Skip to main content
Log in

Quantitative analysis of transgenes in cassava plants using real-time PCR technology

  • Genetic transformation
  • Published:
In Vitro Cellular & Developmental Biology - Plant Aims and scope Submit manuscript

Abstract

To speed up the molecular analysis of cassava transgenic plants, we developed real-time polymerase chain reaction (PCR)-based methods that could be implemented as a tool in the primary scrutiny of putative transgenic plants. We tested for the presence of transgenes, estimated copy number, and quantified messenger RNA (mRNA) levels of genes introduced through Agrobacterium. Copy numbers for the genes ß-glucuronidase and hygromycin phosphortransferase were estimated in 15 transgenic lines. Most lines contained one or two copies of each gene; in some, the copy number was different for the two genes, suggesting rearrangements of the transferred DNA. Six of the 15 lines were analyzed by Southern blot. The copy number so estimated was concordant in most cases. Although real-time PCR was efficient for classifying transgenic lines with one or more transgenes inserted, for conclusive analysis of gene copy number, i.e., in a potential breeding line, the Southern blot may still be required. The transcript levels from both genes were determined in eight lines. High, medium, and low levels of mRNA expression were detected. No direct relationship between copy number and expression level of transgenes was obvious, suggesting that factors like position effects or DNA rearrangements led to differential expression. Quantitative mRNA expression data for the ß-glucuronidase gene agreed with results from histochemical staining. With real-time PCR we could detect high levels of transgene expression in 3-y-old cassava plants maintained and propagated as clones in the greenhouse. This is the first time that real-time PCR is reported to be used for transgene analysis in cassava.

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.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.
Figure 4.

Similar content being viewed by others

References

  • Altschul, S. F.; Madden, T. L.; Schaffer, A. A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389–402; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Bhat, S. R.; Srinivasan, S. Molecular and genetic analyses of transgenic plants: considerations and approaches. Plant Sci. 163: 673–681; 2002.

    Article  CAS  Google Scholar 

  • Bubner, B.; Baldwin, I. T. Use of real-time PCR for determining copy number and zygosity in transgenic plants. Plant Cell. Rep. 23: 263–271; 2004 doi:10.1007/s00299-004-0859-y.

    Article  PubMed  CAS  Google Scholar 

  • Czechowski, T.; Bari, R. P.; Stitt, M.; Scheible, W. R.; Udvardi, M. K. Real-time RT-PCR profiling for over 1400 Arabidopsis transcription factors: unprecedented sensitivity reveals novel root- and shoot-specific genes. Plant J. 38: 366–379; 2004 doi:10.1111/j.1365-313X.2004.02051.x.

    Article  PubMed  CAS  Google Scholar 

  • Dai, S.; Zheng, P.; Marmey, P.; Zhang, S.; Tian, W. Z.; Chen, S. Y.; Beachy, R. N.; Fau, C. Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment. Mol. Breed. 7: 25–33; 2001.

    Article  CAS  Google Scholar 

  • Dellaporta, S. L.; Wood, J.; Hicks, J. B. A plant DNA minipreparation: version II. Plant Mol. Biol. Rep. 1: 19–21; 1983.

    Article  CAS  Google Scholar 

  • Elmayan, T.; Vaucheret, H. Expression of single copies of a strongly expressed 35 s transgene can be silenced post-transcriptionally. Plant J. 9: 787–797; 1996.

    Article  CAS  Google Scholar 

  • Fagard, M.; Vaucheret, H. (Trans) gene silencing in plants: How many mechanisms? Annu. Rev. Plant Physiol. Plant Mol. Biol. 51: 167–194; 2000.

    Article  PubMed  CAS  Google Scholar 

  • Flavell, R. B. Inactivation of gene expression in plants as a consequence of specific sequence duplication. Proc. Natl. Acad. Sci. U.S.A. 91: 3490–3496; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Gachon, C.; Mingam, A.; Charrier, B. Real-time PCR: what relevance to plant studies? J. Expl. Bot. 55: 1445–1454; 2004.

