The project combined molecular biology, genome editing, and plant tissue culture approaches to overcome key bottlenecks in poinsettia biotechnology.
In the first phase, the molecular basis of flower colour formation was investigated through analysis of dihydroflavonol 4-reductase (DFR), a key enzyme in flavonoid biosynthesis. A short list of amino acid residues influencing substrate specificity was identified using comparative analyses and validated through site-directed mutagenesis and enzyme kinetics. These results provided a set of precise molecular targets for genome editing aimed at generating novel orange flower phenotypes.
In a second step, genome editing constructs were developed using CRISPR/Cas9 technology. Binary vectors containing guide RNAs and donor templates for targeted DFR modification were successfully assembled using Gateway cloning, establishing the foundation for precise genetic manipulation in poinsettia.
A major focus of the project was the development of efficient regeneration systems, which represent a critical prerequisite for any genome editing application. Two complementary regeneration pathways, direct shoot organogenesis and somatic embryogenesis, were systematically optimised using internode explants. For organogenesis, specific combinations of plant growth regulators were identified that significantly improved regeneration efficiency. The addition of growth adjuvants such as silver nitrate and myo-inositol further enhanced shoot formation, elongation, and tissue quality by reducing stress responses and improving physiological balance.
Beyond protocol optimisation, the project generated important insights into the molecular regulation of regeneration. Gene expression analyses demonstrated that improved regeneration capacity correlated with the activation of key developmental regulators controlling meristem formation, cellular dedifferentiation, and cell-cycle progression.
Somatic embryogenesis was also successfully established and enhanced through the application of polyamines, which significantly increased embryogenic competence and embryo yield. Molecular analyses revealed the activation of transcription factors associated with embryogenesis and totipotency, as well as autophagy-related genes, indicating that successful cellular reprogramming requires coordinated developmental and metabolic adaptation processes.
As a key outcome, the project established reproducible regeneration platforms compatible with genetic transformation and genome editing. First transgenic embryogenic lines carrying DFR mutation constructs were generated and are currently under development towards phenotypic evaluation.
Overall, the project delivered both applied methodologies and fundamental knowledge, providing a robust framework for future genome editing and precision breeding in poinsettia and other recalcitrant ornamental crops.
