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Summary A procedure to regenerate plants from leaf protoplasts of two micropropagated hybrid species of mint, Mentha × piperita L. and Mentha X citrata Ehrh., has been developed in order to determine whether the in vitro treatment could influence the monoterpene composition. Purified protoplasts were first plated in liquid media containing 3.5 μmol/L BA, 1.25 μmol/L zeatin, 2.5μmol/L NAA and 2.25 or 5.5μmol/L 2,4-D as growth regulators to induce initial divisions. In these conditions, high percentages of protoplast-derived cells divided, especially for M. × piperita clones (ADF ranging from 17 % to 31 % at day 6). Reduction of both medium osmolality and 2,4-D concentration resulted in a sustained development of microcalli for both species. Calli were then transferred onto solidified regeneration media. The first regenerated shoots of M. × piperita were observed 3 months after protoplast isolation on media containing 8.9μmol/L and 13.2μmol/L BA or 1.8μmol/L and 4.5 μmol/L TDZ. Regeneration frequency did not exceed 4%. Regeneration medium sequences were required for shoot regeneration on M. × citrata calli. At first, they were cultured on a 1.8 μmol/L TDZ containing-medium for 1 week. They were then transferred onto media supplemented with various concentrations of TDZ or BA. The highest frequencies of shoot regeneration were around 10 %. Shoot morphology was affected by TDZ for both species, but not by BA. In a field trial, the amount of menthone, menthol and carvone in the regenerated plants of M. × piperita vulgaris was compared with that of the control. Results showed a decrease in the amount of menthone and menthol and an increase of carbone levels in all protoplast-derived plants. |
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