    Article  CAS  Google Scholar 

  • Holland, P. M.; Abramson, R. D.; Watson, R.; Gelfand, D. H. Detection of specific polymerase chain reaction product by utilizing the 5′–3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 88: 7276–7280; 1991.

    Article  PubMed  CAS  Google Scholar 

  • Ingham, D. J.; Beer, S.; Money, S.; Hansen, G. Quantitative real-time PCR assay for determining transgene copy number in transformed plants. Biotechniques 31: 132–140; 2001.

    PubMed  CAS  Google Scholar 

  • Jaimes, H. A. Mejora del protocolo para la transformación genética de yuca (Manihot esculenta Crantz) mediada por Agrobacterium tumefaciens usando callo embriogénico friable. Thesis. Department of Biology, Universidad del Valle, Cali, Colombia, 92 p; 2005.

  • Jefferson, R. A. Assaying chimeric genes in plants: the GUS gene fusion system. Plant. Mol. Biol. Rep. 5: 387–405; 1987.

    Article  CAS  Google Scholar 

  • Jørgensen, K.; Bak, S.; Busk, P. K.; Sørensen, C.; Olsen, C. E.; Puonti-Kaerlas, J.; Møller, B. L. Cassava plants with a depleted cyanogenic glucoside content in leaves and tubers: Distribution of cyanogenic glucosides, their site of synthesis and transport, and blockage of the biosynthesis by RNA interference technology. Plant Physiol. 139: 363–374; 2005.

    Article  PubMed  Google Scholar 

  • Kumpatla, S. P.; Chandrasekharan, M. B.; Iyer, L. M.; Li, G.; Hall, T. C. Genome intruder scanning and modulation systems and transgene silencing. Trends Plant Sci. 3: 97–104; 1998.

    Article  Google Scholar 

  • Li, Z.; Hansen, J. L.; Liu, Y.; Zemetra, R. S.; Berger, P. H. Using real-time PCR to determine copy number in wheat. Plant Mol. Biol. Rep. 22: 179–188; 2004.

    Article  CAS  Google Scholar 

  • Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the \(2^{{\Delta \Delta {\text{CT}}}} \) method. Methods 25: 402–408; 2001 doi:10.1006/meth.2001.1262.

    Article  PubMed  CAS  Google Scholar 

  • Mason, G.; Provero, P.; Vaira, A. M.; Accotto, G. P. Estimating the number of integrations in transformed plants by quantitative real-time PCR. BMC Biotechnol. 2: 20; 2001.

    Article  Google Scholar 

  • Miyamoto, T.; Nakamura, T.; Nagao, I.; Obokata, J. Quantitative analysis of transiently expressed mRNA in particle-bombarded tobacco seedlings. Plant Mol. Biol. Rep. 18: 101–107; 2000.

    Article  CAS  Google Scholar 

  • Olsen, K. M.; Schaal, B. A. Evidence on the origin of cassava: phylogeography of Manihot esculenta. Proc. Natl. Acad. Sci. U.S.A. 96: 5586–5591; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Pfaffl, M. W. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 299: e45; 2001.

    Article  PubMed  CAS  Google Scholar 

  • Sambrook, J.; Fritsch, E. F.; Maniatis, Y. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA1989.

    Google Scholar 

  • Schaart, J. G.; Salentijn, E. M. J.; Krens, F. A. Tissue-specific expression of the β-glucuronidase reporter gene in transgenic strawberry (Fragaria × ananassa) plants. Plant Cell Rep. 21: 313–319; 2002 doi:10.1007/s00299-002-0514-4.

    Article  CAS  Google Scholar 

  • Shou, H.; Frame, B. R.; Whitham, S. A.; Wang, K. Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation. Mol. Breed. 13: 201–208; 2004.

    Article  CAS  Google Scholar 

  • Song, P.; Cai, C. Q.; Skokut, M.; Kosegi, B. D.; Petolino, J. F. Quantitative real-time PCR as a screening tool for estimating transgene copy number in WHISKERS™-derived transgenic maize. Plant Cell Rep. 20: 948–954; 2002 doi:10.1007/s00299-001-0432-x.

