Skip to main content
SpringerLink
Log in

Development and optimization of agroinfiltration for soybean

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

Abstract

Key message

Agroinfiltration is an efficient method to study transgene expression in plant tissue. In this study, sonication followed by vacuum infiltration is shown to increase agroinfiltration-mediated GUS expression in soybean.

Abstract

Agroinfiltration, a valuable tool for rapid analysis of gene function, has been used extensively on leaf tissue of Nicotiana benthamiana and several other plant species. However, the application of this approach for gene functionality studies in soybean has been largely unsuccessful. Improvements in agroinfiltration of many plants have been achieved through a variety of approaches to allow better delivery, penetration and infection of Agrobacterium to interior leaf tissues. In this work, an agroinfiltration approach was developed for transient expression in soybean utilizing sonication followed by vacuum infiltration of intact seedlings. The optimal infiltration buffer, sonication time, and vacuum conditions for agroinfiltration of soybean were evaluated by monitoring expression of an introduced β-glucuronidase (GUS) reporter gene. The developed method included the use of an infiltration buffer (10 mM 2-(N-morpholino)ethanesulfonic acid sodium salt, 10 mM MgCl2, 100 µM acetosyringone) supplemented with the reducing agent dithiothreitol, with 30 s sonication followed by vacuum infiltration. These techniques were further applied to evaluate five different Agrobacterium strains and six different plant genetic backgrounds. Among the Agrobacterium strains examined, J2 produced the highest levels of GUS activity and ‘Peking’ was the most responsive genotype.

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
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Andrieu A, Breitler JC, Siré C, Meynard D, Gantet P, Guiderdoni E (2012) An in planta, Agrobacterium-mediated transient gene expression method for inducing gene silencing in rice (Oryza sativa L.) leaves. Rice 5:1–13

    Article  Google Scholar 

  • Bakshi S, Sadhukhan A, Mishra S, Sahoo L (2011) Improved Agrobacterium-mediated transformation of cowpea via sonication and vacuum infiltration. Plant Cell Rep 30:2281–2292

    Article  CAS  PubMed  Google Scholar 

  • Bendahmane A, Querci M, Kanyuka K, Baulcombe DC (2000) Agrobacterium transient expression system as a tool for the isolation of disease resistance genes: application to the Rx2 locus in potato. Plant J 21:73–81

    Article  CAS  PubMed  Google Scholar 

  • Bernard RL, Cremeens CR (1988) Registration of ‘Williams 82’ soybean. Crop Sci 28:1027

    Google Scholar 

  • Bernard RL, Lindahl DA (1972) Registration of ‘Williams’ soybean. Crop Sci 12:716

    Article  Google Scholar 

  • Bernard RL, Nelson RL, Cremeens CR (1991) USDA Soybean genetic collection: isoline collection. Soybean Genetics Newsletter 18:27–57

    Google Scholar 

  • Bush AL, Pueppke SG (1991) Cultivar-strain specificity between Chrysanthemum morifolium and Agrobacterium tumefaciens. Physiol Mol Plant Pathol 39:309–323

    Article  Google Scholar 

  • Byrne MC, McDonnell RE, Wright MS, Carnes MG (1987) Strain and cultivar specificity in the Agrobacterium-soybean interaction. Plant Cell, Tissue Organ Cult 8:3–15

    Article  CAS  Google Scholar 

  • Cervera M (2004) Histochemical and fluorometric assays for uidA (GUS) Gene Detection. Method Mol Biol 286:203–213

    Google Scholar 

  • Chen X, Equi R, Baxter H, Berk K, Han J, Agarwal S, Zale J (2010) A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings. Biotechnol Biofuels 3:9

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Chiera JM, Bouchard RA, Dorsey SL, Park E, Buenrostro-Nava MT, Ling PP, Finer JJ (2007) Isolation of two highly active soybean (Glycine max (L.) Merr.) promoters and their characterization using a new automated image collection and analysis system. Plant Cell Rep 26:1501–1509

    Article  CAS  PubMed  Google Scholar 

  • Chiu M-H, Chen I-H, Baulcombe DC, Tsai C-H (2010) The silencing suppressor P25 of Potato virus X interacts with Argonaute 1 and mediates its degradation through the proteasome pathway. Mol Plant Pathol 11:641–649

    CAS  PubMed  Google Scholar 

  • Chopra R, Aparna, Saini R (2012) Use of sonication and vacuum infiltration for Agrobacterium-mediated transformation of an Indian lentil (Lens culinaris Medik.) cultivar. Sci Hortic 143:127–134

  • Clapham D, Ekberg I, Eriksson G, Hood EE, Norell L (1990) Within-population variation in susceptibility to Agrobacterium tumefaciens A281 in Picea abies (L.) Karst. Theor Appl Genet 79:654–656

    Article  CAS  PubMed  Google Scholar 

  • de Framond AJ, Barton KA, Chilton MD (1983) Mini–Ti: a new vector strategy for plant genetic engineering. Nat Biotechnol 1:262–269

