Agrobacterium-mediated direct transformation of wheat mature embryos through organogenesis

Abstract

Transgenic plant production in monocotyledonous species has primarily relied on embryogenic callus induction from both immature and mature embryos as the pathway for plant regeneration. We have efficiently regenerated fertile transgenic wheat plants through organogenesis after Agrobacterium-mediated direct transformation of mechanically isolated mature embryos from field-grown seed. Centrifugation of the mature embryos in the presence of Agrobacterium was found to be essential for efficient T-DNA delivery to the relevant regenerable cells. The inoculated mature embryos formed multiple buds/shoots on high-cytokinin medium, which directly regenerated into transgenic shoots on hormone-free medium containing glyphosate for selection. Rooted transgenic plantlets were obtained within 10-12 weeks after inoculation. Further optimization of this transformation protocol resulted in significant reduction of chimeric plants to below 5%, as indicated by leaf GUS staining and T1 transgene segregation analysis. Direct transformation of wheat mature embryos has substantial advantages over traditional immature embryo-based transformation systems, including long-term storability of the mature dry explants, scalability, and greatly improved flexibility and consistency in transformation experiments.

Agrobacterium-mediated transfer of the Fusarium graminearum Tri6 gene into barley using mature seed-derived shoot tips as explants

“…The transformation frequency of the immature embryo-based systems usually ranged from 1 to 5% and has even reached 25% for certain cereal genotypes (Hayta et al 2019 ). However, our barley transformation frequency was 0.34% (15/4,432*100) which was much lower than that of routine transformation systems in cereal crops but it is similar to that observed in wheat shoot tip transformation (~ 0.4%) (Ye et al 2023 ).…”

DNA Delivery by Virus-Like Nanocarriers in Plant Cells

“…The delivery of transgenes into plant species mainly relies on two transformation methods: Agrobacterium tumefaciens -mediated transformation and particle bombardment . However, the Agrobacterium -mediated system has some significant drawbacks such as uncontrollable target gene integration into the host chromosomes causing positional effects on gene expression, and many plant species are inherently resistant to Agrobacterium infection or showed low transformation efficiency (∼5% to 33%). , Biolistics has been utilized in various plant species, as a random gene delivery system into the host nucleus, mitochondria, and chloroplast . Particle bombardment is performed by high-pressure gene gun delivery that damages host genomic DNA and results in random insertions of multiple copies of the gene The particle bombardment system is also expensive, requires labor-intensive tissue culture and selection, has low transformation efficiency often requiring hundreds of transformation attempts to generate a transgenic line, , and has not been successfully implemented in diverse plant species .…”