1997. Genetic engineering approaches to improve agronomic traits in cassava (Manihot esculenta Crantz)

1997. Genetic engineering approaches to improve agronomic traits in cassava (Manihot esculenta Crantz)

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Cassava (Manihot esculenta Crantz) is a tropical tuber crop that is grown for its starchy, thickened roots. The cassava roots are used mainly as a food source or for animal feed. Due to the presence of cyanogenic glycosides, however, cassava is potentially toxic. This and other aspects of cassava are potentially amenable to improvement through genetic manipulation. Cassava is a highly heterozygous plant with low natural fertility which makes genetic manipulation via traditional breeding methods very long and difficult. Genetic engineering is an alternative approach to circumvent this problem and modify some aspects of cassava such as: starch quality and quantity, resistance to pests and diseases, and reduction of cyanogenic potential.

Linamarin, a cyanogenic glycoside, is stored in the root, stems and leaves of cassava and upon tissue damage (such as food preparation) is broken down by linamarase to produce acetone cyanohydrin. Acetone cyanohydrin can break down to produce acetone and hydrogen cyanide either spontaneously or by the action of hydroxynitrile lyase (HNL). Of these three cyanogens (linamarin, acetone cyanohydrin, and hydrogen cyanide) acetone cyanohydrin is the main contributor to consumer cyanide exposure.

Here, we report for the first time the stable transformation of cassava via Agrobacterium-mediated system with a gene of agronomic interest. We have cloned a HNL cDNA into an Agrobacterium binary vector under the control of a double CaMV 35S promoter. The modified binary vector was transformed into two different strains of Agrobacterium, LB A 4404 and EHA105 which were used for stable transformation of cassava. In vitro apical leaves and germinated somatic embryos of a cassava cultivar, Mcol 2215, were used to regenerate transgenic cassava plants resistant to paromomycin after co-cultivation with Agrobacterium. The overall efficiency of transformation was approximately 2.8%, however, when only apical leaves were cocultivated with the modified LBA4404, the transformation efficiency increases to 5.5%. All plant DNA evaluated so far by PCR amplification of specific introduced genes indicates integration of the selectable marker, April, and the gene of interest,
HNL.

Hydroxynitrile lyase from leaves and stems of untransformed plants had an activity of 1.7 mmol HCN/mg protein/h versus 2.4, 3.8, 4.0 mmol HCN/mg protein/h from three different transformed plants. Western blots of untransformed and transformed leaf-stem total protein support the higher activity of hydroxynitrile lyase in at least one of the transformed plants. However, HNL has not yet been detected in root tissues of transformed plants  .