Skip to main content
SpringerLink
Log in

Effect of Agrobacterium strain and plasmid copy number on transformation frequency, event quality and usable event quality in an elite maize cultivar

  • Original Paper
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Key message

Improving Agrobacterium -mediated transformation frequency and event quality by increasing binary plasmid copy number and appropriate strain selection is reported in an elite maize cultivar.

Abstract

Agrobacterium-mediated maize transformation is a well-established method for gene testing and for introducing useful traits in a commercial biotech product pipeline. To develop a highly efficient maize transformation system, we investigated the effect of two Agrobacterium tumefaciens strains and three different binary plasmid origins of replication (ORI) on transformation frequency, vector backbone insertion, single copy event frequency (percentage of events which are single copy for all transgenes), quality event frequency (percentage of single copy events with no vector backbone insertions among all events generated; QE) and usable event quality frequency (transformation frequency times QE frequency; UE) in an elite maize cultivar PHR03. Agrobacterium strain AGL0 gave a higher transformation frequency, but a reduced QE frequency than LBA4404 due to a higher number of vector backbone insertions. Higher binary plasmid copy number positively correlated with transformation frequency and usable event recovery. The above findings can be exploited to develop high-throughput transformation protocols, improve the quality of transgenic events in maize and other plants.

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

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Abbreviations

SC %:

Single copy frequency (percentage of events which are single copy for all transgenes)

BB %:

Backbone minus frequency (percentage of events which no vector backbone insertions among all the events analyzed transgenes)

QE %:

Quality event frequency (percentage of single copy events with no vector backbone insertions among all events analyzed)

UE:

Usable event quality frequency (transformation frequency times QE frequency)

References

  • Al-Abed D, Rudrabhatla S, Talla R, Goldman S (2006) Split-seed: a new tool for maize researchers. Planta 223:1355–1360

    Article  CAS  PubMed  Google Scholar 

  • Anand A, Vaghchhipawala ZE, Mysore KS (2010) Genomics of Agrobacterium–plant interaction: An approach to refine the plant transformation technology. In: Plant transformation technologies. Wiley-Blackwell, pp 31–49

  • Cao S-l, Masilamany P, Li W-b, Pauls KP (2014) Agrobacterium tumefaciens-mediated transformation of corn (Zea mays L.) multiple shoots. Biotechnol Biotec Eq 28:208–216

    Article  CAS  Google Scholar 

  • Cervantes-Rivera R, Pedraza-López F, Pérez-Segura G, Cevallos MA (2011) The replication origin of a repABC plasmid. BMC Microbiol 11:1–14

    Article  Google Scholar 

  • Cho H, Winans SC (2005) VirA and VirG activate the Ti plasmid repABC operon, elevating plasmid copy number in response to wound-released chemical signals. Proc Natl Acad Sci USA 102:14843–14848

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Cho M-J, Wu E, Kwan J, Yu M, Banh J, Linn W, Anand A, Li Z, TeRonde S, Register J III, Jones T, Zhao Z-Y (2014) Agrobacterium-mediated high-frequency transformation of an elite commercial maize (Zea mays L.) inbred line. Plant Cell Rep 33:1767–1777

    Article  CAS  PubMed  Google Scholar 

  • Frame BR, Shou H, Chikwamba RK, Zhang Z, Xiang C, Fonger TM, Pegg SEK, Li B, Nettleton DS, Pei D, Wang K (2002) Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system. Plant Physiol 129:13–22

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Frame BR, McMurray JM, Fonger TM, Main ML, Taylor KW, Torney FJ, Paz MM, Wang K (2006) Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts. Plant Cell Rep 25:1024–1034

    Article  CAS  PubMed  Google Scholar 

  • Frame B, Main M, Schick R, Wang K (2011) Genetic transformation using maize immature zygotic embryos. In: Thorpe TA, Yeung EC (eds) Plant embryo culture. Humana Press, NewYork, pp 327–341

    Chapter  Google Scholar 

  • Fromm ME, Taylor LP, Walbot V (1986) Stable transformation of maize after gene transfer by electroporation. Nature 319:791–793

    Article  CAS  PubMed  Google Scholar 

  • Fromm ME, Morrish F, Armstrong C, Williams R, Thomas J, Klein TM (1990) Inheritance and expression of chimeric genes in the progeny of transgenic maize plants. Nat Biotech 8:833–839

