Callus Induction from Diverse Explants and Genotypes Enables Robust Transformation of Perennial Ryegrass (Lolium perenne L.)

Abstract

Genetic transformation of perennial ryegrass (Lolium perenne L.) is critical for fundamental and translational research in this important grass species. It often relies on Agrobacterium-mediated transformation of callus tissue. However, callus induction is restricted to a few genotypes that respond well to tissue culture. Here, we report callus induction from different perennial ryegrass genotypes and explants, such as shoot tips, seeds, and anthers, which were transformed with several plasmids for functional genomics. β-glucuronidase (GUS) histochemical staining showed the LmdsRNAbp promoter sequence was active in stigmas, spikelets, anthers, and leaves. We also transformed calli with plasmids allowing gene silencing and gene knock-out using RNA interference and CRISPR/Cas9, respectively, for which genotypic and phenotypic investigations are ongoing. Using 19 different constructs, 262 transgenic events were regenerated. Moreover, the protocol regenerated a doubled haploid transgenic event from anther-derived calli. This work provides a proof-of-concept method for expanding the range of genotypes amenable to transformation, thus, serving research and breeding initiatives to improve this important grass crop for forage and recreation.

Comparative Transcriptome Analysis of High- and Low- Embryogenic Hevea brasiliensis Genotypes Reveals Involvement of Phytohormones in Somatic Embryogenesis

“…The genotype YT exhibited a higher callus induction compared to RT (Fig. 1), such differences could be due to the genetic background as reported in cotton [53], ryegrass [54], and Primula ssp. [55] etc.…”

Advances in Molecular Breeding of Forage Crops: Technologies, Applications and Prospects

“…Robust transformation protocols have been developed for perennial ryegrass, facilitating advancements in functional genomics and breeding for this important grass species [48]. A chromosome-level, allele-aware genome assembly for cultivated alfalfa has been developed, alongside the establishment of an efficient CRISPR/Cas9-based genome editing protocol.…”

A protoplast-based transient gene expression assay for the identification of heat and oxidative stress-regulatory genes in perennial ryegrass

“…Perennial ryegrass ( Lolium perenne L.) is an important perennial cool-season grass widely used for turf and forage purposes. Stable genetic transfection of the grass takes at least 7 months from callus initiation to the regeneration of transgenic plants [ 4 ]. Ryegrass leaves covered with shining thick cuticles are not amenable to Agroinfiltration or biolistic-based transient gene expression assays.…”