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

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.

Keywords: Achromobacter; Agave marmorata; clonally propagated; endophytes; germinated seeds; in vitro micropropagation.

Figure 1

Accumulative germination rate and final germination percentage of the two A. marmorata seed lots without plant growth regulators or scarification treatment. (A) The number of germinated seeds and the percentage of accumulative germination, and (B) the germination rates. The L12 reached the maximum rate on day 4, with 87 seeds germinating, and the final percentage was 90% on day 9. The L18 seeds had the best germination rate on day 6, but the selected seedlings were taken from day 3. The final percentage for L18 seeds was 13% lower than for L12. The data are expressed as the mean ± SD of three independent replicates with 200 seeds per replicate.

Figure 2

Seedling responses to MS+BAP (at 22.2 µM) for clonal line selection. Shoot appearance: (A) Completely formed—AM32; (B) Hyperhydric shoots (crystalline)—AM31; (C) Completely formed and hyperhydric (combination)—AM31; Coloring: (D) Totally green—AM32, (E) Purple tones—AM31, AM33, AM34, AM36 and M14D-E; (F) White coloring/chlorotic—AM50 to AM58; Tissue condition: (G) Crust-browning tissue—AM32 and AM34; (H) Rooting—AM20 and AM31; and (I) Senescent tissue (shoots/leaves)—AM36, M06 and AM38. Bar = 1 cm.

Figure 3

Effect of BAP cytokinin (22.2 µM) on axillary bud proliferation of seed-derived clonal lines of A. marmorata on MS medium after 8 weeks of culture. (A) Number of shoots formed, (B) Shoot height in cm, and (C) Number of dry leaves representative of dead tissue. The highest number of shoots was observed in the clonal line AM32, followed by AM33 and AM23. The most stable line was M14d-e which was without senescence tissue. Data are presented as the mean ± SD (top and bottom bars) of results from five repeated experiments. Different letters indicate significant differences, according to Tukey p ≤ 0.05. Bar = 1 cm.

Figure 4

Positive ion MALDI-TOF mass spectra of the hydroalcoholic shoot extracts of the three clonal lines in reflectron mode. (A) AM31, (B) AM32, and (C) AM33. The concentration of each extract was adjusted to 10 mg/mL. The ion fragments at m/z 254.425 and 294.758 correpond to DHB matrix [M+H]⁺ and [M+K+]⁺.

Figure 5

Endophytic bacteria affect the growth of clonal lines AM31, AM32 and AM33 under nutrient limitation. The plant control group was treated with a saline—glucose solution. The positive and negative effects on plant growth must be attributed directly to bacterial inoculation. Data are presented as the mean and standard error of the mean of inoculated plants in forming new leaves, roots, root length and dry weight 30 d post-inoculation. Values and mean represent three replicated experiments. C = control, AX = A. xylosoxydans, PA = P. aeruginosa, BT = B. tequilensis and EC = E. cloacae.

Figure 6

Response of A. marmorata clonal lines to endophytic bacteria inoculation. The figure summarizes the best treatment between bacterial inoculation and control, where A. xylosoxidans was the best treatment for AM31 and AM32, and P. aeruginosa was best for AM33.