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A High-throughput Platform for the Production and Analysis of Transgenic Cassava (Manihot esculenta) Plants

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Abstract

A platform for high-throughput production and analysis of transgenic cassava (Manihot esculenta) has been developed for the variety 60444 and implemented to generate plants expressing traits for nutritional enhancement, modified metabolism, promoter analysis and disease resistance. Over a three and a half year period this system has been utilized to produce more than 3500 independent transgenic plant lines from 50 different genetic constructs within a single laboratory. Plants recovered through this system have proven robust and efficacious for engineered traits under greenhouse conditions and within the first confined field trials of transgenic cassava carried out in Uganda, Kenya, Nigeria and Puerto Rico. Detailed procedures are described for the operation of this platform, including all steps in tissue culture, genetic transformation, copy number estimation, greenhouse establishment for shoot and storage root formation and systems for centralized quality control, transgenic plant tracking and regulatory compliance. In addition to providing reliable transgenic plant production for proof of concept studies in the variety 60444, the systems implemented and described here form the structure for high throughput production of transgenic farmer-preferred cultivars of cassava.

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Abbreviations

BAP:

benzylaminopurine

DDPSC:

Donald Danforth Plant Science Center

FEC:

friable embryogenic callus

GD2:

Greshoff and Doy basal medium containing 20 g/l sucrose

GD2 50P:

GD2 supplemented with 50 μM picloram

GFP:

green fluorescent protein

ILTAB:

International Laboratory for Tropical Agricultural Biotechnology

MS2:

Murashige and Skoog basal medium containing 20 g/l sucrose

MS2 0.5NAA:

MS2 supplemented with 0.5 μM naphthalene acetic acid

MS2 2BAP:

MS2 supplemented with 2 μM benzylaminopurine

MS2 50P:

MS2 supplemented with 50 μM picloram

MS2 5NAA:

MS2 supplemented with 5 μM naphthalene acetic acid

NAA:

naphthalene acetic acid

OES:

organised embryogenic callus

SCV:

settled cell volume

VIRCA:

Virus Resistant Cassava for Africa project

References

  • Ayling S, Ferguson M, Rounsley S et al (2012) Information resources for cassava research and breeding. Trop Plant Biol (this issue)

  • Bartlett JG, Alves SC, Smedley M et al (2008) High-throughput Agrobacterium-mediated barley transformation. Plant Meth 4:22. doi:10.1186/1746-4811-1184-1122

    Article  Google Scholar 

  • Bhatnagar M, Prasad K, Bhatnagar-Mathur P et al (2010) An efficient method for the production of marker-free transgenic plants of peanut (Arachis hypogaea L.). Plant Cell Rep 29:495–502

    Article  PubMed  CAS  Google Scholar 

  • Bull SE, Owiti JA, Niklaus M et al (2009) Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava. Nat Protoc 4:1845–1854

    Article  PubMed  CAS  Google Scholar 

  • Chan AP, Crabtree J, Zhao Q et al (2010) Draft genome sequence of the oilseed species Ricinus communis. Nat Biotechnol 28:951–956

    Article  PubMed  CAS  Google Scholar 

  • Food and Agriculture Organization of the United Nations (2011) FAOSTAT. http://faostat.fao.org/site/339/default.aspx Cited 4 Dec 2011

  • Ferguson M, Rabbi I, Kim D-J et al (2012) Molecular markers and their application to cassava breeding: Past, present and future. Trop Plant Biol (this issue)

  • Greshoff PM, Doy CH (1972) Development and differentiation of haploid Lycopersicon esculentum (tomato). Planta 107:161–170

    Article  Google Scholar 

  • Hiei Y, Komari T (2008) Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat Protoc 3:824–834

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Jansson S, Douglas CJ (2007) Populus: a model system for plant biology. Annu Rev Plant Biol 58:435–458

    Article  PubMed  CAS  Google Scholar 

  • Jørgensen K, Bak S, Busk PK et al (2005) 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

    Article  PubMed  Google Scholar 

  • Kawuki R, Ferguson M, Labuschagne M et al (2009) Identification, characterisation and application of single nucleotide polymorphisms for diversity assessment in cassava (Manihot esculenta Crantz). Mol Breed 23:669–684

    Article  CAS  Google Scholar 

  • Li HQ, Sautter C, Potrykus I et al (1996) Genetic transformation of cassava (Manihot esculenta Crantz). Nat Biotechnol 14:736–740

    Article  PubMed  CAS  Google Scholar 

  • Liu J, Zheng Q, Ma Q et al (2011) Cassava genetic transformation and its application in breeding. J Integr Plant Biol 53:552–569

    Article  PubMed  CAS  Google Scholar 

  • Lokko Y, Anderson J, Rudd S et al (2007) Characterization of an 18, 166 EST dataset for cassava (Manihot esculenta Crantz) enriched for drought-responsive genes. Plant Cell Rep 26:1605–1618

    Article  PubMed  CAS  Google Scholar 

  • Lopez C, Jorge V, Piegu B et al (2004) A unigene catalogue of 5700 expressed genes in cassava. Plant Mol Biol 56:541–554

