Tropical Plant Biology

, Volume 5, Issue 1, pp 127–139 | Cite as

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

  • Nigel Taylor
  • Eliana Gaitán-Solís
  • Theodore Moll
  • Brent Trauterman
  • Tira Jones
  • Amita Pranjal
  • Cindy Trembley
  • Vince Abernathy
  • David Corbin
  • Claude M. Fauquet


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.


Cassava DNA genomic dot blot Friable embryogenic callus Genetic transformation 





Donald Danforth Plant Science Center


friable embryogenic callus


Greshoff and Doy basal medium containing 20 g/l sucrose

GD2 50P

GD2 supplemented with 50 μM picloram


green fluorescent protein


International Laboratory for Tropical Agricultural Biotechnology


Murashige and Skoog basal medium containing 20 g/l sucrose

MS2 0.5NAA

MS2 supplemented with 0.5 μM naphthalene acetic acid


MS2 supplemented with 2 μM benzylaminopurine

MS2 50P

MS2 supplemented with 50 μM picloram


MS2 supplemented with 5 μM naphthalene acetic acid


naphthalene acetic acid


organised embryogenic callus


settled cell volume


Virus Resistant Cassava for Africa project



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.


  1. Ayling S, Ferguson M, Rounsley S et al (2012) Information resources for cassava research and breeding. Trop Plant Biol (this issue)Google Scholar
  2. 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 CrossRefGoogle Scholar
  3. 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 29:495–502PubMedCrossRefGoogle Scholar
  4. Bull SE, Owiti JA, Niklaus M et al (2009) Agrobacterium-mediated transformation of friable embryogenic calli and regeneration of transgenic cassava. Nat Protoc 4:1845–1854PubMedCrossRefGoogle Scholar
  5. Chan AP, Crabtree J, Zhao Q et al (2010) Draft genome sequence of the oilseed species Ricinus communis. Nat Biotechnol 28:951–956PubMedCrossRefGoogle Scholar
  6. Food and Agriculture Organization of the United Nations (2011) FAOSTAT. Cited 4 Dec 2011
  7. 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)Google Scholar
  8. Greshoff PM, Doy CH (1972) Development and differentiation of haploid Lycopersicon esculentum (tomato). Planta 107:161–170CrossRefGoogle Scholar
  9. Hiei Y, Komari T (2008) Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nat Protoc 3:824–834PubMedCrossRefGoogle Scholar
  10. Ishida Y, Hiei Y, Komari T (2007) Agrobacterium-mediated transformation of maize. Nat Protoc 2:1614–1621PubMedCrossRefGoogle Scholar
  11. Jansson S, Douglas CJ (2007) Populus: a model system for plant biology. Annu Rev Plant Biol 58:435–458PubMedCrossRefGoogle Scholar
  12. 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 139:363–374PubMedCrossRefGoogle Scholar
  13. 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 23:669–684CrossRefGoogle Scholar
  14. Li HQ, Sautter C, Potrykus I et al (1996) Genetic transformation of cassava (Manihot esculenta Crantz). Nat Biotechnol 14:736–740PubMedCrossRefGoogle Scholar
  15. Liu J, Zheng Q, Ma Q et al (2011) Cassava genetic transformation and its application in breeding. J Integr Plant Biol 53:552–569PubMedCrossRefGoogle Scholar
  16. 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 26:1605–1618PubMedCrossRefGoogle Scholar
  17. Lopez C, Jorge V, Piegu B et al (2004) A unigene catalogue of 5700 expressed genes in cassava. Plant Mol Biol 56:541–554PubMedCrossRefGoogle Scholar
  18. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  19. Prochnik S, Marri PR, Desany B et al (2012) The cassava genome: current progress, future directions. Trop Plant Biol (this issue)Google Scholar
  20. Raemakers CJJM, Amati M, Staritsky G et al (1993) Cyclic somatic embryogenesis and plant regeneration in cassava. Ann Bot 71:289–294CrossRefGoogle Scholar
  21. Raemakers K, Schreuder M, Pereira I et al (2001) Progress made in FEC transformation of cassava. Euphytica 120:15–24CrossRefGoogle Scholar
  22. 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 PubMedCrossRefGoogle Scholar
  23. Sayre R, Beeching JR, Cahoon EB et al (2011) The BioCassava Plus Program: biofortification of cassava for Sub-Saharan Africa. Annu Rev Plant Biol 62:251–272PubMedCrossRefGoogle Scholar
  24. Schöpke C, Taylor N, Carcamo R et al (1996) Regeneration of transgenic cassava plants (Manihot esculenta Crantz) from microbombarded embryogenic suspension cultures. Nat Biotechnol 14:731–735PubMedCrossRefGoogle Scholar
  25. 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 14:726–730PubMedCrossRefGoogle Scholar
  26. 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 2:200–204Google Scholar
  27. Taylor NJ, Masona MV, Carcamo R et al (2001) Production of embryogenic tissues and regeneration of transgenic plants in cassava (Manihot esculenta Crantz). Euphytica 120:25–34CrossRefGoogle Scholar
  28. Taylor NJ, Chavarriaga P, Raemakers K et al (2004) Development and application of transgenic technologies in cassava. Plant Mol Biol 56:671–688PubMedCrossRefGoogle Scholar
  29. Taylor N, Halsey M, Gaitán-Solís E et al (2012) The VIRCA project: virus resistant cassava for Africa. GM Crops In PressGoogle Scholar
  30. 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 56:555–561PubMedCrossRefGoogle Scholar
  31. Utsumi Y, Sakurai T, Umemura Y et al (2012) RIKEN Cassava initiative: establishment of a cassava functional genomics platform. Trop Plant Biol (this issue)Google Scholar
  32. Yadav J, Ogwok E, Wagaba H et al (2011) RNAi mediated resistance to Cassava brown streak Uganda virus in transgenic cassava. Mol Plant Pathol 12:677–687PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Nigel Taylor
    • 1
  • Eliana Gaitán-Solís
    • 1
  • Theodore Moll
    • 1
  • Brent Trauterman
    • 2
  • Tira Jones
    • 1
  • Amita Pranjal
    • 1
  • Cindy Trembley
    • 1
  • Vince Abernathy
    • 1
  • David Corbin
    • 3
  • Claude M. Fauquet
    • 1
  1. 1.Donald Danforth Plant Science CenterSt. LouisUSA
  2. 2.University of Texas Southwestern Medical CenterDallasUSA
  3. 3.Dow Agro SciencesIndianapolisUSA

Personalised recommendations