Plant Cell Reports

, Volume 32, Issue 3, pp 329–337 | Cite as

Development of an efficient transformation method by Agrobacterium tumefaciens and high throughput spray assay to identify transgenic plants for woodland strawberry (Fragaria vesca) using NPTII selection

  • Christopher J. Pantazis
  • Sarah Fisk
  • Kerri Mills
  • Barry S. Flinn
  • Vladimir Shulaev
  • Richard E. Veilleux
  • Yinghui Dan
Original Paper

Abstract

Key message

We developed an efficient Agrobacterium -mediated transformation method using an Ac/Ds transposon tagging construct for F. vesca and high throughput paromomycin spray assay to identify its transformants for strawberry functional genomics.

Abstract

Genomic resources for Rosaceae species are now readily available, including the Fragaria vesca genome, EST sequences, markers, linkage maps, and physical maps. The Rosaceae Genomic Executive Committee has promoted strawberry as a translational genomics model due to its unique biological features and transformability for fruit trait improvement. Our overall research goal is to use functional genomic and metabolic approaches to pursue high throughput gene discovery in the diploid woodland strawberry. F. vesca offers several advantages of a fleshy fruit typical of most fruit crops, short life cycle (seed to seed in 12–16 weeks), small genome size (206 Mbb/C), small plant size, self-compatibility, and many seeds per plant. We have developed an efficient Agrobacterium tumefaciens-mediated strawberry transformation method using kanamycin selection, and high throughput paromomycin spray assay to efficiently identify transgenic strawberry plants. Using our kanamycin transformation method, we were able to produce up to 98 independent kanamycin resistant insertional mutant lines using a T-DNA construct carrying an Ac/Ds transposon Launchpad system from a single transformation experiment involving inoculation of 22 leaf explants of F. vesca accession 551572 within approx. 11 weeks (from inoculation to soil). Transgenic plants with 1–2 copies of a transgene were confirmed by Southern blot analysis. Using our paromomycin spray assay, transgenic F. vesca plants were rapidly identified within 10 days after spraying.

Keywords

Agrobacterium tumefaciens-mediated transformation Woodland strawberry Kanamycin selection Paromomycin spray assay 

Notes

Acknowledgments

This work was supported through funds from the “High Value Horticulture and Forestry-Virginia” Special Grants (Project No. 2003-38891-02112) from the United States Department of Agriculture CSREES, USDA/NRI Award (Project No. 2008-35300-04458), the Department of Horticulture, the Virginia Tobacco Indemnification and Community Revitalization Commission and operating funds from the Commonwealth of Virginia to the Institute for Advanced Learning and Research.