    Article  CAS  Google Scholar 

  • Gallie DR, Zaitlin D, Perry KL, Kado CI (1984) Characterization of the replication and stability regions of Agrobacterium tumefaciens plasmid pTAR. J Bacteriol 157:739–745

    PubMed Central  CAS  PubMed  Google Scholar 

  • Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the “Gene-Jockeying” tool. Microbiol Mol Biol Rev 67:16–37

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Gordon-Kamm WJ, Spencer TM, Mangano ML, Adams TR, Daines RJ, Start WG, O’Brien JV, Chambers SA, Adams WR, Willetts NG, Rice TB, Mackey CJ, Krueger RW, Kausch AP, Lemaux PG (1990) Transformation of maize cells and regeneration of fertile transgenic plants. Plant Cell 2:603–618

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Henkel CV, den Dulk-Ras A, Zhang X, Hooykaas PJJ (2014) Genome sequence of the octopine-type Agrobacterium tumefaciens strain Ach5. Genome Announc 2:e00225–14. doi:10.1128/genomeA.00225-14

  • Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282

    Article  CAS  PubMed  Google Scholar 

  • Hood EE, Fraley RT, Chilton M-D (1987) Virulence of Agrobacterium tumefaciens strain A281 on legumes. Plant Physiol 83:529–534

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotech 14:745–750

    Article  CAS  Google Scholar 

  • Ishida Y, Saito H, Hiei Y, Komari T (2003) Improved protocol for transformation of maize (zea mays l.) mediated by Agrobacterium tumefaciens. Plant Biotech 20:57–66

    Article  CAS  Google Scholar 

  • Ishida Y, Hiei Y, Komari T (2007) Agrobacterium-mediated transformation of maize. Nat Protocols 2:1614–1621

    Article  CAS  Google Scholar 

  • Jayne S, Barbour E, Meyer T (2000) Methods for improving transformation efficiency. In. US patent 6096947 A

  • Kant P, Gulati A, Harris L, Gleddie S, Singh J, P.K P (2012) Transgenic corn plants with modified ribosomal protein L3 show decreased ear rot disease after inoculation with Fusarium graminearum. Aust J Crop Sci 6:1598–1605

  • Komari T (1990) Transformation of cultured cells of Chenopodium quinoa by binary vectors that carry a fragment of DNA from the virulence region of pTiBo542. Plant Cell Rep 9:303–306

    Article  CAS  PubMed  Google Scholar 

  • Komari T, Hiei Y, Saito Y, Murai N, Kumashiro T (1996) Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J 10:165–174

    Article  CAS  PubMed  Google Scholar 

  • Koziel MG, Beland GL, Bowman C, Carozzi NB, Crenshaw R, Crossland L, Dawson J, Desai N, Hill M, Kadwell S, Launis K, Lewis K, Maddox D, McPherson K, Meghji MR, Merlin E, Rhodes R, Warren GW, Wright M, Evola SV (1993) Field performance of elite transgenic maize plants expressing an insecticidal protein derived from Bacillus thuringiensis. Nat Biotech 11:194–200

    Article  CAS  Google Scholar 

  • Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Nat Biotech 9:963–967

    Article  CAS  Google Scholar 

  • Lee L-Y, Gelvin SB (2004) Osa protein constitutes a strong oncogenic suppression system that can block vir-dependent transfer of incQ plasmids between Agrobacterium cells and the establishment of incQ plasmids in plant cells. J Bacteriol 186:7254–7261

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Lee L-Y, Gelvin SB (2008) T-DNA binary vectors and systems. Plant Physiol 146:325–332

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • McBride KE, Summerfelt KR (1990) Improved binary vectors for Agrobacterium-mediated plant transformation. Plant Mol Biol 14:269–276

    Article  CAS  PubMed  Google Scholar 

  • Negrotto D, Jolley M, Beer S, Wenck AR, Hansen G (2000) The use of phosphomannose-isomerase as a selectable marker to recover transgenic maize plants (Zea mays L.) via Agrobacterium transformation. Plant Cell Rep 19:798–803

    Article  CAS  Google Scholar 

  • Oltmanns H, Frame B, Lee L-Y, Johnson S, Li B, Wang K, Gelvin SB (2010) Generation of backbone-free, low transgene copy plants by launching T-DNA from the Agrobacterium chromosome. Plant Physiol 152:1158–1166

