Skip to main content
Log in

Agroinfiltration as a technique for rapid assays for evaluating candidate insect resistance transgenes in plants

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


Functional analysis of candidate transgenes for insect resistance in stably transformed plants is a time-consuming task that can take months to achieve in even the fastest of plant models. In this study, a rapid screening technique is described, which employs candidate transgene transient expression using agroinfiltration in Nicotiana benthamiana combined with a simple insect bioassay. Using this system the known insecticidal protein Cry1Ac is demonstrated to effectively control Helicoverpa zea. Insects fed tissue with synthesized GFP (green fluorescent protein) as a positive control were shown to have enhanced growth and development. Additionally, a Brassica oleracea proteinase inhibitor (BoPI), a less characterized insect resistance candidate, demonstrated effectiveness to decrease the growth and development of H. zea at high levels of transient expression. Bioassays performed on stable transformants showed that BoPI had a low level of insect resistance at the more typical levels of gene transcription found in stably transformed plants. This agroinfiltration-insect bioassay procedure can give a rapid assessment of insect resistance significantly decreasing the time needed for evaluation of candidate genes.

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

Access this article

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

Instant access to the full article PDF.

Fig. 1
Fig. 2

Similar content being viewed by others


  • Abbott WS (1925) A method of computing the effectiveness of an insecticide. J Econ Entomol 18:265–276

    CAS  Google Scholar 

  • Barber GW (1936) The cannibalistic habits of the corn ear worm. US Dep Agric Tech Bull 499:1–19

    Google Scholar 

  • Broadway RM (1995) Are insects resistant to plant proteinase inhibitors? J Insect Physiol 41:107–116

    Article  CAS  Google Scholar 

  • Chilcutt CF (2006) Cannibalism of Helicoverpa zea (Lepidoptera: Noctuidae) from Bacillus thuringiensis (Bt) transgenic corn versus non-Bt corn. J Econ Entomol 99:728–732

    Article  PubMed  Google Scholar 

  • Daly H (1985) Insect morphometrics. Annu Rev Entomol 30:415–438

    Article  Google Scholar 

  • De Leo F, Bonadé-Bottino MA, Ceci LR, Gallerani R et al (1998) Opposite effects on Spodoptera littoralis larvae of high expression level of a trypsin proteinase inhibitor in transgenic plants. Plant Physiol 118:997–1004

    Article  PubMed  Google Scholar 

  • Estruch JJ, Carozzi NB, Desai N, Duck NB et al (1997) Transgenic plants: an energing approach to pest control. Nat Biotechnol 15:137–141

    Article  CAS  PubMed  Google Scholar 

  • Girard C, Le Métayer M, Zaccomer B, Bartlet E et al (1998) Growth stimulation of beetle larvae reared on a transgenic oilseed rape expressing a cysteine proteinase inhibitor. J Insect Physiol 44:263–270

    Article  CAS  PubMed  Google Scholar 

  • Gleba Y, Klimyuk V, Marillonnet S (2007) Viral vectors for the expression of proteins in plants. Curr Opin Cell Biol 18:134–141

    CAS  Google Scholar 

  • Harper BK, Mabon SA, Leffel SM, Halfhill MD et al (1999) Green fluorescent protein in transgenic plants indicates the presence and expression of a second gene. Nat Biotechnol 17:1125–1129

    Article  CAS  PubMed  Google Scholar 

  • Janssen B, Gardner R (1989) Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium. Plant Mol Biol 14:61–72

    Article  Google Scholar 

  • Lacomme C, Hrubikova K, Hein I (2003) Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats. Plant J 34:543–553

    Article  CAS  PubMed  Google Scholar 

  • Lawrence SD, Novak NG (2001) A rapid method for the production and characterization of recombinant insecticidal proteins in plants. Mol Breed 8:139–146

    Article  CAS  Google Scholar 

  • Li J, Chen M, Liu X, Zhang H et al (2007) Transient expression of an active human interferon-beta in lettuce. Sci Hortic 112:258–265

    Article  CAS  Google Scholar 

  • McManus MT, White DWR, McGregor PG (1994) Accumulation of a chymotrypsin inhibitor in transgenic tobacco can affect the growth of insect pests. Transgenic Res 3:50–58

