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

SIMPLE SUMMARY: The wild Agave marmorata Roezl has been classified as an endangered species. Extracting these plants from the forest for commercial purposes and long maturation periods of close to 30 years have contributed to their loss. A. marmorata interacts with pollinators and other Agaves species to maintain genetic variability. Thus, the conservation and restoration of the agave ecosystem is an ecological challenge. Typically, agave micropropagation use meristem or leaves as explants to rapidly produce uniform agave plants in age and size on a large scale leading to homogeneous plantations. However, introducing these clones to the field reduces genetic variability. This study evaluated in vitro micropropagation of A. marmorata from seeds to generate clonal lines. The selected seedlings exhibited variations in multiplication capacity and stable tissue formation. Variations in clonal lines could be exploited to produce high-quality plants with different capacities, such as faster propagation, enhanced stress adaptation, and continued growth under nutrient limitation conditions, consequently maintaining genetic variability. Furthermore, some clonal lines were inoculated with four endophytic bacteria to identify other differences among these plants, including endophyte-host compatibility. Variable responses to inoculation were observed among clonal lines. We found that Achromobacter xylosoxidans was compatible, unlike Enterobacter cloacae which caused plant death. ABSTRACT: A. marmorata is the raw material used for tepextate mescal production but is classified as an endangered species. In the present study, we obtain and multiply clonal lines of Agave marmorata Roezl by selecting seedlings derived from seeds. Ten seedlings from two lots of 400 germinated seeds were selected for axillary bud proliferation induced by BAP 5 mg/L in vitamin-free Murashige and Skoog’s medium. Differences in shoot numbers, heights and senescent tissue formation were observed. Notably, the AM32 line formed 84 shoots and presented low senescent tissue after 60 d of culture. We also selected the AM31 and AM33 clonal lines. Four-month shoots were extracted with 80% methanol in water to determine the total content of saponins, flavonoids, and phenolic acids and compare the three clonal lines. Some bioactive molecules were identified using HPLC techniques and MALDI-TOF mass spectrometry none showed significant differences in content. Additionally, plants derived from the clonal lines were inoculated with four endophytic bacteria. Among these, Achromobacter xylosoxidans supported plant growth of AM32. A notable effect of plant death was observed after inoculation with Enterobacter cloacae, an endophyte of A. tequilana. Additionally, Pseudomonas aeruginosa, an endophyte from A. marmorata, reduced biomass. Our results demonstrate the incompatibility of A. marmorata to E. cloacae and specialization between the host plant and its endophytes. The compatibility of the plant-endophyte could be exploited to boost the establishment and stability of mutualisms to benefit plant development, stress tolerance and pathogen resistance. The differences in multiplication capacity, stable tissue formation, and endophyte biotization responses may indicate genetic variability. Clonal selection and micropropagation from seed-derived plants could contribute to conserving the endangered A. marmorata plant for reforestation in their natural habitats, thus, assuring mass propagation for sustainable industrial production of mescal, bioactive compounds, and prebiotics.