Genetic transformation method for tef and other recalcitrant orphan crops using GRF-GIF technology

Overview

Tef is a highly nutritious orphan crop with strong potential for food security, yet its productivity is limited by lodging, terminal drought, small seed size, and weed susceptibility. Conventional breeding and available transformation methods have struggled to improve tef due to its recalcitrance to tissue culture and regeneration. This project develops a genetic transformation method based on the simultaneous expression of GROWTH-REGULATING FACTORs (GRFs) and their cofactors GRF-INTERACTING FACTORs (GIFs). The GRF-GIF approach has been shown to dramatically improve regeneration efficiency in other difficult-to-transform species such as wheat and citrus, and is being adapted here for tef with the goal of extending the technology to additional orphan crops including millets.

Technical specifications

• Core approach: Co-expression of GRF and GIF chimeric constructs under a constitutive ubiquitin promoter to stimulate callus regeneration and accelerate genetic modification.
• Construct design: Chimeric GRF-GIF proteins, including a wheat GRF4-GIF1 chimera previously validated in recalcitrant wheat and citrus, are being generated for tef.
• Delivery system: Constructs are introduced via Agrobacterium tumefaciens strain EHA 105 into embryogenic calli derived from mature seeds and from leaf sheath tissue of two-to-three-week-old tef plants.
• Genome resources: Several endogenous GRF and GIF genes have already been identified in the tef genome to support construct development.
• Prior related work: The team previously generated Baby boom (Bbm) and Wuschel2 (Wus2) constructs, which yielded only a limited number of regenerants; the GRF-GIF strategy builds on and aims to improve upon those results.
• Future pipeline: Once transformation is optimized, the platform will be used to co-express trait-improvement genes for lodging, drought tolerance, seed size, and weed competitiveness.

Technology readiness level

The technology is at an early-to-mid stage of development. GRF and GIF gene candidates have been identified in the tef genome, and chimeric constructs are currently being generated. Agrobacterium-mediated delivery protocols using embryogenic calli and leaf sheath explants are being optimized, building on prior experience with Bbm-Wus2 constructs. Proof-of-concept validation in tef is ongoing, with the goal of establishing a reliable regeneration and transformation pipeline that can then be transferred to other recalcitrant orphan crops such as millets. The approach is ready for collaborative partnerships to advance construct optimization, trait gene integration, and broader crop application.