    Article  PubMed  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Prochnik S, Marri PR, Desany B et al (2012) The cassava genome: current progress, future directions. Trop Plant Biol (this issue)

  • Raemakers CJJM, Amati M, Staritsky G et al (1993) Cyclic somatic embryogenesis and plant regeneration in cassava. Ann Bot 71:289–294

    Article  Google Scholar 

  • Raemakers K, Schreuder M, Pereira I et al (2001) Progress made in FEC transformation of cassava. Euphytica 120:15–24

    Article  CAS  Google Scholar 

  • Sakurai T, Plata G, Rodriguez-Zapata F et al (2007) Sequencing analysis of 20,000 full-length cDNA clones from cassava reveals lineage specific expansions in gene families related to stress response. BMC Plant Biol 7:66. doi:10.1186/1471-2229-1187-1166

    Article  PubMed  Google Scholar 

  • Sayre R, Beeching JR, Cahoon EB et al (2011) The BioCassava Plus Program: biofortification of cassava for Sub-Saharan Africa. Annu Rev Plant Biol 62:251–272

    Article  PubMed  CAS  Google Scholar 

  • Schöpke C, Taylor N, Carcamo R et al (1996) Regeneration of transgenic cassava plants (Manihot esculenta Crantz) from microbombarded embryogenic suspension cultures. Nat Biotechnol 14:731–735

    Article  PubMed  Google Scholar 

  • Taylor NJ, Edwards M, Kiernan RJ et al (1996) Development of friable embryogenic callus and suspension culture systems in cassava (Manihot esculenta Crantz). Nat Biotechnol 14:726–730

    Article  PubMed  CAS  Google Scholar 

  • Taylor N, Kiernan R, Davey CDM et al (1997) Improved procedures for the production of embryogenic tissues across a range of African cassava cultivars: implications for genetic transformation. Afr J Root Tuber Crops 2:200–204

    Google Scholar 

  • Taylor NJ, Masona MV, Carcamo R et al (2001) Production of embryogenic tissues and regeneration of transgenic plants in cassava (Manihot esculenta Crantz). Euphytica 120:25–34

    Article  CAS  Google Scholar 

  • Taylor NJ, Chavarriaga P, Raemakers K et al (2004) Development and application of transgenic technologies in cassava. Plant Mol Biol 56:671–688

    Article  PubMed  CAS  Google Scholar 

  • Taylor N, Halsey M, Gaitán-Solís E et al (2012) The VIRCA project: virus resistant cassava for Africa. GM Crops In Press

  • Tomkins J, Fregene M, Main D et al (2004) Bacterial artificial chromosome (BAC) library resource for positional cloning of pest and disease resistance genes in cassava (Manihot esculenta Crantz). Plant Mol Biol 56:555–561

    Article  PubMed  CAS  Google Scholar 

  • Utsumi Y, Sakurai T, Umemura Y et al (2012) RIKEN Cassava initiative: establishment of a cassava functional genomics platform. Trop Plant Biol (this issue)

  • Yadav J, Ogwok E, Wagaba H et al (2011) RNAi mediated resistance to Cassava brown streak Uganda virus in transgenic cassava. Mol Plant Pathol 12:677–687

    Article  PubMed  CAS  Google Scholar 

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Acknowledgements

The work described was funded by the United States Agency for International Development (USAID) from the people of the United States of America, the Monsanto Fund and The Bill & Melinda Gates Foundation. The authors are appreciative of support provided by Kevin Lutke, DDPSC Plant Tissue Culture and Transformation Facility, Dale Burkhart, Plant Growth Facility and Howard Berg, DDPSC Integrated Microscopy Facility.

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Authors and Affiliations

  1. Donald Danforth Plant Science Center, 975 North Warson Rd., St. Louis, MO, 63132, USA

    Nigel Taylor, Eliana Gaitán-Solís, Theodore Moll, Tira Jones, Amita Pranjal, Cindy Trembley, Vince Abernathy & Claude M. Fauquet

  2. University of Texas Southwestern Medical Center, Dallas, TX, USA

    Brent Trauterman

  3. Dow Agro Sciences, Indianapolis, IN, USA

    David Corbin

Authors
  1. Nigel Taylor
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  2. Eliana Gaitán-Solís
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  3. Theodore Moll
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  4. Brent Trauterman
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  5. Tira Jones
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  6. Amita Pranjal
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  7. Cindy Trembley
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  8. Vince Abernathy
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  9. David Corbin
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  10. Claude M. Fauquet
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Corresponding author

Correspondence to Nigel Taylor.

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Communicated by: Paul Moore

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Taylor, N., Gaitán-Solís, E., Moll, T. et al. A High-throughput Platform for the Production and Analysis of Transgenic Cassava (Manihot esculenta) Plants. Tropical Plant Biol. 5, 127–139 (2012). https://doi.org/10.1007/s12042-012-9099-4

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  • DOI: https://doi.org/10.1007/s12042-012-9099-4

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