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ooms G, Hooykaas PJJ, Van Veen RJM, Van Beelen P, Regensburg-Tuïnk TJG, Schilperoort RA (1982) Octopine Ti-plasmid deletion mutants of Agrobacterium tumefaciens with emphasis on the right side of the T-region. Plasmid 7:15–29

    Article  CAS  PubMed  Google Scholar 

  • Petti C, Wendt T, Meade C, Mullins E (2009) Evidence of genotype dependency within Agrobacterium tumefaciens in relation to the integration of vector backbone sequence in transgenic Phytophthora infestans-tolerant potato. J Bio and Bioeng 107:301–306

    Article  CAS  Google Scholar 

  • Que Q, Elumalai S, Li X, Zhong H, Nalapalli S, Schweiner M, Fei X, Nuccio M, Kelliher T, Gu W, Chen Z, Chilton M-DM (2014) Maize transformation technology development for commercial event generation. Front Plant Sci 5:379. doi:10.3389/fpls.2014.00379

    Article  PubMed Central  PubMed  Google Scholar 

  • Rhodes CA, Pierce DA, Mettler IJ, Mascarenhas D, Detmer JJ (1988) Genetically transformed maize plants from protoplasts. Science 240:204–207

    Article  CAS  PubMed  Google Scholar 

  • Sidorov V, Gilbertson L, Addae P, Duncan D (2006) Agrobacterium-mediated transformation of seedling-derived maize callus. Plant Cell Rep 25:320–328

    Article  CAS  PubMed  Google Scholar 

  • Sripriya R, Sangeetha M, Parameswari C, Veluthambi B, Veluthambi K (2011) Improved Agrobacterium-mediated co-transformation and selectable marker elimination in transgenic rice by using a high copy number pBin19-derived binary vector. Plant Sci 180:766–774

    Article  CAS  PubMed  Google Scholar 

  • USDA (2013) Major crops grown in the United States. Environmental Protection Agency, United States Department Agriculture. http://www.epa.gov/agriculture/ag101/cropmajor.html

  • USDA (2014) Adoption of Genetically Engineered Crops. U.S Economic Research Service, United States Department of Agriculture. http://www.ers.usda.gov/data-products/adoption-of-genetically-engineered-crops-in-the-us/recent-trends-in-ge-adoption.aspx

  • Veluthambi K, Jayaswal RK, Gelvin SB (1987) Virulence genes A, G, and D mediate the double-stranded border cleavage of T-DNA from the Agrobacterium Ti plasmid. Proc Natl Acad Sci USA 84:1881–1885

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Wu E, Lenderts B, Glassman K, Berezowska-Kaniewska M, Christensen H, Asmus T, Zhen S, Chu U, Cho M-J, Zhao Z-Y (2014) Optimized Agrobacterium-mediated sorghum transformation protocol and molecular data of transgenic sorghum plants. In Vitro Cell Dev Biol-Plant 50:9–18

    Article  Google Scholar 

  • Zhao Z-Y, Gu W, Cai T, Tagliani L, Hondred D, Bond D, Schroeder S, Rudert M, Pierce D (2002) High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize. Mol Breeding 8:323–333

    Article  Google Scholar 

Download references

Acknowledgments

The event quality analysis was supported by molecular characterization group. We would like to thank Andrea Carzoli for her help with plasmid copy number determination assays. The authors also thank Steven Bass for reviewing the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. DuPont Agricultural Biotechnology, DuPont-Pioneer, 8305 NW 62nd Avenue, P. O. Box 7060, Johnston, IA, 50131, USA

    Li Zhi, Susan TeRonde, Sandra Meyer, Maren L. Arling, James C. Register III, Zuo-Yu Zhao, Todd J. Jones & Ajith Anand

Authors
  1. Li Zhi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Susan TeRonde
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sandra Meyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maren L. Arling
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. James C. Register III
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Zuo-Yu Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Todd J. Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Ajith Anand
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ajith Anand.

Additional information

Communicated by Prakash P. Kumar.

L. Zhi and S. TeRonde contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 35 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhi, L., TeRonde, S., Meyer, S. et al. Effect of Agrobacterium strain and plasmid copy number on transformation frequency, event quality and usable event quality in an elite maize cultivar. Plant Cell Rep 34, 745–754 (2015). https://doi.org/10.1007/s00299-014-1734-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-014-1734-0

Keywords

Search

Navigation