, Volume 217, Issue 3, pp 367–373 | Cite as

Generation of cyanogen-free transgenic cassava

  • Dimuth Siritunga
  • Richard T. SayreEmail author
Original Article


Cassava (Manihot esculenta Crantz.) is the major source of calories for subsistence farmers in sub-Saharan Africa. Cassava, however, contains potentially toxic levels of the cyanogenic glucoside, linamarin. The cyanogen content of cassava foods can be reduced to safe levels by maceration, soaking, rinsing and baking; however, short-cut processing techniques can yield toxic food products. Our objective was to eliminate cyanogens from cassava so as to eliminate the need for food processing. To achieve this goal we generated transgenic acyanogenic cassava plants in which the expression of the cytochrome P450 genes (CYP79D1 and CYP79D2), that catalyze the first-dedicated step in linamarin synthesis, was inhibited. Using a leaf-specific promoter to drive the antisense expression of the CYP79D1/CYP79D2 genes we observed up to a 94% reduction in leaf linamarin content associated with an inhibition of CYP79D1 and CYP79D2 expression. Importantly, the linamarin content of roots also was reduced by 99% in transgenic plants having between 60 and 94% reduction in leaf linamarin content. Analysis of CYP79D1/CYP79D2 transcript levels in transgenic roots indicated they were unchanged relative to wild-type plants. These results suggest that linamarin is transported from leaves to roots and that a threshold level of leaf linamarin production is required for transport.


Manihot Cyanogenic glycoside Cyanide Linamarin Cytochrome P450 



Special thanks to Dr. Johnnie Brown of the Ohio State University, Campus Chemical Instrument Center for GC–MS method development for linamarin quantification and Dr. J.C. Jang of the Ohio State University, Horticulture and Crop Science department for critical comments on this manuscript. This research has been supported by grants from the Rockefeller Foundation, the Consortium for Plant Biotechnology Research, the Cassava Biotechnology Network, and the Ohio State University (to R.T.S.). This paper is dedicated to Dr. Chusa Ginés and Verónica Mera.


