<p>This study presents the first comprehensive investigation into the ex situ conservation of the newly discovered and endangered endemic plant species <i>Ochradenus lakhpatensis</i> using biotechnological tools. Special emphasis was placed on evaluating the morpho-structural characteristics of regenerated plantlets to ensure effective conservation. The cultures were initiated using seeds inoculated on Murashige and Skoog (MS) medium supplemented with 2.0 mg L^− 1 Gibberellic acid (GA₃), resulting in a germination rate of 16.7%. The seedlings obtained were utilized for shoot proliferation on media containing various combinations of cytokinins and auxins. Among the tested formulations, MS medium fortified with 1.2 mg L^− 1 6-benzylaminopurine (BAP) + 0.4 mg L^− 1 α-Naphthaleneacetic acid (NAA) proved to be the most effective, producing 13.0 ± 1.53 shoots with 4.8 ± 0.12&#xa0;cm length per seedling within 8 weeks. This performance was superior to the kinetin (Kn) and indole-3-acetic acid (IAA) combination, which yielded 8.0 ± 1.00 shoots with 3.0 ± 0.15&#xa0;cm length. The shoot system developed on BAP and IAA exhibited significantly enhanced tissue differentiation, including better-developed cuticle layer deposition, dermal, ground, and vascular tissues, compared to those treated with Kn and IAA. In contrast, Kn and IAA treatment resulted in weak shoot development, poorly differentiated tissues, and higher frequencies of morpho-anatomical disorders. Nutrient medium (½ strength MS) supplemented with 1.2 mg L^− 1 indole-3-butyric acid (IBA) promoted rooting (83.3% response with 6.0 ± 1.52 roots per shoot) within 4 weeks, and the rooted plantlets exhibited a 79.2% survival rate during ex vitro acclimatization in a greenhouse. In conclusion, this study establishes an effective in vitro regeneration protocol for <i>O. lakhpatensis</i>, offering a valuable tool for its ex situ conservation and reintroduction into natural habitats to support populations recovery.</p>