Success and limitations in adaptation of Fast-TrACC tissue culture-independent transformation in coffee, cotton, and tree tobacco.

Plant transformation is a critical process for generating transgenic and genome-edited plants for use in research and agriculture. For most plant species, this process has traditionally involved genomic insertion of DNA in tissue culture and regeneration of transformed plants through hormonal induction. Recently, methods for plant transformation in a tissue culture-independent manner, through the expression of growth regulators, have been published. We attempted to adapt this promising approach to three woody species, coffee (Coffea arabica), cotton (Gossypium hirsutum), and tree tobacco (Nicotiana glauca), using a combination of Agrobacterium strains, plasmid systems, and different promoters driving the expression of ZmWUS2 and AtIPT, which were originally adapted for this purpose in Nicotiana benthamiana. We found that tree tobacco was amenable to tissue culture-independent transformation but had difficulty developing transgenic seeds. Coffee was not receptive to this transformation method, and cotton was amenable to callus formation but did not exhibit gene insertions in the newly-formed shoots. These limitations are partially technical, such as maize WUS not affecting coffee similarly to other plants, but are in part fundamental setbacks in the use of growth regulators to drive tissue culture-independent transformation. We suggest how these drawbacks can be overcome in the future through the use of inducible or tissue-specific promoters and other means.