References

  1. Abdal-Aziz SA, Pliego-Alfaro F, Quesada MA, Mercado JA (2006) Evidence of frequent integration of non-T-DNA vector backbone sequences in transgenic strawberry plant. J Biosci Bioeng 101:508–510PubMedCrossRefGoogle Scholar
  2. Ayliffe MA, Pallotta M, Langridge P, Pryor AJ (2007) A barley activation tagging system. Plant Mol Biol 64:329–347PubMedCrossRefGoogle Scholar
  3. Cervera M, Juarez J, Navarro A, Pina JA, Duran-Vila N, Navarro L, Pena L (1998) Genetic transformation and regeneration of mature tissue of woody fruit plants bypassing the juvenile stage. Transgenic Res 7:51–59CrossRefGoogle Scholar
  4. Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM, Duncan Dr, Conner TW, Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115:971–980PubMedGoogle Scholar
  5. Cheng M, Lowe BA, Spencer MT, Ye X, Armstrong CL (2004) Factors influencing Agrobacterium-mediated transformation of monocotyledonous species. In Vitro Cell Dev Biol Plant 40:31–45CrossRefGoogle Scholar
  6. Cordero de Mesa M, Jimnez-Bermudez S, Pliego-Alfaro F, Quesada MA, Mercado JA (2000) Agrobacterium cells as microprojectile coating: a novel approach to enhance stable transformation rates in strawberry. Aust J Plant Physiol 27:1093–1100Google Scholar
  7. Cordero de Mesa M, Santiago-Doménech N, Pliego-Alfaro F, Quesada MA, Mercado JA (2004) The CaMV 35S promoter is highly active on floral and pollen of transgenic strawberry plants. Plant Cell Rep 23:32–38PubMedGoogle Scholar
  8. Folta KM, Dhingra A (2006) Transformation of strawberry: the basis for translational genomics in rosaceae. In Vitro Cell Dev Biol Plant 42:482–490CrossRefGoogle Scholar
  9. Ipek A, Masson P, Simon PW (2006) Genetic transformation of an Ac/Ds-based transposon tagging system in carrot (Daucus carota L.). Eur J Hort Sci 71:245–251Google Scholar
  10. James DJ, Passey AJ, Barbara DJ, Bevan M (1989) Genetic transformation of apple (Malus pumila Mill) using a disarmed Tibinary vector. Plant Cell Rep 7:658–661Google Scholar
  11. Kolesnik T, Szeverenyi I, Bachmann D, Kumar CS, Jiang S, Ramamoorthy R, Cai M, Ma ZG, Sundaresan V, Ramachandran S (2004) Establishing an efficient Ac/Ds tagging system in rice: large-scale analysis of Ds flanking sequences. Plant J 37:301–314PubMedCrossRefGoogle Scholar
  12. Kuta DD, Tripathi L (2005) Agrobacterium-induced hypersensitive necrotic reaction in plant cells: a resistance response against Agrobacterium-mediated DNA transfer. Afr J Biotechnol 4(8):752–757Google Scholar
  13. Moore GA, Jacono CC, Neidigh JL, Lawrence SD, Cline K (1992) Agrobacterium-mediated transformation of citrus stem segments and regeneration of transgenic plants. Plant Cell Rep 11:238–242CrossRefGoogle Scholar
  14. Mourgues F, Chevreau E, Lambert C, Bondt A (1996) Efficient Agrobacterium-mediated transformation and recovery of transgenic plants from pear (Pyrus communis L.). Plant Cell Rep 16:245–249Google Scholar
  15. Murkute A, Patil A, Mayakumari S (2003) Exudation and browning in tissue culture of pomegranate. Agric Sci Dig 23(1):29–31Google Scholar
  16. Oosumi T, Gruszewski HA, Blischak LA, Baxter AJ, Wadl PA, Shuman JL, Veilleux RE, Shulaev V (2006) High-efficiency transformation of the diploid strawberry (Fragaria vesca) for functional genomics. Planta 223:1219–1230PubMedCrossRefGoogle Scholar
  17. Oosumi T, Ruiz-Rojas JJ, Veilleux RE, Dickerman A, Shulaev V (2010) Implementing reverse genetics in Rosaceae: analysis of T-DNA flanking sequences of insertional mutant lines in the diploid strawberry, Fragaria vesca. Physiol Plant 140:1–9PubMedCrossRefGoogle Scholar
  18. Pastori GM, Huttly A, West J, Sparks C, Pieters A, Luna CM, Jones HD, Foyer CH (2007) The maize activator/dissociation system is functional in hexaploid wheat through successive generations. Func Plant Biol 34:835–843CrossRefGoogle Scholar
