Agave tequilana Weber cultivar ‘Chato’ represents an important genetic supply of wild severely in decline populations of ‘Chato’ for breeding and transformation programs. In this work, the indirect somatic embryogenesis and cryopreservation of Somatic Embryos (SEs) were investigated using the ‘Chato’ cultivar as a study case. Methods: Embryogenic calli were induced by the cultivation of 1 cm of young leaves from in vitro plants on MS semisolid medium supplemented with 24.84, 33.13, 41.41, 49.69, and 57.98 μM 4-amino-3,5,6-trichloro-2- pyridinecarboxylic acid (picloram) in combination with 2.21, 3.32, and 4.43 μM 6-benzylaminopurine (BAP). The origin and structure of formed SEs were verified by histological analysis. Cryopreservation studies of SEs were performed following the V-cryoplate technique and using for dehydration two vitrification solutions (PVS2 and PVS3). Results: The highest average (52.43 ± 5.74) of produced SEs and the Embryo Forming Capacity (estimated index 52.43) were obtained using 49.69 µM picloram and 3.32 µM BAP in the culture medium. The highest post-cryopreservation regrowth (83%) and plant conversion rate (around 70%) were achieved with PVS2 at 0 °C for 15 min. Conclusion: Our work provides new advances about somatic embryogenesis in Agave and reports the first results on cryopreservation of SEs of this species.

The objective of this work was to assess the suitability of the Droplet-vitrification protocol previously developed with Agave peacockii shoot tips for the cryopreservation of six Agave species. Shoot tips were precultured for 1 day on a medium with 0.3 M sucrose in the dark, loaded in a solution with 1.6 M glycerol and 0.4 M sucrose for 20 min, and dehydrated by exposure to Plant Vitrification Solution 2 (PVS2) at 0 °C for 20 min. Complementary studies using histological analysis, Differential scanning calorimetry (DSC), and evaluation of morphological characteristics in cryo-derived plants were performed. Survival rates ranged from 84% to 100% and from 76% to 97% before and after cryopreservation regardless of the Agave species belonging to two taxonomic subgenera. Thermal analysis of shoot tips subjected to the successive steps of the Droplet-vitrification protocol identified ice crystal formation after loading treatment and glass transition after osmotic dehydration with PVS2. The average glass transition temperature (Tg) was −55.44 °C based on the results of four Agave species. The histological studies showed the anatomical differences that could be found in the meristematic structures depending on the loss of apical dominance. This is the most advanced research on cryopreservation of Agave shoot tips.

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.