HOW PLANT-REGENERATION FROM MENTHA X PIPERITA L AND MENTHA X CITRATA EHRH LEAF PROTOPLASTS AFFECTS THEIR MONOTERPENE COMPOSITION IN-FIELD CONDITIONS

A procedure to regenerate plants from leaf protoplasts of two micropro pagated hybrid species of mint, Mentha x piperita L. and Mentha x citr ata Ehrh., has been developed in order to determine whether the in vit ro treatment could influence the monoterpene composition. Purified pro toplasts were first plated in liquid media containing 3.5 mu mol/L BA, 1.25 mu mol/L zeatin, 2.5 mu mol/L NAA and 2.25 or 5.5 mu mol/L 2,4-D as growth regulators to induce initial divisions. In these conditions , high percentages of protoplast-derived cells divided, especially for M. x piperita clones (ADF ranging from 17% to 31% at day 6). Reductio n of both medium osmolality and 2,4-D concentration resulted in a sust ained development of microcalli for both species. Calli were then tran sferred onto solidified regeneration media. The first regenerated shoo ts of M. x piperita were observed 3 months after protoplast isolation on media containing 8.9 mu mol/L and 13.2 mu mol/L BA or 1.8 mu mol/L and 4.5 mu mol/L TDZ. Regeneration frequency did not exceed 4 %. Regen eration medium sequences were required for shoot regeneration on M. x citrata calli. At first, they were cultured on a 1.8 mu mol/L TDZ cont aining-medium for 1 week. They were then transferred onto media supple mented with various concentrations of TDZ or BA. The highest frequenci es of shoot regeneration were around 10 %. Shoot morphology was affect ed by TDZ for both species, but not by BA. In a field trial, the amoun t of menthone, menthol and carvone in the regenerated plants of M. x p iperita vulgaris was compared with that of the control. Results showed a decrease in the amount of menthone and menthol and an increase of c arbone levels in all protoplast-derived plants.