    Article  CAS  Google Scholar 

  • Millwood RJ, Halfhill MD, Harkins D, Rusotti R et al (2003) Instrumentation and methodology for quantifying GFP fluorescence in intact plant organs. BioTechniques 34:638–643

    CAS  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473

    Article  CAS  Google Scholar 

  • Neunzig HH (1969) The biology of the tobacco budworm and the corn earworm in North Carolina with particular reference to tobacco as a host. N C Agric Exp Station Tech Bull 196:1–63

    Google Scholar 

  • Pulliam DA, Williams DL, Broadway RM, Stewart CN Jr (2001) Isolation and characterization of a serine proteinase inhibitor cDNA from cabbage and its antibiosis in transgenic tobacco plants. Plant Cell Biotechnol Mol Biol 2:19–32

    Google Scholar 

  • Santos-Rosa M, Poutaraud A, Merdinoglu D, Mestre P (2008) Development of a transient expression system in grapevine via agro-infiltration. Plant Cell Rep 27:1053–1063

    Article  CAS  PubMed  Google Scholar 

  • SAS Institute (2003) SAS/STAT version 9.1. SAS Institute, Inc, Cary

  • Shelton AM, Zhao J, Roush RT (2002) Economic, ecological, food safety, and social consequences of the deployment of Bt transgenic plants. Ann Rev Entomol 47:845–881

    Article  CAS  Google Scholar 

  • Sheludko YV, Sindarovska YR, Gerasymenko IM, Bannikova MA et al (2006) Comparison of several Nicotiana species as host for high-scale Agrobacterium-mediated transient expression. Biotechnol Bioeng 96:608–614

    Article  Google Scholar 

  • Sparkes IA, Runions J, Kearns A, Hawes C (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 1:2019–2025

    Article  CAS  PubMed  Google Scholar 

  • Stewart CN Jr, Adang MJ, All JN, Raymer PL et al (1996) Insect control and dosage effects in transgenic canola, Brassica napus L. (Brassicaceae), containing a synthetic Bacillus thuringiensis CryIA(c) gene. Plant Physiol 112:115–120

    Article  CAS  Google Scholar 

  • Van der Hoorn RAL, Laurent F, Roth R, De Wit PJ (2000) Agroinfiltration is a versatile tool that facilitates comparative analysis of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis. Mol Plant Microbe Interact 13:439–446

    Article  PubMed  Google Scholar 

  • VanderGheynst JS, Guo HY, Simmons CW (2008) Response surface studies that elucidate the role of infiltration conditions on Agrobacterium tumefaciens-mediated transient transgene expression in harvested switchgrass (Panicum virgatum). Biomass Bioenergy 32:372–379

    CAS  Google Scholar 

  • Voinnett O, Rivas S, Mestre P, Baulcombe D (2003) An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 3:259–273

    Google Scholar 

  • Wroblewski T, Tomczak A, Michelmore R (2005) Optimization of Agrobacterium-mediated transient assays of gene expression in lettuce, tomato, and Arabidopsis. Plant Biotechnol J 3:259–273

    Article  CAS  PubMed  Google Scholar 

  • Yuan J, Wang D, Stewart CN Jr (2008) Statistical methods for efficiency adjusted real-time PCR analysis. Biotechnol J 3:112–123

    Article  CAS  PubMed  Google Scholar 

  • Zhu-Salzman K, Zeng RS (2008) Molecular mechanisims of insect adaptation to plant defense: lessons learned from a Bruchid beetle. Insect Sci 15:477–481

    Article  CAS  Google Scholar 

Download references


We would like to thank Joshua Yuan for his help in real-time RT-PCR analysis, Laura Abercrombie for providing the GFP primers, and Jason Abercrombie for his help with agroinfiltration. This study was funded by the University of Tennessee Racheff Chair of Excellence Graduate Student and Research Funds.

Conflict of interest

The authors have declared no conflict of interests.

Author information

Authors and Affiliations


Corresponding author

Correspondence to C. Neal Stewart Jr.

Additional information

Communicated by P. Lakshmanan.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Leckie, B.M., Neal Stewart, C. Agroinfiltration as a technique for rapid assays for evaluating candidate insect resistance transgenes in plants. Plant Cell Rep 30, 325–334 (2011).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: