New Agrobacterium tumefaciens plasmids for cassava transformation
1995
Arias Garzon, D.I. | Sarria, Rodrigo A.
We aimed to identify Agrobacterium tumefaciens strains and Ti plasmids that are optimal for transformation efficiency in cassava. We screened a variety of strains, using transient expression assays for the Ti-plasmid-encoded uidA gene product. Small pieces (10 mm(2)) of germinated somatic embryos were co-cultivated with pre-induced bacterial cultures for 24 h. After co-cultivation, explants were washed with somatic embryo induction medium plus cefotaxime to eliminate bacterial contamination. The explants were then cultured under normal conditions for embryo induction on solid somatic embryo induction medium plus cefotaxime. At this point, some explants were evaluated histochemically for transient expression of the uidA gene. Regions expressing gus activity were restricted to the borders (wounded tissue) and midveins of the explants. No explant expressed gus activity when treated with control strains that contained the binary vector, but no Ti plasmids. The highest numbers of transient transformation events (manifested as blue spots) were induced by two different Agrobacterium strains. Although the strains had different binary vectors, they had the same type of binary vector borders (nopaline) and Ti plasmids (agropine). Based on these results, a new Agrobacterium plasmid was constructed, combining the apparent best features of the screened plasmids and new traits under study. This construct will be used for the stable transformation of cassava somatic embryos.
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