    Article  Google Scholar 

  • Delzer BW, Somers DA, Orf JH (1990) Agrobacterium tumefaciens susceptibility and plant regeneration of 10 soybean genotypes in maturity groups 00 to II. Crop Sci 30:320–322

    Article  Google Scholar 

  • English JJ, Davenport GF, Elmayan T, Vaucheret H, Baulcombe DC (1997) Requirement of sense transcription for homology-dependent virus resistance and trans-inactivation. Plant J 12:597–603

    Article  CAS  Google Scholar 

  • Fehr WR, Caviness CE, Burmood DT, Pennington JS (1971) Stage of development descriptions for soybeans, Glycine max (L.) Merr. Crop Sci 11:929–931

    Article  Google Scholar 

  • Finer JJ, Larkin KM (2008) Genetic transformation of soybean using particle bombardment and SAAT approaches. In: Kirti PB (ed) Handbook of new technologies for genetic improvement of legumes. CRC Press, Boca Raton, pp 103–125

    Google Scholar 

  • Fujioka Y, Utsumi M, Ohba Y, Watanabe Y (2007) Location of a possible miRNA processing site in SmD3/SmB nuclear bodies in Arabidopsis. Plant Cell Physiol 48:1243–1253

    Article  CAS  PubMed  Google Scholar 

  • Gaba V, Kathiravan K, Amutha S, Singer S, Xiaodi X, Ananthakrishnan G (2006) The uses of ultrasound in plant tissue culture. Plant Tissue Cult Eng 6:417–426

    Google Scholar 

  • Govindarajulu M, Elmore JM, Taylor CG (2008) Evaluation of constitutive viral promoters in transgenic soybean roots and nodules. Mol Plant Microbe Interact 21:1027–1035

    Article  CAS  PubMed  Google Scholar 

  • Hobbs SLA, Jackson JA, Mahon JD (1989) Specificity of strain and genotype in the susceptibility of pea to Agrobacterium tumefaciens. Plant Cell Rep 8:274–277

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Hwang H-H, Wu ET, Liu S-Y, Chang S-C, Tzeng K-C, Kado CI (2013) Characterization and host range of five tumorigenic Agrobacterium tumefaciens strains and possible application in plant transient transformation assays. Plant Pathol 62:1384–1397

    Article  CAS  Google Scholar 

  • Islam R, Malik T, Husnain T, Riazuddin S (1994) Strain and cultivar specificity in the Agrobacterium-chickpea interaction. Plant Cell Rep 13:561–563

    Article  CAS  PubMed  Google Scholar 

  • Kapila J, De Rycke R, Van Montagu M, Angenon G (1997) An Agrobacterium-mediated transient gene expression system for intact leaves. Plant Sci 122:101–108

    Article  CAS  Google Scholar 

  • Klapwijk PM, Scheulderman T, Schilperoort RA (1978) Coordinated regulation of octopine degradation and conjugative transfer of Ti plasmids in Agrobacterium tumefaciens: evidence for a common regulatory gene and separate operons. J Bacteriology 136:775–785

    CAS  Google Scholar 

  • Krasileva KV, Zheng C, Leonelli L, Goritschnig S, Dahlbeck D, Staskawicz BJ (2011) Global analysis of Arabidopsis/downy mildew interactions reveals prevalence of incomplete resistance and rapid evolution of pathogen recognition. PLoS ONE 6:e28765

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Manavella PA, Chan RL (2009) Transient transformation of sunflower leaf discs via an Agrobacterium-mediated method: applications for gene expression and silencing studies. Nat Protoc 4:1699–1707

    Article  CAS  PubMed  Google Scholar 

  • Marion J, Bach L, Bellec Y, Meyer C, Gissot L, Faure JD (2008) Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings. Plant J 56:169–179

    Article  CAS  PubMed  Google Scholar 

  • Mauro AO, Pfeiffer TW, Collins GB (1995) Inheritance of soybean susceptibility to Agrobacterium tumefaciens and its relationship to transformation. Crop Sci 35:1152–1156

    Article  Google Scholar 

  • Meurer CA, Dinkins RD, Collins GB (1998) Factors affecting soybean cotyledonary node transformation. Plant Cell Rep 18:180–186

    Article  CAS  Google Scholar 

  • Moffett P, Farnham G, Peart J, Baulcombe DC (2002) Interaction between domains of a plant NBS-LRR protein in disease resistance-related cell death. The EMBO Journal 21:4511–4519

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Nagamatsu A, Masuta C, Senda M, Matsuura H, Kasai A, Hong JS, Kitamura K, Abe J, Kanazawa A (2007) Functional analysis of soybean genes involved in flavonoid biosynthesis by virus-induced gene silencing. Plant Biotechnol J 5(6):778–790

