Organic nitrogen composition of the tissue culture medium influences Agrobacterium tumefaciens growth and the recovery of transformed Pinus radiata embryonal masses after cocultivation

Repeated attempts to genetically transform Pinus radiata embryonal masses through cocultivation with Agrobacterium tumefaciens on MSG medium were unproductive due to Agrobacterium overgrowth. Timentin at either 200 or 400 mg l−1 was ineffective in inhibiting bacterial growth after cocultivation. In this study, the causes of the abundant bacterial growth were investigated by comparing MSG medium with two other media (mLV and DCR) commonly used in conifer somatic embryogenesis. Statistical analysis of the growth data (optical density and number of cell-forming units) showed that bacterium grew significantly more on MSG than on mLV or DCR during the 48-h cocultivation. This enhanced growth was attributed to the higher concentration of L-glutamine in MSG. Lowering the concentration of L-glutamine in MSG to 0.5 g l−1 resulted in similar growth of Agrobacterium compared with the other two media. MSG was also superior for the growth of radiata pine cells, with a statistically significant difference after 14 d of culture. Hence, to avoid bacterial overgrowth during and after cocultivation, a two-medium protocol was developed in which cocultivation was carried out on mLV, followed by 5 d on mLV with 400 mg l−1 Timentin. Selection for transformed cells and further control of bacterial growth was then performed using MSG with Timentin and Geneticin. By sequential application of these two media, 2,096 cell colonies were selected; of these, 94 were analyzed and 49 were transgenic. These results highlight yet another factor that might be critical for the success of transformation experiments but has not been sufficiently studied until now: the growth dynamics and ability to eliminate A. tumefaciens on various plant tissue culture media.