Skip to main content
SpringerLink
Log in

Evaluation of factors influencing Agrobacterium-mediated spinach transformation and transformant selection by EGFP fluorescence under low-selective pressure

  • Embryogenesis/Somatic Embryogenesis
  • Published:
In Vitro Cellular & Developmental Biology - Plant Aims and scope Submit manuscript

Abstract

We developed an efficient Agrobacterium-mediated transformation protocol for spinach (Spinacia oleracea L.) that uses root-derived callus. Evaluation of this protocol was based on the systematic evaluation of factors that influence transformation efficiency. Four of the five factors that were tested significantly affected the transformation efficiency, including spinach cultivar, Agrobacterium tumefaciens strain and density, and the duration of co-cultivation. Transgenic spinach plants were generated based on optimized conditions, consisting of callus explants of the cultivar Gyeowoonae, A. tumefaciens strain EHA105 with OD600 of 0.2, a co-cultivation period of 4 d, and 100 μM acetosyringone supplemented in the inoculation and co-cultivation media. After co-cultivation with A. tumefaciens, explants were cultured in low-selective and then non-selective conditions to enhance the growth of putative transgenic explants. Visualization of the fluorescent marker, enhanced green fluorescent protein (EGFP), was used to select transgenic explants at several stages, including callus, somatic cotyledonary embryo, and plantlet. The best results for fluorescence visualization screening were obtained at the somatic cotyledonary embryo stage. On average, 24.96 ± 6.05% of the initial calli regenerated shoots that exhibited EGFP fluorescence. The putative transgenic plants were subjected to β-glucuronidase (GUS)-staining assay, phosphinothricin acetyltransferase (PAT) strip test, and molecular analyses to assess the transgene incorporation into plant genome and its expression. All EGFP-positive plants tested were confirmed to be transgenic by GUS-staining assay, PAT strip test, and molecular analyses. The transformation system described in this study could be a practical and powerful technique for functional genetic analysis and genetic modification of spinach.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.

Similar content being viewed by others

References

  • Aileni M, Abbagani S, Zhang P (2011) Highly efficient production of transgenic Scoparia dulcis L. mediated by Agrobacterium tumefaciens: Plant regeneration via shoot organogenesis. Plant Biotechnol Rep 5:147–156

    Article  Google Scholar 

  • Bao JH, Chin DP, Fukami M, Ugaki M, Nomura M, Mii M (2009) Agrobacterium-mediated transformation of spinach (Spinacia oleracea) with Bacillus thuringiensis cry1Ac gene for resistance against two common vegetable pests. Plant Biotechnol 26:249–254

    Article  CAS  Google Scholar 

  • Chin DP, Bao JH, Mii M (2009) Transgenic spinach plants produced by Agrobacterium-mediated method based on the low temperature-dependent high plant regeneration ability of leaf explants. Plant Biotechnol 26:243–248

    Article  CAS  Google Scholar 

  • Christianson J, McPherson M, Topinka D, Hall L, Good AG (2008) Detecting and quantifying the adventitious presence of transgenic seeds in safflower, Carthamus tinctorius L. J Agric Food Chem 56:5506–5513

    Article  PubMed  CAS  Google Scholar 

  • Duque AS, Araujo SS, Cordeiro MA, Santos DM, Fevereiro MP (2007) Use of fused gfp and gus reporters for the recovery of transformed Medicago truncatula somatic embryos without selective pressure. Plant Cell Tissue Organ Cult 90:325–330

    Article  CAS  Google Scholar 

  • Dutt M, Grosser JW (2009) Evaluation of parameters affecting Agrobacterium-mediated transformation of citrus. Plant Cell Tissue Organ Cult 98:331–340

    Article  CAS  Google Scholar 

  • Elmayan T, Tepfer M (1995) Evaluation in tobacco of the organ specificity and strength of the rolD promoter, domain A of the 35S promoter and the 35S2 promoter. Transgenic Res 4:388–396

    Article  PubMed  CAS  Google Scholar 

  • Hoekema A, Hirsch P, Hooykaas J, Schilperoort R (1983) A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180

    Article  CAS  Google Scholar 

  • Hood E, Gelvin S, Melchers L, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218