  1. Anderssen M, Bush P, Svendsen I, Moller B (2000) Cytochromes P450 from cassava catalyzing the first steps in the biosynthesis of the cyanogenic glycosides linamarin and lotaustralin. J Biol Chem 275:1966–1975CrossRefPubMedGoogle Scholar
  2. Arias-Garzon D, Sayre R (1993) Tissue specific inhibition of transient gene expression in cassava (Manihot esculenta Crantz). Plant Sci 93:121–130CrossRefGoogle Scholar
  3. Arias-Garzon D, Sayre R (2000) Genetic engineering approaches to reducing the cyanide toxicity in cassava (Manihot esculenta, Crantz). In: Carvalho L, Thro AM, Vilarinhos AD (eds) Proceedings of the fourth international scientific meeting of the Cassava Biotechnology Network, Embrapa, Brasilia, pp 213–221Google Scholar
  4. Arias-Garzon D, Sarria R, Gelvin S, Sayre R (1994) New Agrobacterium tumefaciens plasmids for cassava transformation. In: Roca WM, Thro AM (eds) Proceedings of the second international scientific meeting of the Cassava Biotechnology Network, Bogor, Indonesia, pp 245–256Google Scholar
  5. Bediako M, Tapper B, Pritchard G (1981) Metabolism synthetic site and translocation of cyanogenic glucoside in cassava. In: Terry ER (ed) Proceedings of the first triennial root crops symposium of the International Society for Tropical Root Crops. IDRC, Canada, pp 143–148Google Scholar
  6. Best R, Hargrove T (1994) Casssava: the latest facts about an ancient crop. CIAT Publication, Cali, ColombiaGoogle Scholar
  7. Bevan W, Barnes W, Chilton M (1983) Structure and transcription of the nopaline synthase gene region of T-DNA. Nucleic Acids Res 11:369–385PubMedGoogle Scholar
  8. Brusslan J, Tobin E (1992) Light-independent developmental regulation of cab gene expression in Arabidopsis thaliana seedlings. Proc Natl Acad Sci USA 89:7791–7795PubMedGoogle Scholar
  9. Doyle J, Doyle J (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  10. Du L, Bokanga M, Moller B, Halkier B (1995) The biosynthesis of cyanogenic glucosides in roots of cassava. Phytochemistry 39:323–326CrossRefGoogle Scholar
  11. Gamborg O, Miller R, Ojima K (1968) Nutrient requirement of suspension cultures of soybean and carrot cells. Exp Cell Res 50:151–158PubMedGoogle Scholar
  12. Hughes J, Carvalho F, Hughes M (1994) Purification, characterization and cloning of hydroxynitrile lyase from cassava (Manihot esculenta Crantz). Arch Biochem Biophys 311:496–502CrossRefPubMedGoogle Scholar
  13. Ikoigbo B (1980) Nutritional implications of projects giving high priority to the production of staples of low nutritive quality. The case of cassava in the humid tropics of West Africa. Food and Nutrition Bulletin, United Nations University, Tokyo, vol 2, pp 1–10Google Scholar
  14. Kahn R, Bak S, Svendsen I, Halkier B, Moller B (1997) Isolation and reconstitution of cytochrome P450ox and in vitro reconstitution of the entire biosynthetic pathway of the cyanogenic glucoside dhurrin from sorghum. Plant Physiol 115:1661–1670CrossRefPubMedGoogle Scholar
  15. Kakes P (1990) Properties and functions of the cyanogenic system in higher plants. Euphytica 48:25–43Google Scholar
  16. Koch B, Nielsen V, Halkier B, Olsen C, Møller B (1992) The biosynthesis of cyanogenic glycosides in seedlings of cassava (Manihot esculenta Crantz). Arch Biochem Biophys 292:141–150PubMedGoogle Scholar
  17. Latham M (1979) Human nutrition in tropical Africa. Food and Agriculture Organization of the United Nations, RomeGoogle Scholar
  18. Li H-Q, Sautter C, Potrykus I, Puonti-Kaerlas J (1996) Genetic transformation of cassava (Manihot esculenta Crantz). Nature Biotechnol 14:736–740Google Scholar
  19. Makame M, Akoroda M, Hahn S (1987) Effects of reciprocal stem grafts on cyanide translocation in cassava. J Agric Sci Cambridge 109:605–608Google Scholar
  20. McMahon J, Sayre R (1994) Regulation of cyanogenic potential in cassava (Manihot esculenta Crantz). In: Roca WM, Thro AM (eds) Proceedings of the second international scientific meeting of the Cassava Biotechnology Network, Bogor, Indonesia, pp 423–438Google Scholar
  21. McMahon J, White W, Sayre R (1995) Cyanogenesis in cassava (Manihot esculenta Crantz). J Exp Bot 46:731–741Google Scholar
  22. Mkpong O, Yan H, Chism G, Sayre R (1990) Purification, characterization, and localization of linamarase in cassava. Plant Physiol 93:176–181Google Scholar
  23. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue culture. Physiol Plant 15:473–497Google Scholar
  24. Osuntokun B (1991) Cassava diet, chronic cyanide intoxification and neuropathy in Nigerian Africans. World Rev Nutr Diet 36:141–173Google Scholar
  25. Ramanujam T, Indira P (1984) Effect of girdling on the distribution of total carbohydrates and hydrocyanic acid in cassava. Indian J Plant Phys 27:355–360Google Scholar
  26. Rosling H (1974) Cassava cyanide and epidemic spastic paraparesis: a study in Mozambique on dietery cyanide exposure. ACTA Universitatis Upsalienis, Ph.D. thesis, Uppsala University, SwedenGoogle Scholar
  27. Rosling H, Mlingi N, Tylleskar T, Banea M (1992) Causal mechanisms behind human diseases induced by cyanide exposure from cassava. In: Roca WM, Thro AM (eds) Proceedings of the first international scientific meeting of the Cassava Biotechnology Network, Cartegena, Cali, Colombia, pp 366–375Google Scholar
  28. Sambrook J, Fritsch E, Maniatis J (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  29. Selmar D (1993) Transport of cyanogenic glucosides: linustatin uptake in Hevea cotyledons. Planta 191:191–199Google Scholar
  30. Selmar D, Lieberei R, Biehl R (1988) Mobilization and utilization of cyanogenic glycosides: the linustatin pathway. Plant Physiol 86:711–716Google Scholar
  31. Sibbensen O, Koch B, Halkier B, Moller B (1994) Isolation of a heme-thiolate enzyme cytochrome P450TYR which catalyzes the committed step in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench. Proc Natl Acad Sci USA 91:9740–9744PubMedGoogle Scholar
  32. Soni R, Murray A (1994) Isolation of intact DNA and RNA from plant tissue. Anal Biochem 218:474–476CrossRefPubMedGoogle Scholar
  33. Stitt M, Sonnewald U (1995) Regulation of metabolism in transgenic plants. Annu Rev Plant Physiol Plant Mol Biol 46:341–368Google Scholar
  34. Tylleskar T, Banea M, Bikangi N, Cooke R, Poulter N, Rosling H (1992) Cassava cyanogens and konzo, an upper motor neuron disease found in Africa. Lancet 339:208–211PubMedGoogle Scholar
  35. Vincent J (1985) A manual for the practical study of root-nodule bacteria. IBP handbook No. 15Google Scholar
  36. Wheatley C, Orrego J, Sanchez T, Granados E (1993) Quality evaluation of cassava core collection at CIAT. In: Roca WM, Thro AM (eds) Proceedings of the first international scientific meeting of the Cassava Biotechnology Network, Cartegena, Cali, Colombia, pp 255–264Google Scholar
  37. White W, McMahon J, Sayre R (1994) Regulation of cyanogenesis in cassava. Acta Hort 375:69–77Google Scholar
  38. White W, Arias-Garzon D, McMahon J, Sayre R (1998) Cyanogenesis in cassava: the role of hydroxynitrile lyase in root cyanide production. Plant Physiol 116:1219–1225PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  1. 1.Department of Plant BiologyThe Ohio State UniversityColumbusUSA

Personalised recommendations