Co-transformation using T-DNA genes from Agrobacterium strain 82.139 enhances regeneration of transgenic shoots in Populus.

Barriers to transformation and regeneration continue to hamper the application of recombinant DNA-based biotechnologies in most crops, including for gene editing. To tackle this problem, there has been increasing interest in morphogenic regulator genes, which aid in regeneration and are often plant developmental master regulator genes. Using a set of six genes from the T-DNA of a 'shooty' Agrobacterium tumefaciens strain first discovered by researchers at INRA (France) in the 1990s, we developed a co-transformation ('altruistic') system where these genes promote the recovery and rate of regeneration of transgenic poplars without the use of exogenous plant growth regulators. This method was more efficient (2.3x) at regenerating transgenic shoots in poplar and reduced the culturing time by approximately 6 weeks relative to the conventional approach. Resulting transgenic shoots were positive for GFP and antibiotic resistance genes but did not integrate the altruistic morphogenic genes from the second strain and were phenotypically normal. Deletion testing revealed that the hormone biosynthesis genes alone were insufficient to induce altruistic shoot production in poplar. Further mutational analysis of each gene identified 6b, in combination with iaaH, iaaM and ipt, as the major factor required for non-cell autonomous shoot proliferation. Altogether, our approach highlights the utility of leveraging Agrobacterium genes for transformation, especially through co-transformation, to avoid retaining morphogenic genes in the genomes of clonally propagated plants.