Earth laughs in flowers: Research training in horticultural precision breeding addressing flower colour as established model

Project Information

COLORnamental

Grant agreement ID: 101065228

DOI

10.3030/101065228

Project closed

EC signature date 1 July 2022

Start date 1 April 2023

End date 31 May 2026

Funded under

• Marie Skłodowska-Curie Actions (MSCA)

Total cost

No data

EU contribution

€ 249 301,20

Coordinated by

TECHNISCHE UNIVERSITAET WIEN
Austria

Periodic Reporting for period 1 - COLORnamental (Earth laughs in flowers: Research training in horticultural precision breeding addressing flower colour as established model)

Reporting period: 2023-04-01 to 2026-05-31

Summary of the context and overall objectives of the project

The Marie Skłodowska-Curie project COLORnamental addresses key scientific and technological challenges in the improvement of Euphorbia pulcherrima (poinsettia), one of the most economically important ornamental crops worldwide. Despite its high market value, especially during the Christmas season, the species remains difficult to improve through modern breeding approaches due to limited genetic variability, long breeding cycles, and, critically, its low regeneration capacity in vitro. This recalcitrance significantly constrains the application of advanced biotechnological tools such as genome editing, which rely on efficient and reproducible plant regeneration systems. Overcoming these barriers is essential not only for poinsettia but also for a wide range of horticultural species with similar limitations.

The project was developed in the context of increasing demand for sustainable, resource-efficient, and innovation-driven horticultural production systems in Europe. Enhancing regeneration efficiency and enabling precision breeding approaches can accelerate the development of new varieties with improved ornamental traits, such as novel flower colours, as well as increased resilience and production efficiency.

The overall objective of COLORnamental was therefore to establish robust and reproducible in vitro regeneration systems for poinsettia and to enable targeted modification of flower colour through CRISPR/Cas9-mediated genome editing. In addition, the project aimed to elucidate the molecular and physiological mechanisms underlying cellular reprogramming during regeneration, including the regulatory networks controlling dedifferentiation and organ formation. By combining plant tissue culture, molecular biology, biochemistry, and gene expression analysis, the project sought to generate both applied methodologies and fundamental insights. These outcomes provide a foundation for future genetic transformation and precision breeding strategies in ornamental crops and contribute to advancing plant biotechnology within a sustainable agricultural framework.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The project delivered several advances that significantly extend the current state of the art in ornamental plant biotechnology and regeneration biology.

First, it established one of the most comprehensive regeneration frameworks for poinsettia by integrating physiological optimisation with detailed molecular characterisation. While previous studies largely relied on empirical tissue culture approaches, this project directly linked regeneration efficiency to transcriptional regulation and cellular reprogramming processes, providing a mechanistic understanding of regeneration in a recalcitrant species.

Second, the work demonstrated that non-hormonal growth adjuvants, such as silver nitrate and myo-inositol, can substantially improve regeneration efficiency through modulation of stress responses and endogenous hormonal balance. This represents a shift beyond conventional auxin–cytokinin-based protocols and introduces new strategies for improving regeneration outcomes in difficult-to-transform crops.

Third, the project identified a coordinated activation of key developmental regulators and autophagy-related pathways during somatic embryogenesis. The involvement of autophagy in regeneration has not previously been characterised in poinsettia, and the findings highlight the importance of cellular quality-control and metabolic adaptation mechanisms during the transition to embryogenic competence.

Fourth, the study revealed that polyamines act as important modulators of regeneration by linking stress physiology, developmental signalling, and autophagic processes. This integrated regulatory framework advances current understanding of how cellular plasticity is controlled during plant regeneration.

Finally, the project combined these advances with targeted CRISPR/Cas9-mediated modification of DFR, enabling the generation of genome-edited poinsettia lines with the potential for novel flower colour traits.

Together, these results provide both conceptual and technological breakthroughs that support the development of efficient regeneration and precision breeding platforms for poinsettia and other recalcitrant ornamental species.