    Article  CAS  Google Scholar 

  • Ishizaki T, Hoshino Y, Masuda K, Oosawa K (2002) Explants of Ri-transformed hairy roots of spinach can develop embryogenic calli in the absence of gibberellic acid, an essential growth regulator for induction of embryogenesis from non-transformed roots. Plant Sci 163:223–231

    Article  CAS  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

    PubMed  CAS  Google Scholar 

  • Jian B, Hou W, Wu C, Liu B, Liu W, Song S, Bi Y, Han T (2009) Agrobacterium rhizogenes-mediated transformation of Superroot-derived Lotus corniculatus plants: A valuable tool for functional genomics. BMC Plant Biol. doi:10.1186/1471-2229-9-78

    PubMed  Google Scholar 

  • Jung M, Shin SH, Park JM, Lee SN, Lee MY, Ryu KH, Paek KY, Harn CH (2011) Detection of transgene in early developmental stage by GFP monitoring enhances the efficiency of genetic transformation of pepper. Plant Biotechnol Rep 5:157–167

    Article  PubMed  Google Scholar 

  • Karimi M, Inzé D, Depicker A (2002) Gateway vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195

    Article  PubMed  CAS  Google Scholar 

  • Karthikeyan A, Pandian SK, Ramesh M (2011) Agrobacterium-mediated transformation of leaf base derived callus tissues of popular indica rice (Oryza sativa L. sub sp. indica cv. ADT 43). Plant Sci 181:258–268

    Article  PubMed  CAS  Google Scholar 

  • Knoll KA, Short KC, Curtis IS, Power JB, Davey MR (1997) Shoot regeneration from cultured root explants of spinach (Spinacia oleracea L.): A system for Agrobacterium transformation. Plant Cell Rep 17:96–101

    Article  CAS  Google Scholar 

  • Lee SH, Lee DG, Woo HS, Lee KW, Kim DH, Kwak SS, Kim JS, Kim H, Ahsan N, Choi MS, Yang JK, Lee BH (2006) Production of transgenic orchardgrass via Agrobacterium-mediated transformation of seed-derived callus tissues. Plant Sci 171:408–414

    Article  PubMed  CAS  Google Scholar 

  • Mishra M, Devi S, McCormac A, Scott N, Chen D, Elliott M, Slater A (2010) Green fluorescent protein as a visual selection marker for coffee transformation. Biologia 65:639–646

    Article  CAS  Google Scholar 

  • Mukeshimana G, Ma Y, Walworth AE, Song G-q, Kelly JD (2012) Factors influencing regeneration and Agrobacterium tumefaciens mediated transformation of common bean Phaseolus vulgaris L. Plant Biotechnol Rep. doi:10.1007/s11816-012-0237-0

    Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Mußmann V, Serek M, Winkelmann T (2011) Selection of transgenic Petunia plants using the green fluorescent protein (GFP). Plant Cell Tissue Organ Cult 107:483–392

    Article  Google Scholar 

  • Neuhaus-Url G, Neuhaus G (1993) The use of the nonradioactive digoxigenin chemiluminescent technology for plant genomic Southern blot hybridization: A comparison with radioactivity. Transgenic Res 2:115–120

    Article  Google Scholar 

  • Nguyen QV, Sun HJ, Boo KH, Lee D, Lee J-H, Lim PO, Lee HY, Riu K-Z, Lee D-S (2013) Effect of plant growth regulator combination and culture period on in vitro regeneration of spinach (Spinacia oleracea L.). Plant Biotechnol Rep 7:99–108. doi:10.1007/s11816-012-0242-3

    Article  Google Scholar 

  • Padilla IMG, Golis A, Gentile A, Damiano C, Scorza R (2006) Evaluation of transformation in peach Prunus persica explants using green fluorescent protein (GFP) and beta-glucuronidase (GUS) reporter genes. Plant Cell Tissue Organ Cult 84:309–314. doi:10.1007/s11240-005-9039-1

    Article  CAS  Google Scholar 

  • Prasher D, Eckenrode V, Ward W, Prendergast F, Cormier M (1992) Primary structure of the Aequorea victoria green fluorescent protein. Gene 111:229–233

    Article  PubMed  CAS  Google Scholar 

  • Ribas AF, Dechamp E, Champion A, Bertrand B, Combes MC, Verdeil JL, Lapeyre F, Lashermes P, Etienne H (2011) Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures. BMC Plant Biol. doi:10.1186/1471-2229-11-92

