Recombinant Wuschel Protein for Enhanced Sorghum Regeneration and DNA-Free Gene Editing
A recombinant Wuschel protein designed to improve regeneration rates in sorghum tissue culture, enabling DNA-free gene editing when combined with Cas9 ribonucleoprotein. This approach eliminates transgene removal steps, accelerates breeding timelines, and reduces pre-transformation preparation compared to conventional Agrobacterium and biolistic methods.
Overview
This solution offers a recombinant Wuschel protein derived from a sorghum homolog of the Zea mays Wuschel2 gene, intended to enhance the regeneration of Sorghum bicolor explants during tissue culture. By using a protein-based approach instead of delivering a Wuschel transgene via Agrobacterium, this technology enables DNA-free gene editing when paired with Cas9 ribonucleoprotein (RNP). This eliminates the need for subsequent transgene removal, significantly expediting breeding timelines for sorghum and potentially other monocot crops.
The technology addresses a critical bottleneck in sorghum biotechnology: low and inconsistent regeneration rates that limit transformation efficiency. If the protein demonstrates stability during long-term storage, it could substantially reduce the preparation work required immediately before or on the day of transformation compared to traditional biolistic or Agrobacterium-mediated methods.
Technical specifications
Key features:
- Recombinant Wuschel protein expressed in E. coli and purified using immobilized metal affinity chromatography (IMAC)
- Delivered to leaf whorl explants via vacuum infiltration, avoiding the use of Agrobacterium or biolistic delivery
- Compatible with Cas9 ribonucleoprotein for DNA-free gene editing, eliminating the need for transgene removal in subsequent generations
- Potential for long-term storage stability, reducing same-day preparation requirements
- Tunable regeneration optimization through adjustment of protein concentration and infiltration duration
How it works:
The protein is produced recombinantly in E. coli, purified, and its identity and quantity confirmed before application. Fresh sorghum leaf whorl explants are vacuum infiltrated with the protein solution. Regeneration rates are optimized by varying protein concentration and infiltration duration, and results are benchmarked against hormone-only controls and Agrobacterium-delivered transgene methods. Subsequent experiments will validate the protein's effectiveness for enhancing regeneration of gene-edited plants.
Technology readiness level
The technology is at an early stage of development. A recombinant GFP-Wuschel fusion protein has been successfully expressed in E. coli, but it has not yet been tested in plant systems. The next validation phase involves producing and purifying the protein, confirming its identity and concentration, and conducting vacuum infiltration experiments on fresh sorghum leaf whorls to measure regeneration enhancement. Success will be defined by improved regeneration rates relative to hormone-only treated explants, with subsequent comparisons to Agrobacterium-based transgene delivery and validation in gene-editing workflows.
About University of Florida
The University of Florida is a comprehensive public research university with a broad academic and research portfolio and a statewide presence. Industry collaborates through co-located labs and shared core facilities and through an integrated academic health system that accelerates clinical translation. A statewide extension network and multiple research and education sites connect companies with field-scale testing and rapid deployment, while incubators and an adjacent innovation district provide pathways from lab to market. Research is supported by competitive funding from major federal agencies such as NIH, NSF, USDA, and DOE. A dedicated technology transfer office supports IP, licensing, and startup formation.
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