Micropropagation of the Moroccan Endemic Plant <i>Thymus broussonetii</i> Boiss. with Aromatic-Medicinal Value and Conservation Concern

Micropropagation from shoot tips and nodal segments\r\nwas carried out for the conservation and domestication of spontaneous Moroccan\r\nthyme, Thymus broussonetii Boiss.\r\nsubsp. broussonetii (endemic threatened). The mineral composition of the\r\nculture medium, as well as the succession of different growth regulators,\r\ninfluenced the in vitro growth of\r\nthis species. Sterilized achenes of T. broussonetii were able to germinate on an agar medium containing Gautheret\r\nmacronutrients with a rate of 25% and a degree of contamination of less than\r\n4%. Shoot apices of 15-day seedlings (two cotyledon leaves) were cultivated on\r\nSD + 0.46 μM Kin medium and the explants obtained were transplanted every\r\nmonth. Six macronutrients (MS, B5, SH, SD, MSm and N30K) were tested and N30K\r\nwas chosen for the following experiments. Seven cytokinins (Kin, BAP, 2iP, DPU,\r\nadenine, Zeat and TDZ) at 0.46, 0.93 and 2.32 μM/l were evaluated and the\r\naddition of 0.93 μM adenine to N30K medium favored significantly the\r\ninduction of buds and the elongation of explants. Three polyamines (putrescine,\r\nspermidine and spermine) at 2, 5, 10 and 20 μM/l were tested. A better\r\nmultiplication of buds, shoots and roots was noted for N30K + 10 μM\r\nspermine. Cytokinin-auxin combinations led to better root multiplication and an\r\nincrease in the number of buds and the length of explants, particularly\r\nfor 0.46 μM Kin + 2.85 μM IAA. Acclimatization was successfully carried out\r\nusing vitroplants developing a good root system. One month after the start of\r\nacclimatization, 97% of T. broussonetii plantlets were healthy. Three months later, they were transplanted\r\ninto larger pots. 100% of the acclimatized plants developed flowers in the 2nd year between June and August. Re-initiation of the in vitro culture was carried out from sterilized twig segments\r\ncollected from the acclimatized plants of T. broussonetii with 1 - 2 nodes on the medium N30K\r\n+ 0.46 μM Kin, and 52.1% of the explants healthily proliferated. Finally, two\r\nmicropropagation prototypes were developed: shoot tip culture from seedlings\r\nobtained after germination of achenes and node culture from acclimatized\r\nplants.