Small Helper Ti-plasmid Coexisting With the A281virF Gene Encoding an F-Box-Like Protein Improves the Efficiency of...

Transient transformation is a convenient and less time-consuming method for investigating gene functions compared with production of stable transformants. Most systems for Agrobacterium-mediated plant transformation, regardless of transient or stable transformation, utilize a combination of Agrobacterium strains carrying a helper Ti-plasmid and a binary vector. However, the helper Ti-plasmids which are mega-plasmids with sizes of 200-250 kbp and are difficult to manipulate directly with conventional molecular cloning techniques due to their large sizes.

A small helper Ti-plasmid, pCU307D with a size of 46 kbp was constructed from pTiEHA101 which is commonly used for plant genetic engineering. They consist of the virulence (vir) region, the mutated replication origin from pTiEHA101, the gentamicin resistance marker and the replication origin for E. coli cells. The abilities of T-DNA transfer were examined by transient expression in Arabidopsis cultured cells.

Efficiencies to transfer T-DNA to plant cells by EHA101 and C58C1 carrying pCU307D were similar, although pCU307D had 2.8-fold higher copy numbers than EHA101. The construction processes of pCU307D eliminated the A281virF gene encoding F-box-like protein which was located outside of the vir region. The A281virF gene was cloned into a binary vector and it was introduced into C58C1 cells with pCU307D which was named as CU307DF. T-DNA transfer efficiencies by EHA101 or CU307DF were examined by GUS activities from transiently transformed T-DNA with the CaMV35S::NLS-GUS cassette in Arabidopsis cells. Co-culture with CU307DF carrying the GUS gene conferred 3.2-fold higher GUS activities, compared to that with its parental strain EHA101.

A new Agrobacterium strain CU307DF has higher capacity for T-DNA transfer, compared with its parental strain EHA101. pCU307D is as small as 46 kbp and can propagate in E. coli, and that may enable to add further modification to it for further improvement of T-DNA transfer by CU307DF.