Casanova-project_Molecular breeding of acyanogenic cassava

Africa is the only continent on Earth where food availability per capita has declined over the last 30 years. Cassava provides a secure carbohydrate supply in Sub-Saharan Africa. Unfortunately, when cassava is the main or sole staple crop, extensive processing is required to avoid cronic cyanide intoxication from the ingestion of cyanogenic glucosides or their degradation products present in all cassava tissues. Unfortunately, efficient processing results in a simultaneous loss of proteins, vitamin A, iron and zinc. The availability of acyanogenic cassava plants would make extensive processing superfluous and thus at least partly replace costly biofortification programs that rarely reach the very poor rural populations. Acyanogenic lines carrying a high content of pro-Vitamin A would help to alleviate vitamin A deficiency. Needless to say, all transgenic lines obtained will be subjected to extensive testing under different growth conditions to ensure that the acyanogenic trait is maintained. Likewise, all biosafety protocols will be adhered to. Our research group has patented the cassava genes and licenses are provided free of charge for uses that benefits the Third World countries.

Transgenic cassava (Manihot esculenta Crantz, cv MCol22) plants with a 92% reduction in cyanogenic glucoside content in tubers and acyanogenic (<1% of wild type) leaves have been obtained using RNA interference technology to block expression of CYP79D1 and CYP79D2, the two paralogous genes which encode the two isoforms that catalyze the first committed step in linamarin and lotaustralin synthesis. About 180 independent lines with acyanogenic (<1% of wild type) leaves were obtained. Only a few of these were depleted with respect to cyanogenic glucoside content in tubers. In agreement with this observation, girdling experiments demonstrated that cyanogenic glucosides are essentially synthesized in the shoot apex and transported to the root, resulting in a negative concentration gradient basipetal in the plant with the concentration of cyanogenic glucosides being highest in the shoot apex and the petiole of the first unfolded leaf. Supply of nitrogen increased the cyanogenic glucoside concentration in the shoot apex. In situ polymerase chain reaction studies have demonstrated that CYP79D1 and CYP79D2 were preferentially expressed in leaf mesophyll cells positioned adjacent to the epidermis. In young petioles, preferential expression was observed in the epidermis, in the two first cortex cell layers, and in the endodermis together with pericycle cells and specific parenchymatic cells around the laticifers. These data demonstrate that it is possible to drastically reduce the linamarin and lotaustralin content in cassava tubers by blockage of cyanogenic glucoside synthesis in leaves and petioles. The reduced flux to the roots of reduced nitrogen in the form of cyanogenic glucosides did not prevent tuber formation. The fitness of the nearly acyanogenic plants when grown in their natural environment will be assessed in field trials as soon as biosafety regulations in the relevant countries are in place.

We have managed to adapt the transformation method used above to obtain efficient transformation of African elite cultivars. In combination with the use of endogenous promoters to optimize tissue-specificity of RNAi construct expression, we are in the progress of generating acyanogenic lines from these elite African cassava cultivars. Work to transform high beta-carotene lines and to identify cassava cultivars with high protein content is also in progress.

Researchers involved: Kirsten Jørgensen, Charlotte Sørensen, Marc Morant, Anne Vinther Rasmussen, Søren Bak, Birger Lindberg Møller

 

Foreign collaborators: Johanna Puonti-Kaerlas, Laboratory of Plant Biotechnology, Swiss Federal Institute of Technology, Eidgenössische Technische Hochschule (ETH), Zürich, Switzerland

 

Financial support: Danish National Research Foundation, Marie Curie Training Grant, DANIDA, Research Council for Technology and Production