An indirect organogenesis-based citrus transformation system monitored using visible reporter markers

An innovative genetic transformation method for citrus has been developed, aiming to enhance functional genomics and trait improvement. This indirect organogenesis-based system, specifically applied to Carrizo citrange, overcomes the challenges usually encountered in citrus transformation, such as long regeneration cycles and inefficient screening of transformed tissues.

To monitor the progress of this transformation process, two visible reporter systems were tested: ROSEA1, responsible for anthocyanin regulation, and RUBY, a betalain biosynthetic cassette. Both reporters proved compatible with callus development and shoot regeneration during the indirect organogenesis workflow. Within seven days of pre-culture treatment, visible callus initiation was observed, and PCR-confirmed transgenic plantlets were recovered within five months.

During callus proliferation, the visible pigmentation caused by these reporters corresponded well with green fluorescent protein (GFP) fluorescence, allowing rapid and non-destructive identification of transformed tissues. However, the performance of the two reporters differed during shoot regeneration. While RUBY maintained stable pigmentation throughout this stage, ROSEA1-associated pigmentation gradually diminished despite the continuous presence of GFP fluorescence.

Consequently, RUBY demonstrated a significantly higher pigment-based detection rate (4.7 times) than ROSEA1 and showed better agreement with GFP-based detection.

Both constructs were confirmed by PCR in independent lines, together with GFP fluorescence in leaves and root tips. In conclusion, this study establishes an efficient indirect organogenesis-based transformation platform for citrus. Moreover, it demonstrates that both ROSEA1 and RUBY are effective visual reporters during callus proliferation, with RUBY providing more reliable visual identification during shoot regeneration and plant recovery.