When performing Gateway vector cloning and transforming Agrobacterium cells for plant transformation or transient expression of a gene of interest in tobacco leaves:

  • Is it necessary to generate 3–4 independent Gateway clones and prepare separate Agrobacterium transformants for expression analysis, since in some cases transformed plants or infiltrated tobacco leaves fail to show the enzyme activity of the target gene?
  • I have already tested GFP fusion constructs, but certain genes still do not produce detectable protein, even after multiple optimization attempts. I also tried using a truncated version of the gene fused with GFP, but expression in tobacco leaves was still not observed.

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