A simple seed-piercing transformation protocol for pearl millet and finger millet

Pearl millet and finger millet face challenges in adopting transgenic or editing approaches due to their inherent recalcitrance to genetic transformation protocols. To overcome these limitations, the current study aims to streamline the genetic transformation protocol for pearl millet and finger millet. We targeted mature embryos as explants to assess transformation efficiencies, gain in time, and overall feasibility. Notably, a transformation efficiency of 17.74% and 18.79%, for pearl millet and finger millet, respectively, was observed using a method that involved directly piercing the mature seeds at the embryonic region with a needle dipped in Agrobacterium suspension, followed by vacuum infiltration. After infection, the seeds were allowed to produce calli and differentiate into shoots and roots, resulting in the development of PCR-positive plants. The induction of infected explants to form calli and subsequent differentiation into shoots and roots, leading to putatively transformed plants, was achieved within 60-66 days. Chi-square analysis of hygromycin selection in T₁ progeny showed a 3:1 segregation, indicating single-locus inheritance, and PCR of T₁ plants with Cas9 and HptII primers confirmed intact T-DNA transmission. Furthermore, as a proof-of-concept for transformation leading to gene editing, a grain-specific phospholipase-d delta1 (PgPLD-delta1-7a), previously identified in our study, was successfully targeted in pearl millet using the CRISPR/Cas9 approach. This seed-piercing protocol has been successfully evaluated in two genotypes of pearl millet and one genotype of finger millet, resulting in the generation of putative-transformed plants.