PlantTransform: Defining the Mechanisms Responsible for the Recalcitrance of Cultivated Sunflower to Transformation and Tissue Regeneration

Genome engineering holds great promise for producing new plant varieties better able to feed the increasing global population. In many species, this approach requires selectively coaxing small clumps of cells carrying DNA sequences of interest to regenerate into whole plants with well-organized shoots and roots. Although regeneration is readily achieved and allows for successful genome engineering in some plant species, regeneration is still too low-throughput or prohibitively difficult in many crops. A critical challenge then is defining and overcoming the barriers that frustrate regeneration in these species. The proposed research will address this problem by investigating why whole plant regeneration is so hard to achieve in sunflower, a globally significant oil and confectionary seed crop. Specifically, the responses of cultivated sunflower cells and tissues to various experimental regeneration conditions will be characterized, and these observations will be compared to the responses of more easily regenerated relatives in the sunflower family. These results will then be leveraged to design experimental interventions with the goal of overcoming sunflower’s barriers to regeneration. Thus, the proposed research will deliver a toolkit of solutions for a major crop that currently lacks modern biotechnology tools to validate gene functions and accelerate breeding for traits essential to sunflower biology and agricultural production. The findings and methods from the proposed research will be communicated to the sunflower research and plant biotechnology communities through a workshop and online materials. Multiple early-career researchers will receive cross-disciplinary training over the course of the project. Recent successes in overcoming barriers to plant regeneration have been achieved by directly manipulating the transcription factors that promote shoot initiation and patterning. These gains have come largely in transformable species, rather than in species recalcitrant to regeneration, in which additional undetermined inhibitory mechanisms may be active. The proposed research seeks to define the gene regulatory programs that inhibit regeneration and test experimental means to overcome them in Helianthus annuus, the common sunflower. Using both tissue-level analyses and single nucleus transcriptomics, the project will characterize the meristematic cell types and gene expression patterns that arise during regeneration in the absence and presence of various regulators of shoot development. A comparative experimental approach will be taken to assess how growth and transcriptional responses to treatments with regeneration-promoting developmental regulators differ between regeneration-recalcitrant sunflower and two readily regenerable relatives in the Asteraceae family. Experiments will also be performed with both wild and domesticated sunflower accessions to test whether changes that evolved in developmental gene networks during domestication are a major contributor to cultivated sunflower’s recalcitrance to regeneration. Insights gained from identifying the molecular mechanisms that inhibit regeneration in sunflower and from additional plans for methods development will contribute to broader efforts in plant biotechnology and support transformation in other recalcitrant species. Together, the proposed research will not only deliver long sought biotechnology solutions for plant breeding in a major crop, but the findings will also support development of new techniques to engineer other species important to the bioeconomy. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.