PlantTransform: TRTech-PGR: Genotype-independent Regeneration for Recalcitrant Species Through Induced Totipotent Plant Cells

The application of biotechnology for crop trait improvement holds great promise for the implementation of more sustainable and resilient agricultural practices. For instance, the generation of crops that have been modified to require less water and fertilizer supplementation or that are more tolerant of drought conditions and resistant to plant diseases can help improve crop yields in the face of a changing climate and increasing population size.

Although we have made great strides in our ability to modify the plant genome (that encodes the heritable traits of our crops) in individual cells or tissues, turning those cells or tissues back into whole plants presents a significant bottleneck in the application of biotechnology for crop trait improvement. The aim of this project is to find effective ways to reprogram cells in order to facilitate their transformation and regeneration into whole plants.

To that end, we will perform studies in a model plant system, thale cress, to better understand the cellular and molecular regulation of regeneration and identify factors that can enhance the process. As proof of principle, we will subsequently implement the use of such factors in an important crop species known to be difficult to transform and regenerate, namely wheat.

In addition to the planned research, we will also advance public understanding and youth involvement through the development of a lecture series for high school students in the Virginia Summer Residential Governor's School for Agriculture titled The Past, Present, and Future of Bioengineering for Crop Trait Improvement.

Regeneration is a bottleneck for plant transformation, and we know little about the molecular regulatory networks that govern regeneration. This project brings together a multi-disciplinary research team to develop technology that can advance the field of plant transformation and further understanding the fundamental aspects of cell totipotence. The focus will be to enhance regeneration efficiency through ectopic expression of morphogenic transcription factors, with an emphasis on regeneration from protoplast culture.

The application of genome editing technology through transient transformation of protoplasts holds great promise for the rapid generation of transgene-free edited plants, especially in highly heterozygous, outcrossing, or vegetatively propagated crop species. The project utilizes Arabidopsis as a model to investigate regeneration from protoplast culture.

We will use automated digital image analysis and single-cell transcript profiling of developing microcalli as a high-throughput, information-rich platform. A collection of 20 morphogenic transcription factors will be comparatively screened in Arabidopsis for their effects on regeneration through conventional somatic tissue culture and protoplast culture.

The gene regulatory networks governed by transcription factors that promote regeneration from protoplast culture will be studied to give mechanistic insight into their function. Effective transcription factors will be tested in winter wheat, a species recalcitrant to transformation and tissue culture. This project will advance our foundational understanding of totipotency and cell fate determination.

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.