[39] Assay and partial purification of the cytokinin biosynthetic enzyme dimethylallylpyrophosphate: 5′-AMP transferase

Publisher Summary This chapter describes the techniques required for the growth of crown gall tumor tissues, the preparation of dimethylallyl pyrophosphate, an assay method for DMA transferase and a protocol for isolation and partial purification of the enzyme. The enzyme, dimethylallyl pyrophosphate:5'-AMP dimethylallyltransferase (DMA transferase), had a strict requirement for 5'- AMP and could not utilize adenine or adenosine. Subsequently, adenosine was proposed as a substrate. The most convenient source of DMA transferase has been found to be the tobacco crown gall tumor line, (15955/01) described by Gelvin et al. The tumor was incited originally in Nicotiana tabacum cv. Xanthi by infection with Agrobacterium tumefaciens, strain, and has been cloned. It grows readily upon Murashige and Skoog medium in the absence of added phytohormones and is stable in culture. Under high light intensities, it forms an undifferentiated mass of dark green callus.