Efficient co-transformation of Nicotiana tabacum by two independent T-DNAs, the effect of T-DNA size and implications for genetic separation

The co-transformation of a single plant genome with two independent T-DNA r egions provides opportunities for genetic separation in subsequent generati ons. In an effective strategy, co-delivery events must form a high proporti on of the total transformed population. In this study, using the model plan t species tobacco (Nicotiana tabacum), it was shown that the frequency of c o-transformation within a given T-0 population could be as high as 100% and this was found to be dependent, at least in part, on designing the plasmid vectors so that the kbp size of the first selected T-DNA region was >2-fol d that of the designated T-DNA region for co-transfer. Overall, 40-50% of T -0 lines demonstrated the capacity for segregational separation of co-trans formed T-DNA regions. Hence, the estimate of the required number of total t ransformants for such an independent strategy may seem to be as little as 2 -fold that for a conventional, single T-DNA strategy, but we strongly tempe r such estimates with indications that high co-transformation frequencies m ay be associated with a higher incidence of linkage. In this co-transformat ion study we used a single (Agrobacterium) strain system in which a single binary plasmid contained either two or three T-DNA regions, each with a sel ectable marker. This arrangement could reveal that 'read-through' events wi thin the Agrobacterium cells, resulting in the co-transfer of adjacent T-DN A regions as a single linked unit, accounted for up to 20% of co-transforme d plant lines. Such read-through co-delivery appeared to be more frequent f rom the 'supervirulent' EHA101 A. tumefaciens strain, compared to the 'ordi nary' LBA4404 strain. By using the binary plasmid with three selectable T-D NA regions, we have been able to consider the frequency of co-integration o f a third independent T-DNA within a T-0 subpopulation of co-transformants. This was found to be higher than expected. These observations were applied to the co-transfer of (unwanted) plasmid backbone sequences and showed tha t screening against such sequences may add a significant factor in achievin g the desired, final genotype.