Regeneration of fertile plants from protoplasts of different Arabidopsis thaliana genotypes

Summary

A protocol for obtaining regenerated fertile plants from mesophyll protoplasts of Arabidopsis thaliana is reported. Protoplasts were isolated from leaves of 21-to 28-day-old Arabidopsis plants grown in a controlled environment. Sustained divisions were achieved when protoplasts were embedded in beads formed by 1.4% sodium alginate in the presence of 50mM CaCl₂ in 0.4 mannitol, which was then exchanged againts modified B5 medium. About 0.4%–0.6% of the protoplasts developed into colonies of which 80%–90% formed shoots and subsequently regenerated to fertile plants. Seeds harvested from more than 200 independently regenerated plants were sown and germination frequencies of more than 95% were obtained. Furthermore, the F₁ plants did not show any evidence of somaclonal variation on visual inspection. This protocol was originally developed for Arabidopsis thaliana “Columbia”; however it was shown to be applicable also for the genotypes “Wassilewskija”, “Landsberg erecta” and “Estland” though with differing efficiencies.