Batch-to-batch variations in protein expression levels pose an intractable and often unreported technical hurdle in the development of rapid plant-based heterologous protein expression platforms. Our group is developing protein expression in non-transgenic plant tissue culture which is grown and then co-cultured with Agrobacterium tumefaciens to rapidly deliver heterologous transfer DNA (T-DNA) to the nucleus of the plant cells (Curtis 2004). The Agrobacterium strain we utilize is an auxotroph that prevents overgrowth within the tissue culture system due to its inability to grow in the absence of a supplied amino acid (Collens, 2004). Since T-DNA transcripts can be rapidly cloned into binary vectors, it is possible to produce kilogram quantities of plant tissue transiently expressing the gene of interest in a matter of days. We have successfully demonstrated this technology at the 50-L pilot scale (O'Neill, 2008) with productivity superior to shake-flask controls. However, even after systematically improving reproducibility via rigor in culture maintenance and synchronization, the concentration of the reporter gene product β-glucuronidase has been observed to vary by orders of magnitude from one experiment to the next. Similar large variations have been observed in plant leaves infiltrated with Agrobacterium.^
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