Monitoring genetic transformation with RUBY visible reporter in Nicotiana tabaccum L.

Plant genetic transformation has emerged as a key platform for both fundamental and translational research. The reporter genes are widely used to visualize gene expression patterns, promoter analysis, localization, and stable transformation studies. Marker genes would be expected to exhibit characteristics such as robustness, chemical affordability, visualization without any special equipment, and a non-destructive detection approach. Betalains are tyrosine-derived red-yellow pigments found in the caryophyllale plant group that are capable of being used as a visual reporter marker to identify plant transformation. In this study, we transformed Nicotiana tabacum with a RUBY vector construct consisting of three gene clusters, CYP76AD1, DODA, and 5-GT, along with a hygromycin selective marker for visible reporter study. After three weeks of transformation, the betalain pigments were easily detected in the explant leaf tissue. The RUBY-transformed shoots displayed a reddish color that could easily be distinguished from the non-transgenic green shoots during tissue culture. Besides the reddish color, RUBY transformed shoots displayed phenotypically similar to nontransformed shoots and did not show any negative effect during multiple shoots differentiation, proliferation, regeneration, and in the initial stage of plant development. However, the RUBY-expressed plants exhibited slightly reduced plant height and delayed flowering time compared to the wild-type plant. Transgenic plants were further confirmed by PCR amplification of a 341 bp inter DNA fragment of the gene construct. The betalain pigments were observed during all stages of plant development (stem, root, and leaves), beginning with the in vitro shoot buds of genetically transformed explants. Additionally, microstructural analysis of the leaf clearly indicates that betalain pigments accumulated more in the vascular bundle, lamina, trichome, and especially stomata guard cell. A comparative cross-section of the stem and root clearly revealed the presence of betalain pigment within the cortex, endodermis, epidermis, xylem, and phloem tissues of the RUBY-transformed plant. Our findings provide conclusive evidence for the potential application of the RUBY for the visual screening of genetic transformations. The transgenic tobacco (Nicotiana tabacum L.) plants were produced with a non-invasive visual reporter (RUBY) gene. The accumulation of betalin pigment was observed in various plant parts during the different developmental stages.