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

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, B5, SH, SD, MSm and N30K) were tested and N30K 
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 adenine to N30K 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 N30K + 10 μM 
spermine. Cytokinin-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 2nd 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 N30K 
+ 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.