song Y, Canli FA, Meerja F, Wang X, Henry HAL, An L, Tian L (2011) Evaluation of factors affecting European plum (Prunus domestica L.) genetic transformation. Genetic Transformation, Alvarez M (Ed.), InTech, Rijeka, Available: https://www.intechopen.com/books/genetic-transformation/evaluation-of-factors-affecting-european-plum-prunus-domestica-l-genetic-transformation
  59. Sreeramanan S, Vinod B, Sashi S, Xavier R (2008) Optimization of the transient gusA gene transfer of Phalaenopsis violacea orchid via Agrobacterium tumefaciens: an assessment of factors influencing the efficiency of gene transfer mechanisms. Adv Nat Appl Sci 2:77–88Google Scholar
  60. Stevens ME, Pijut PM (2014) Agrobacterium-mediated genetic transformation and plant regeneration of the hardwood tree species Fraxinus profunda. Plant Cell Rep 33:861–870CrossRefPubMedGoogle Scholar
  61. Subramanyam K, Subramanyam K, Sailaja KV, Srinivasulu M, Lakshmidevi K (2011) Highly efficient Agrobacterium-mediated transformation of banana cv. Rasthali (AAB) via sonication and vacuum infiltration. Plant Cell Rep 30:425–436CrossRefPubMedGoogle Scholar
  62. Takata N, Eriksson ME (2012) A simple and efficient transient transformation for hybrid aspen (Populus tremula × P. tremuloides). Plant Methods 8:30CrossRefPubMedPubMedCentralGoogle Scholar
  63. Tang W, Tian Y (2003) Transgenic loblolly pine (Pinus taeda L.) plants expressing a modified δ-endotoxin gene of Bacillus thuringiensis with enhanced resistance to Dendrolimus punctatus Walker and Crypyothelea formosicola Staud. J Exp Bot 54:835–844CrossRefPubMedGoogle Scholar
  64. Tang W, Newton RJ, Weidner DA (2007) Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine. J Exp Bot 58:545–554CrossRefPubMedGoogle Scholar
  65. Telander AC, Slesak RA, D’Amato AW, Palik BJ, Brooks KN, Lenhart CF (2015) Sap flow of black ash in wetland forests of northern Minnesota, USA: hydrologic implications of tree mortality due to emerald ash borer. Agric For Meteorol 206:4–11CrossRefGoogle Scholar
  66. Trick HN, Finer JJ (1997) SAAT: sonication-assisted Agrobacterium-mediated transformation. Transgenic Res 6:329–336CrossRefGoogle Scholar
  67. USDA–APHIS/ARS/FS (2017) Emerald ash borer biological control release and recovery guidelines. USDA–APHIS–ARS-FS, Riverdale, MarylandGoogle Scholar
  68. van Lijsebettens M, Vanderhaeghen R, Van Montagu M (1991) Insertional mutagenesis in Arabidopsis thaliana: isolation of a T-DNA-linked mutation that alters leaf morphology. Theor Appl Genet 81:277–284CrossRefPubMedGoogle Scholar
  69. van Sambeek JW, Preece JE, Navarrete-Tindall NE (2001) Comparative in vitro culture of white and green ash from seed to plantlet production. Comb Proc Int Plant Prop Soc 51:526–534Google Scholar
  70. Wang Y, Pijut PM (2014) Improvement of Agrobacterium-mediated transformation and rooting of black cherry. In Vitro Cell Dev Biol Plant 50:307–316CrossRefGoogle Scholar
  71. Weng H, Pan A, Yang L, Zhang C, Liu Z, Zhang D (2004) Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with HMG I/Y as an endogenous reference gene. Plant Mol Biol Rep 22:289–300CrossRefGoogle Scholar
  72. Whitehill JGA, Opiyo SO, Koch JL, Herms DA, Cipollini DF, Bonello P (2012) Interspecific comparison of constitutive ash phloem phenolic chemistry reveals compounds unique to manchurian ash, a species resistant to emerald ash borer. J Chem Ecol 38:499–511CrossRefPubMedGoogle Scholar
  73. Willow AJ (2011) Indigenizing invasive species management: native North Americans and the emerald ash borer (EAB) beetle. Cult Agric Food Environ 33:70–82CrossRefGoogle Scholar
  74. Yong WTL, Abdullah JO, Mahmood M (2006) Optimization of Agrobacterium-mediated transformation parameters for Melastomataceae spp. using green fluorescent protein (GFP) as a reporter. Sci Hort 109:78–85CrossRefGoogle Scholar
  75. Zhang X, Henriques R, Lin S-S, Niu Q-W, Chua N-H (2006) Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nat Protoc 1:641–646CrossRefPubMedGoogle Scholar

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© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2018

Authors and Affiliations

  1. 1.Department of Forestry and Natural Resources, Hardwood Tree Improvement and Regeneration Center (HTIRC)Purdue UniversityWest LafayetteUSA
  2. 2.USDA Forest Service, Northern Research StationHTIRCWest LafayetteUSA

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