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

Abstract

Micropropagation from shoot tips and nodal segments was carried out for the conservation and domestication of spontaneous Moroccan thyme, Thymus broussonetii Boiss. subsp. broussonetii (endemic threatened). The mineral composition of the culture medium, as well as the succession of different growth regulators, influenced the in vitro growth of this species. Sterilized achenes of T. broussonetii were able to germinate on an agar medium containing Gautheret macronutrients with a rate of 25% and a degree of contamination of less than 4%. Shoot apices of 15-day seedlings (two cotyledon leaves) were cultivated on SD + 0.46 μM Kin medium and the explants obtained were transplanted every month. Six macronutrients (MS, B 5 , SH, SD, MS m and N 30 K) were tested and N 30 K was chosen for the following experiments. Seven cytokinins (Kin, BAP, 2iP, DPU, adenine, Zeat and TDZ) at 0.46, 0.93 and 2.32 µM/l were evaluated and the addition of 0.93 µM ade-nine to N 30 K medium favored significantly the induction of buds and the elongation of explants. Three polyamines (putrescine, spermidine and spermine) at 2, 5, 10 and 20 µM/l were tested. A better multiplication of buds, shoots and roots was noted for N 30 K + 10 µM spermine. Cytoki-nin-auxin combinations led to better root multiplication and an increase in the number of buds and the length of explants, particularly for 0.46 µM Kin + 2.85 µM IAA. Acclimatization was successfully carried out using vitroplants developing a good root system. One month after the start of acclimatization, 97% of T. broussonetii plantlets were healthy. Three months later, they were transplanted into larger pots. 100% of the acclimatized plants developed flowers in the 2 nd year between June and August. Re-initiation of the in vitro culture was carried out from sterilized twig segments collected from the acclimatized plants of T. broussonetii with 1-2 nodes on the medium N 30 K + 0.46 µM Kin, and 52.1% of the explants healthily proliferated. Finally, two micropropagation prototypes were developed: shoot tip culture from seedlings obtained after germination of achenes and node culture from acclimatized plants.