  19. Pena L, Cervera M, Juarez J, Ortega C, Pina JA, Duran-Vila N, Navarro L (1995a) High-efficiency Agrobacterium-mediated transformation and regeneration of citrus. Plant Sci 104:183–191CrossRefGoogle Scholar
  20. Pena L, Cervera M, Juarez J, Navarro A, Pina JA, Duran-Vila N, Navarro L (1995b) Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants. Plant Cell Rep 14:616–619CrossRefGoogle Scholar
  21. Pena L, Cervera M, Juarez J, Navarro A, Pina JA, Navarro L, Rep PC (1997) Genetic transformation of lime (Citrus aurantifolia Swing.): factors affecting transformation and regeneration. Plant Cell Rep 16:731–737CrossRefGoogle Scholar
  22. Perl A, Lotan O, Abu-Abied M, Holland D (1996) Establishment of an Agrobacterium-mediated transformation system for grape (Vitis vinifera L.): establishment of an Agrobacterium-mediated transformation system for grape (Vitis vinifera L.): the role of antioxidants during grape-Agrobacterium interactions. Nat Biotechnol 14(5):624–628PubMedCrossRefGoogle Scholar
  23. Qin Y, Teixeira da Silva JA, Zhang L, Zhang S (2008) Transgenic strawberry: state of the art for improved traits. Biotechnol Adv 26:219–232PubMedCrossRefGoogle Scholar
  24. Qin YH, Teixeira da Silva JA, Bi JH, Zhang SL, Hu GB (2011) Response of in vitro strawberry to antibiotics. Plant Growth Regul 65:183–193CrossRefGoogle Scholar
  25. Sheikholeslam SN, Weeks DP (1987) Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens. Plant Mol Bio 8:291–298CrossRefGoogle Scholar
  26. Shulaev V, Sargent DJ, Crowhurst RN et al (2011) The genome of woodland strawberry (Fragaria vesca). Nat Genet 43:109–116PubMedCrossRefGoogle Scholar
  27. Stipic M, Rotino GL, Piro F (2000) Regeneration and genetic transformation attempts in the cauliflower ‘Tardivo di Fano’. Italus Hortus 7(2):20–26Google Scholar
  28. Veilleux RE, Mills KP, Baxter AJ, Upham KT, Ferguson TJ, Holt SH, Lu N, Ruiz-Rojas JJ, Pantazis C, Davis CM, Lindsey RC, Powell FL, Dan Y, Dickerman A, Oosumi T, Shulaev V (2012) Transposon tagging in diploid strawberry. Plant Biotechnol J 10:985–994PubMedCrossRefGoogle Scholar
  29. Woody ST, Austin-Phillips S, Amasino RM, Krysan PJ (2007) The WiscDsLox T-DNA collection: an arabidopsis community resource generated by using an improved high-throughput T-DNA sequencing pipeline. J Plant Res 120:157–165PubMedCrossRefGoogle Scholar
  30. Wu H, Sparks C, Amoah B, Jones HD (2003) Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell Rep 21:659–668PubMedGoogle Scholar
  31. Zale JM, Agarwal S, Loar S, Steber CM (2009) Evidence for stable transformation of wheat by floral dip in Agrobacterium tumefaciens. Plant Cell Rep 28:903–913PubMedCrossRefGoogle Scholar
  32. Zhang HM, Wang JL (2005) Establishment of genetic transformation system of “Allstar” strawberry leaf. Biotechnology 15:68–70Google Scholar
  33. Zhao Y, Liu Q, Davis RE (2004) Transgene expression in strawberries driven by a heterologous phloem-specific promoter. Plant Cell Rep 23:224–230PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Christopher J. Pantazis
    • 1
  • Sarah Fisk
    • 1
  • Kerri Mills
    • 2
  • Barry S. Flinn
    • 1
    • 2
    • 3
  • Vladimir Shulaev
    • 4
  • Richard E. Veilleux
    • 2
  • Yinghui Dan
    • 1
    • 2
    • 3
  1. 1.Institute for Advanced Learning and ResearchDanvilleUSA
  2. 2.Department of HorticultureVirginia Polytechnic Institute and State UniversityBlacksburgUSA
  3. 3.Department of Forest Resources and Environmental ConservationVirginia Polytechnic Institute and State UniversityBlacksburgUSA
  4. 4.Department of Biological SciencesUniversity of North TexasDentonUSA

Personalised recommendations