    Article  CAS  Google Scholar 

  • Soni, R.; Murray, J. A. H. Isolation of intact DNA and RNA from plant tissues. Anal. Biochem. 218: 474–476; 1994.

    Article  PubMed  CAS  Google Scholar 

  • Taylor, N. J.; Edwards, M.; Kiernan, R. J.; Davey, C.; Blakesley, D.; Henshaw, G. G. Development of friable embryogenic callus and embryogenic suspension cultures in cassava (Manihot esculenta Crantz). Nature Biotech. 14: 726–730; 1996.

    Article  CAS  Google Scholar 

  • Taylor, N.; Chavarriaga, P.; Raemakers, K.; Siritunga, D.; Zhang, P. Development and application of transgenic technologies in cassava. Plant Mol. Biol. 56: 671–688; 2004.

    Article  PubMed  CAS  Google Scholar 

  • Toplak, N.; Okršlar, V.; Stanič-Racman, D.; Gruden, K.; Zěl, J. A high-throughput method for quantifying transgene expression in transformed plants with real-time PCR analysis. Plant Mol. Biol. Rep. 22: 237–250; 2004.

    Article  CAS  Google Scholar 

  • Travella, S.; Ross, S. M.; Harden, J.; Everett, C.; Snape, J. W.; Harwood, W. A. A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques. Plant Cell Rep. 23: 780–789; 2005.

    Article  PubMed  CAS  Google Scholar 

  • Tzu-Ming, P. Current status and detection of genetically modified organism. Journal of Food and Drug Analysis 10: 229–241; 2002.

    Google Scholar 

  • Vaucheret, H.; Fagard, M. Transcriptional gene silencing in plants: targets, inducers and regulators. Trends Genet. 17: 29–35; 2001.

    Article  PubMed  CAS  Google Scholar 

  • Vaucheret, H.; Béclin, C.; Elmayan, T.; Feuerbach, F.; Godon, C.; Morel, J. B.; Mourrain, P.; Palauqui, J. C.; Vernhettes, S. Transgene induced gene silencing in plants. Plant J. 16: 651–659; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Weng, H.; Pan, A.; Yang, L.; Zhang, C.; Liu, Z.; Zhang, D. Estimating number of transgene copies in transgenic rapeseed by real-time PCR assay with HMG I/Y as an endogenous reference gene. Plant Mol. Biol. Rep. 22: 289–300; 2004.

    Article  CAS  Google Scholar 

  • Wilkening, S.; Bader, A. Quantitative real-time polymerase chain reaction: methodical analysis and mathematical model. J. Biomol. Tech. 15: 107–111; 2004.

    PubMed  Google Scholar 

  • Yang, L.; Ding, J.; Zhang, C.; Jia, J.; Weng, H.; Liu, W.; Zhang, D. Estimating the copy number of transgenes in transformed rice by real-time quantitative PCR. Plant Cell Rep. 23: 759–763; 2005 doi:10.1007/s00299-004-0881-0.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors wish to thank Pablo Herrera and Carlos Dorado for their collaboration in maintaining the transgenic cassava lines in the greenhouse. This study was funded by the Harvest-Plus program, under the auspices of the Bill & Melinda Gates Foundation, Swedish International Development Assistance (SIDA), Danish International Development Assistance (DANIDA), The US Agency for International Development (USAID), and The World Bank.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to P. Chavarriaga.

Additional information

Editor: Marc C. Jordan

Rights and permissions

Reprints and permissions

About this article

Cite this article

Beltrán, J., Jaimes, H., Echeverry, M. et al. Quantitative analysis of transgenes in cassava plants using real-time PCR technology. In Vitro Cell.Dev.Biol.-Plant 45, 48–56 (2009). https://doi.org/10.1007/s11627-008-9159-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11627-008-9159-5

Keywords

Navigation