Skip to main content

Knockdown of Polyphenol Oxidase Gene Expression in Potato (Solanum tuberosum L.) with Artificial MicroRNAs

Part of the Methods in Molecular Biology book series (MIMB,volume 1405)

Abstract

It is of great importance and interest to develop crop varieties with low polyphenol oxidase (PPO) activity for the food industry because PPO-mediated oxidative browning is a main cause of post-harvest deterioration and quality loss of fresh produce and processed foods. We recently demonstrated that potato tubers with reduced browning phenotypes can be produced by inhibition of the expression of several PPO gene isoforms using artificial microRNA (amiRNA) technology. The approach introduces a single type of 21-nucleotide RNA population to guide silencing of the PPO gene transcripts in potato tissues. Some advantages of the technology are: small RNA molecules are genetically transformed, off-target gene silencing can be avoided or minimized at the stage of amiRNA designs, and accuracy and efficiency of the processes can be detected at every step using molecular biological techniques. Here we describe the methods for transformation and regeneration of potatoes with amiRNA vectors, detection of the expression of amiRNAs, identification of the cleaved product of the target gene transcripts, and assay of the expression level of PPO gene isoforms in potatoes.

Key words

  • Artificial microRNA (amiRNA)
  • Gene knockdown
  • Polyphenol oxidase (PPO)
  • Potato genetic transformation
  • RNA silencing
  • Solanum tuberosum
  • StuPPO

This is a preview of subscription content, access via your institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Holderbaum DF, Kon T, Kudo T, Guerra MP (2010) Enzymatic browning, polyphenol oxidase activity, and polyphenols in four apple cultivars: dynamics during fruit development. HortScience 45:1150–1154

    Google Scholar 

  2. Mayer AM (2006) Polyphenol oxidases in plants and fungi: going places? A review. Phytochemistry 67:2318–2331

    CrossRef  CAS  PubMed  Google Scholar 

  3. Tran LT, Taylor JS, Constabel CP (2012) The polyphenol oxidase gene family in land plants: lineage-specific duplication and expansion. BMC Genomics 13:395

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  4. Chi M, Bhagwat B, Lane WD, Tang G, Su Y, Sun R, Oomah BD, Wiersma PA, Xiang Y (2014) Reduced polyphenol oxidase gene expression and enzymatic browning in potato (Solanum tuberosum L.) with artificial microRNAs. BMC Plant Biol 14:62

    CrossRef  PubMed Central  PubMed  Google Scholar 

  5. Bachem CW, Speckmann G-J, van der Linde PC, Verheggen FT, Hunt MD, Steffens JC, Zabeau M (1994) Antisense expression of polyphenol oxidase genes inhibits enzymatic browning in potato tubers. Nat Biotechnol 12:1101–1105

    CrossRef  CAS  Google Scholar 

  6. Coetzer C, Corsini D, Love S, Pavek J, Tumer N (2001) Control of enzymatic browning in potato (Solanum tuberosum L.) by sense and antisense RNA from tomato polyphenol oxidase. J Agric Food Chem 49:652–657

    CrossRef  CAS  PubMed  Google Scholar 

  7. Murata M, Nishimura M, Murai N, Haruta M, Homma S, Itoh Y (2001) A transgenic apple callus showing reduced polyphenol oxidase activity and lower browning potential. Biosci Biotechnol Biochem 65:383–388

    CrossRef  CAS  PubMed  Google Scholar 

  8. Richter C, Dirks ME, Gronover CS, Prüfer D, Moerschbacher BM (2012) Silencing and heterologous expression of ppo-2 indicate a specific function of a single polyphenol oxidase isoform in resistance of dandelion (Taraxacum officinale) against Pseudomonas syringae pv. tomato. Mol Plant Microbe Interact 25:200–210

    CrossRef  CAS  PubMed  Google Scholar 

  9. Bhagwat B, Chi M, Su L, Tang H, Tang G, Xiang Y (2013) An in vivo transient expression system can be applied for rapid and effective selection of artificial microRNA constructs for plant stable genetic transformation. J Genet Genomics 40:261–270

    CrossRef  CAS  PubMed  Google Scholar 

  10. Ossowski S, Schwab R, Weigel D (2008) Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J 53:674–690

    CrossRef  CAS  PubMed  Google Scholar 

  11. Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D (2006) Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell 18:1121–1133

    CrossRef  PubMed Central  CAS  PubMed  Google Scholar 

  12. Beaujean A, Sangwan RS, Lecardonnel A, Sangwan-Norreel BS (1998) Agrobacterium-mediated transformation of three economically important potato cultivars using sliced internodal explants: an efficient protocol of transformation. J Exp Bot 49:1589–1595

    CrossRef  CAS  Google Scholar 

  13. Bhagwat B, Chi M, Han D, Tang H, Tang G, Xiang Y (2015) Design, construction and validation of artificial microRNA vectors using Agrobacterium-mediated transient expression system. Methods Mol Biol

    Google Scholar 

  14. Nicot N, Hausman JF, Hoffmann L, Evers D (2005) Housekeeping gene selection for real-time RT-PCR normalization in potato during biotic and abiotic stress. J Exp Bot 56:2907–2914

    Google Scholar 

  15. Millam S (2006) Potato (Solanum tuberosum L.). Methods Mol Biol 344:25–36

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Yu Xiang .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and Permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Chi, M., Bhagwat, B., Tang, G., Xiang, Y. (2016). Knockdown of Polyphenol Oxidase Gene Expression in Potato (Solanum tuberosum L.) with Artificial MicroRNAs. In: Fett-Neto, A. (eds) Biotechnology of Plant Secondary Metabolism. Methods in Molecular Biology, vol 1405. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3393-8_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3393-8_15

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3391-4

  • Online ISBN: 978-1-4939-3393-8

  • eBook Packages: Springer Protocols