A protocol for Agrobacterium-mediated genetic transformation of Lens culinaris Medik (lentil)

A reliable protocol for Agrobacterium-mediated genetic transformation of Lens culinaris Medik (lentil) was developed. Using cultivar Laird, the protocol yielded rooted shoots from an average of 6.8 independent events per hundred seeds. The protocol utilized longitudinal slices of embryo axes from imbibed mature seed as a starting explant and a plasmid containing a beta-glucuronidase:neomycin phosphotransferase (gus:nptII) fusion gene in Agrobacterium strain EHA105. A series of four media, each with appropriate levels of kanamycin selection were identified and other factors tested included the optical density of the Agrobacterium suspension, and type and concentration of plant growth regulators. The expression of the gus reporter gene was visualized through histochemical staining, and further molecular analysis through PCR, qPCR, ddPCR and Southern hybridization confirmed transformation and provided copy number. The inserted genes were inherited into the T-1 generation and chimaeras were not identified. The time from co-cultivation to the planting of rooted shoots ranged from 4 to 7 months, as transgenic clusters continue to produce additional clonal shoots. Key messageA protocol for Agrobacterium-mediated genetic transformation in with a higher frequency than previous reports, multiple shoots per transgenic event, consistent rooting, minimal escapes, no evidence of chimaeras and confirmed inheritance.

Automated summary

A reliable protocol was developed for Agrobacterium-mediated genetic transformation of Lens culinaris Medik with a higher frequency than previous reports, multiple shoots per transgenic event, consistent rooting, minimal escapes, no evidence of chimaeras and confirmed inheritance. The protocol utilized longitudinal slices of embryo axes from imbibed mature seed as a starting explant and a plasmid containing a beta-glucuronidase:neomycin phosphotransferase (gus:nptII) fusion gene in Agrobacterium strain EHA105. Various factors such as the optical density of the Agrobacterium suspension and type and concentration of plant growth regulators were tested. The gus reporter gene expression was confirmed through histochemical staining and molecular analysis.