    Article  CAS  PubMed  Google Scholar 

  • Nickell CD, Noel GR, Thomas DJ, Waller R (1990) Jack soybean. Crop Sci 30:1365

    Article  Google Scholar 

  • Olhoft PM, Lin K, Galbraith J, Nielsen NC, Somers DA (2001) The role of thiol compounds in increasing Agrobacterium-mediated transformation of soybean cotyledonary-node cells. Plant Cell Rep 20:731–737

    Article  CAS  Google Scholar 

  • 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 28:387–395

    Article  CAS  PubMed  Google Scholar 

  • Owens LD, Cress DE (1985) Genotypic variability of soybean response to Agrobacterium strains harboring the Ti or Ri plasmids. Plant Physiol 77:87–94

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Pruss GJ, Nester EW, Vance V (2008) Infiltration with Agrobacterium tumefaciens induces host defense and development-dependent responses in the infiltrated zone. Mol Plant Microbe In 21:1528–1538

    Article  CAS  Google Scholar 

  • Rasband WS (1997–2011) ImageJ, US National Institutes of Health, Bethesda, Maryland, USA. http://imagej.nih.gov/ij/

  • Santarém ER, Trick HN, Essig JS, Finer JJ (1998) Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons: optimization of transient expression. Plant Cell Rep 17:752–759

    Article  Google Scholar 

  • Schmutz J, Cannon SB, Schlueter J et al (2010) Genome sequence of the palaeopolyploid soybean. Nature 463:178–183

    Article  CAS  PubMed  Google Scholar 

  • Sciaky D, Montoya AL, Chilton MD (1978) Fingerprints of Agrobacterium Ti Plasmids. Plasmid 1:238–253

    Article  CAS  PubMed  Google Scholar 

  • Simmons CW, VanderGheynst JS, Upadhyaya SK (2009) A model of Agrobacterium tumefaciens vacuum infiltration into harvested leaf tissue and subsequent in planta transgene transient expression. Biotechnol Bioeng 102:965–970

    Article  CAS  PubMed  Google Scholar 

  • Simmons CW, Nitin N, VanderGheynst JS (2012) Rapid, in situ detection of Agrobacterium tumefaciens attachment to leaf tissue. Biotechnol Progr 28:1321–1328

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Trick HN, Finer JJ (1997) SAAT: Sonication-assisted Agrobacterium-mediated Transformation. Transgenic Res 6:329–337

    Article  CAS  Google Scholar 

  • Trick HN, Finer JJ (1998) Sonication-assisted Agrobacterium-mediated transformation of soybean (Glycine max [L.] Merrill) embryogenic suspension culture tissue. Plant Cell Rep 17:482–488

    Article  CAS  Google Scholar 

  • Van der Hoorn RAL, Laurent F, Roth R, De Wit PJGM (2000) Agroinfiltration is a versatile tool that facilitates analyses 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 H-Y, 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 

  • 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 

  • Wroblewski T, Caldwell KS, Piskurewicz U et al (2009) Comparative large-scale analysis of interactions between several crop species and the effector repertoires from multiple pathovars of Pseudomonas and Ralstonia. Plant Physiol 150:1733–1749

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Yang Y, Li R, Qi M (2000) In vivo analysis of plant promoters and transcription factors by agroinfiltration of tobacco leaves. Plant J 22:543–551

    Article  CAS  PubMed  Google Scholar 

  • Zhang C, Yang C, Whitham SA, Hill JH (2009) Development and use of an efficient DNA-based viral gene silencing vector for soybean. Mol Plant Microbe Interact 22:123–131

    Article  CAS  PubMed  Google Scholar 

  • Zhang C, Bradshaw JD, Whitman SA, Hill JH (2010) The development of an efficient multipurpose Bean Pod Mottle Virus viral vector set for foreign gene expression and RNA silencing. Plant Physiol 153:52–65

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Zottini M, Barizza E, Costa A, Formentin E, Ruberti C, Carimi F, Lo Schiavo F (2008) Agroinfiltration of grapevine leaves for fast transient assays of gene expression and for long-term production of stable transformed cells. Plant Cell Rep 27:845–853

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Angela Parker for assistance in conducting infiltrations. Salaries and research support for this work were provided by the US soybean farmers’ checkoff through the United Soybean Board and State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University, including an OARDC Competitive Research Enhancement Seed Grant (Grant Number 2010-063).

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Horticulture and Crop Science, The Ohio State University, Columbus, OH, 43210, USA

    Jessica L. King & Leah K. McHale

  2. Department of Horticulture and Crop Science, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH, 44691, USA

    John J. Finer

Authors
  1. Jessica L. King
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. John J. Finer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Leah K. McHale
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Leah K. McHale.

Additional information

Communicated by Hiroyasu Ebinuma.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

King, J.L., Finer, J.J. & McHale, L.K. Development and optimization of agroinfiltration for soybean. Plant Cell Rep 34, 133–140 (2015). https://doi.org/10.1007/s00299-014-1694-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-014-1694-4

Keywords

Search

Navigation