    PubMed  Google Scholar 

  • Song G-q, Walworth A, Hancock JF (2012) Factors influencing Agrobacterium-mediated transformation of switchgrass cultivars. Plant Cell Tissue Organ Cult 108:445–453. doi:10.1007/s11240-011-0056-y

    Article  CAS  Google Scholar 

  • Stave J (2002) Protein immunoassay methods for detection of biotech crops: applications, limitations, and practical considerations. J AOAC Int 85:780–786

    PubMed  CAS  Google Scholar 

  • Sun H-J, Kang H-G, Bae T-W, Cho T-G, Kim J, Lim P-O, Riu K-Z, Lee H-Y (2010) Assessment of phosphinothricin acetyltransferase (PAT) degradation from transgenic zoysiagrass digested with simulated gastric fluid (SGF). J Plant Biol 53:113–120

    Article  CAS  Google Scholar 

  • Van den Bulcke M, Schrijver AD, Bernardi DD, Devos Y, MbongoMbella G, Casi AL, Moens W, Sneyers M (2007) Detection of genetically modified plant products by protein strip testing: An evaluation of real-life samples. Eur Food Res Technol 225:49–57

    Article  Google Scholar 

  • Wakasa Y, Ozawa K, Takaiwa F (2012) Agrobacterium-mediated co-transformation of rice using two selectable marker genes derived from rice genome components. Plant Cell Rep. doi:10.1007/s00299-012-1318-9

    PubMed  Google Scholar 

  • Yang Y, Bao M, Liu G (2010) Factors affecting Agrobacterium-mediated genetic transformation of embryogenic callus of Parthenocissus tricuspidata Planch. Plant Cell Tissue Organ Cult 102:373–380

    Article  CAS  Google Scholar 

  • Yang YM, Al-Khayri JM, Anderson EJ (1997) Transgenic spinach plants expressing the coat protein of cucumber mosaic virus. In Vitro Cell Dev Biol Plant 33:200–204

    Article  CAS  Google Scholar 

  • Zhang HX, Zeevaart JAD (1999) An efficient Agrobacterium tumefaciens-mediated transformation and regeneration system for cotyledons of spinach (Spinacia oleracea L.). Plant Cell Rep 18:640–645

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the grants from Priority Research Centers Program (2012048080) and Mid-career Researcher Program (2011–0027519) through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology, and the Next-Generation BioGreen 21 Program (PJ009094 & PJ008122), Rural Development Administration, Republic of Korea.

Author information

Authors and Affiliations

  1. College of Applied Life Science, Jeju National University, Jeju, 690-756, Republic of Korea

    Quyen Van Nguyen, Kyung Hwan Boo, Hyeon Jin Sun, Dang Viet Cao, Doseung Lee, Seung Hee Ko, Seungtae Kang, Seonyoung Yoon, Seong Cheol Kim, Se Pill Park, Key-Zung Riu & Dong-Sun Lee

  2. Center for Environmental Research and Education, Hanoi National University of Education, Hanoi, Vietnam

    Quyen Van Nguyen

  3. Subtropical Horticulture Research Institute, Jeju National University, Jeju, 690-756, Republic of Korea

    Kyung Hwan Boo, Hyeon Jin Sun, Key-Zung Riu & Dong-Sun Lee

  4. Agricultural Research Center for Climate Change, National Institute of Horticultural & Herbal Science, Rural Development Administration, Jeju, 690-150, Republic of Korea

    Seong Cheol Kim

Authors
  1. Quyen Van Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kyung Hwan Boo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hyeon Jin Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dang Viet Cao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Doseung Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Seung Hee Ko
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Seungtae Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Seonyoung Yoon
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Seong Cheol Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Se Pill Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Key-Zung Riu
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Dong-Sun Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Key-Zung Riu or Dong-Sun Lee.

Additional information

Editor: J. Forster

Quyen Van Nguyen and Kyung Hwan Boo contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nguyen, Q.V., Boo, K.H., Sun, H.J. et al. Evaluation of factors influencing Agrobacterium-mediated spinach transformation and transformant selection by EGFP fluorescence under low-selective pressure. In Vitro Cell.Dev.Biol.-Plant 49, 498–509 (2013). https://doi.org/10.1007/s11627-013-9534-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11627-013-9534-8

Keywords

Search

Navigation