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Ephedra foliata Boiss. & Kotschy ex Boiss., (family - Ephedraceae), is an ecologically and economically important threatened Gymnosperm of the Indian Thar Desert. A method for micropropagation of E. foliata using nodal explant of mature female plant has been developed. Maximum bud-break (90 %) of the explant was obtained on MS medium supplemented with 1.5 mg l(-1) of benzyl adenine (BA) + additives. Explant produces 5.3 +/- 0.40 shoots from single node with 3.25 +/- 0.29 cm length. The multiplication of shoots in culture was affected by salt composition of media, types and concentrations of plant growth regulators (PGR’s) and their interactions, time of transfer of the cultures. Maximum number of shoots (26.3 +/- 0.82 per culture vessel) were regenerated on MS medium modified by reducing the concentration of nitrates to half supplemented with 200 mg l(-1) ammonium sulphate {(NH4) 2SO4} (MMS3) + BA (0.25 mg l(-1)), Kinetin (Kin; 0.25 mg l(-1)), Indole-3-acetic acid (IAA; 0.1 mg l(-1)) and additives. The in vitro produced shoots rooted under ex vitro on soilrite moistened with one-fourth strength of MS macro salts in screw cap bottles by treating the shoot base (s) with 500 mg l(-1) of Indole-3-butyric acid (IBA) for 5 min. The micropropagated plants were hardened in the green house. The described protocol can be applicable for (i) large scale plant production (ii) establishment of plants in natural habitat and (iii) germplasm conservation of this endemic Gymnosperm of arid regions.