An improved method for preparing Agrobacterium cells that simplifies the Arabidopsis transformation protocol.
BACKGROUND
The Agrobacterium vacuum (Bechtold et al 1993) and floral-dip (Clough and Bent 1998) are very efficient methods for generating transgenic Arabidopsis plants. These methods allow plant transformation without the need for tissue culture. Large volumes of bacterial cultures grown in liquid media are necessary for both of these transformation methods. This limits the number of transformations that can be done at a given time due to the need for expensive large shakers and limited space on them. Additionally, the bacterial colonies derived from solid media necessary for starting these liquid cultures often fail to grow in such large volumes. Therefore the optimum stage of plant material for transformation is often missed and new plant material needs to be grown.


RESULTS
To avoid problems associated with large bacterial liquid cultures, we investigated whether bacteria grown on plates are also suitable for plant transformation. We demonstrate here that bacteria grown on plates can be used with similar efficiency for transforming plants even after one week of storage at 4 degrees C. This makes it much easier to synchronize Agrobacterium and plants for transformation. DNA gel blot analysis was carried out on the T1 plants surviving the herbicide selection and demonstrated that the surviving plants are indeed transgenic.


CONCLUSIONS
The simplified method works as efficiently as the previously reported protocols and significantly reduces the workload, cost and time. Additionally, the protocol reduces the risk of large scale contaminations involving GMOs. Most importantly, many more independent transformations per day can be performed using this modified protocol.
Logemann
Elke
E
Max-Planck-Institute for Plant Breeding Research, Department of Plant Microbe Interactions, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany.
Birkenbihl
Rainer P
RP
Max-Planck-Institute for Plant Breeding Research, Department of Plant Microbe Interactions, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany.
Ülker
Bekir
B
Current address: School of Biological and Biomedical Sciences, Durham University, Science Site, South Road, Durham DH1 3LE, UK.
Somssich
Imre E
IE
Max-Planck-Institute for Plant Breeding Research, Department of Plant Microbe Interactions, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany.