Ziziphora tenuior L. (Lamiaceae) is an aromatic herb used for its medicinal values against fungi, bacteria. Micropropagation can be used for large-scale multiplication of essential oil producing plants thus avoiding an overexploitation of natural resources. This work aims to develop a reliable protocol for the in vitro propagation of Z. tenuior, and to compare the antioxidant activity between in vitro propagated and wild plants. The explants were sterilized and cultured on MS medium containing different concentrations of growth regulators naphthalene acetic acid (NAA) or indole-3-butyric acid (IBA) with 0.5 mg/L of kinetin (Kin) callus formation was 70.2% after 45 days of incubation in dark on medium supplemented with 1.5 mg/L of NAA. After one month of callus culture on medium supplemented with 2 mg/L BA the shoot number was 5.12 and for the multiplication stage. The shoot number was 4.21 and length was 6.17 cm on medium supplemented with 1 mg/L Kin + 0.1 mg/L NAA. DPPH• reagent was used to test the antioxidant activity. The aqueous and methanol extracts of in vitro plants which were treated with 1.5 and 1 mg/L of kin plus 0.1 mg/L of NAA showed a strong DPPH• scavenging activity where IC50 was 0.307 and 0.369 mg/ml, respectively, while the IC50 of aqueous and methanol extracts of wild plants was 0.516 and 9.229 mg/ml, respectively. Our results suggested that plant growth regulators and in vitro culture conditions increased the antioxidant activity.

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Ziziphora tenuior L. and Ziziphora canescens Benth. are two of plants which using in folk medicine in the Kalamoon Mountains areas of Syria for cough, stomachache and dysentery. Samples of Ziziphora genotypes were collected from four different locations (Assal Al-Ward, Yabroud, Qarah and Maaraba), 20 RAPD and 12 ISSR primers were used to assess the genetic diversity of 10 genotypes. Amplified fragments were polymorphic with percentage 93.4% and 100% when RAPD and ISSR markers were used respectively. The first cluster depending on RAPD data formed by the grouping of all Ziziphora tenuior L. and the second cluster formed by grouping of all Ziziphora canescens Benth. genotypes. While the first cluster based on ISSR data formed by two genotypes Ziziphora tenuior L. (Maaraba), the second cluster formed by three genotypes Ziziphora tenuior L. and five genotypes Ziziphora canescens Benth. (Assal AlWard, Yabroud and Qarah). ISSR markers were recorded a high degree of biodiversity among Ziziphora tenuior L. genotype collected from Maaraba and other genotypes. Genetic stability of Ziziphora tenuior L. and Ziziphora canescens Benth. was confirmed among mother plant and shoots that underwent one to nine cycles of in vitro subculturing by RAPD markers that produced monomorphic bands, while ISSR bands were polymorphic especially in 7,8 and 9 subcultures. Also Genetic variations of Ziziphora canescens Benth. that resulted from salinity (1,2,3,4 and 5 g/L) and pH (7,8 and 9) media were detected, both of RAPD and ISSR bands were polymorphic compared with control except some RAPD primers produced monomorphic bands. Callus from Ziziphora tenuior L. that induction on different media (MS + 1.5 mg/L NAA + 0.5 mg/L Kin) or (MS + 2 mg/L IBA + 0.5 mg/L Kin) showed high variations compare with micropropagated plants from apical mirestem on media (MS + 1 mg/L Kin + 0.1 mg/L NAA).

Explants excised from adult shrubs were surface sterilized and cultured on Murashige and Skoog (MS) basal medium in the presence of plant growth regulators (PGRs) at different concentrations. A high multiplication rate of 7.2-fold was achieved every four weeks on MS medium supplemented with 4.44 μM BA, 0.49 μM IBA and 0.58 μM GA3. Rooting was achieved with 73% efficiency within 2-4 weeks on agar-gelled MS basal medium free of PGRs. Rooted plantlets were gradually acclimatized to field conditions over 5-6 weeks with 65% efficiency. For in vitro selection for salt tolerance, MS medium was supplemented with increasing concentrations of NaCl ranging between 25 and 1000 mM. This study has demonstrated that in vitro shoots could tolerate up to 600 mM NaCl with optimal growth at 200 mM, while higher concentrations of NaCl affected growth negatively. Growth and shoot number decreased with increasing NaCl concentration with all plantlets died at 1000 mM NaCl.

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