Agave guiengola Gentry is a microendemic ornamental species native to Oaxaca, México, with restricted distribution and threatened by human activities, placing it at risk of extinction. Two culture media were evaluated: Murashige and Skoog (MS) medium and modified MS medium, together with medium consistency (semisolid and liquid) and plant growth regulators (PGRs): 6-benzylaminopurine (BA) (5.0 mg L⁻¹) for MS media and a combination of kinetin (KIN) and 3-indoleacetic acid (IAA) (5.0–9.0 and 0.1–1.0 mg L⁻¹ respectively) for modified media. Shoot clusters were grown in two consecutive 30-day phases, with transfer to opposite consistency after the first month of cultivation (first phase). Growth, hyperhydricity, rooting and callus formation were evaluated. Treatments using modified formulation + KIN + IAA under both consistencies had the greatest growth (5.5 and 5.0 cm²). Hyperhydricity was more frequent in liquid MS media (up to 100%), while adjusted MS medium had lower incidence with or without PGRs (0.0% and 6.6%). Transfer from liquid-to-semisolid medium reduced hyperhydricity (10.47%) compared with the reverse (31.35%). Rooting occurred frequently with modified formulation media. Callogenesis occurred mainly using MS + BA media (up to 93.2%), associated with adventitious shoot formation. A synergistic biphasic effect is proposed using semisolid MS + BA followed by modified MS liquid + KIN + IAA, optimizing in vitro growth for conservation.