New approaches for micropropagation and cryopreservation of Agave peacockii, an endangered species

Abstract

More than 50% of Agave species are endemic to Mexico. Among them, Agave peacockii is listed within the list of threatened species that require special protection. In this work, we aimed at developing new supplementary strategies to achieve micropropagation and perform cryopreservation of in vitro-grown shoot-tips of A. peacockii. For multiplication, the addition of two cytokinins, 6-benzylaminopurine (26.6 μM) and kinetin (27.84 μM) to MS semisolid medium signi cantly favoured the morphogenetic response and produced the highest shoot generation (87.00±17.18) after 60 d of culture. This interaction was more effective than using the same growth regulators separately.Propagated and rooted plantlets were successfully acclimated with 100% survival and a normal morphological development during greenhouse performance. For cryopreservation, an optimized protocol following droplet-vitri cation approach allowed obtaining 98% and 96% regrowth before and after cryopreservation, respectively. Shoot-tips were excised of in vitro-propagated plants, subjected to preculture on MS semisolid medium with 0.3 M sucrose for 1d, loaded in solution with 0.4 M sucrose and 1.6 M glycerol for 20 min, exposed to vitri cation solution PVS2 for 15 min, and then, immersed in liquid nitrogen in droplets of PVS2 placed on aluminium foil strips. The vegetative growth of cryo-derived plants and of the in vitro propagated plants was compared under greenhouse culture conditions. No signi cant differences were detected in most assessed characteristics after 120 d of acclimatization. The results presented here constitute new viable biotechnological approaches for the in vitro propagation and long-term conservation of endangered Agave germplasm. Key MessageAgave peacockii shoot micropropagation was induced combining 6-benzylaminopurine and 6-furfuryl-aminopurine. A droplet-vitri cation protocol was optimized to cryopreserve shoot-tips. Greenhouse performance of in vitro and cryo-derived plants was similar.

Advances in the Micropropagation and Genetic Transformation of Agave Species

“…The reported numbers of shoots generated under these conditions vary extensively from 87 to 4, respectively [30,44] Regarding regeneration efficiency, the system with the highest regeneration rate for the Agave genus is that of somatic embryogenesis, both direct and indirect. Table 2 shows the formation up to 556 somatic embryos for A. tequilana [35], by organogenesis, the number of regenerated shoots was up to 87 in A. peacockii [43]. Nonetheless, when somatic embryogenesis is carried out, more time is needed to achieve shoot regeneration, and a balance must be reached for each specific project in relation to timeframe and funding.…”

Micropropagation of Seed-Derived Clonal Lines of the Endangered Agave marmorata Roezl and Their Compatibility with Endophytes

“…On the other hand, not all agave species respond efficiently to plant growth regulators to enhance shoot multiplication, and it also depends on the type of explant used [ 32 , 33 , 34 , 35 ]. Delgado-Aceves et al [ 36 ] proposed that the protocols for the multiplication of endangered agave species require modifications of other species to increase the adaptation and propagation effectiveness.…”

“…On the other hand, plants derived from in vitro culture to ex vitro conditions must withstand stressful biotic or abiotic conditions. As recently reviewed, plant inoculation with benefic microorganisms positively impacts micropropagation and acclimatization [ 36 ]. The inoculation with endophytic microorganisms under in vitro and ex vitro conditions, known as “plant biotization”, can improve stress alleviation, plant growth and support under limited nutrient situations [ 37 , 38 ].…”