An optimising protocol for protoplast regeneration of three peppermint cultivars (Mentha x piperita)

An efficient protocol for plant regeneration from protoplasts of peppermint 'Mitcham Digne38', 'Mitcham Ribecourt 19' and 'Todd's' was developed by st epwise optimization of first cell division, microcalli formation and shoot differentiation. The rate of first cell divisions was strongly dependent on the addition of 2,4-D to callus induction medium. Best results were obtain ed with 1 mu M 2,4-D in combination with NAA (2.5 mu M) and BA (4 mu M). Al though liquid medium was more efficient to support first protoplast divisio ns, solid medium was clearly more suitable to sustain subsequent cell divis ions leading to the formation of microcalli. Shoot organogenesis was induce d from protoplast-derived calli by using reduced auxin concentration (0.5 m u M NAA) and high concentration of cytokinins. Addition of 2.3 mu M thidiaz uron increased bud formation, allowing a regeneration frequency of more tha n 50% from calli of 'Mitcham Digne 38' and 'Todd's'. Genotypic differences were noticed for regeneration capability and the pathway of shoot regenerat ion.