Cytocompatible cellulose nanofibers from invasive plant species Agave americana L. and Ricinus communis L.: a renewable green source of highly crystalline nanocellulose.

In this study, the fibers of invasive species Agave americana L. and Ricinus communis L. were successfully used for the first time as new sources to produce cytocompatible and highly crystalline cellulose nanofibers. Cellulose nanofibers were obtained by two methods, based on either alkaline or acid hydrolysis. The morphology, chemical composition, and crystallinity of the obtained materials were characterized by scanning electron microscopy (SEM) together with energy-dispersive X-ray spectroscopy (EDX), dynamic light scattering (DLS), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) spectroscopy. The crystallinity indexes (CIs) of the cellulose nanofibers extracted from A. americana and R. communis were very high (94.1% and 92.7%, respectively). Biological studies evaluating the cytotoxic effects of the prepared cellulose nanofibers on human embryonic kidney 293T (HEK293T) cells were also performed. The nanofibers obtained using the two different extraction methods were all shown to be cytocompatible in the concentration range assayed (i.e., 0|‒|500 µg/mL). Our results showed that the nanocellulose extracted from A. americana and R. communis fibers has high potential as a new renewable green source of highly crystalline cellulose-based cytocompatible nanomaterials for biomedical applications.

Physiological differences and changes in global DNA methylation levels in Agave angustifolia Haw. albino variant somaclones during the micropropagation process.

KEY MESSAGE: Global DNA methylation changes caused by in vitro conditions are associated with the subculturing and phenotypic variation in Agave angustifolia Haw. While the relationship between the development of albinism and in vitro culture is well documented, the role of epigenetic processes in this development leaves some important questions unanswered. During the micropropagation of Agave angustifolia Haw., we found three different phenotypes, green (G), variegated (V) and albino (A). To understand the physiological and epigenetic differences among the somaclones, we analyzed several morphophysiological parameters and changes in the DNA methylation patterns in the three phenotypes during their in vitro development. We found that under in vitro conditions, the V plantlets maintained their CAM photosynthetic capacity, while the A variant showed no pigments and lost its CAM photosynthetic ability. Epigenetic analysis revealed that global DNA methylation increased in the G phenotype during the first two subcultures. However, after that time, DNA methylation levels declined. This hypomethylation correlated with the appearance of V shoots in the G plantlets. A similar correlation occurred in the V phenotype, where an increase of 2 % in the global DNA methylation levels was correlated with the generation of A shoots in the V plantlets. This suggests that an "epigenetic stress memory" during in vitro conditions causes a chromatin shift that favors the generation of variegated and albino shoots.

Morphological features of different polyploids for adaptation and molecular characterization of CC-NBS-LRR and LEA gene families in Agave L.

Polyploidy has been widely described in many Agave L. species, but its influence on environmental response to stress is still unknown. With the objective of knowing the morphological adaptations and regulation responses of genes related to biotic (LEA) and abiotic (NBS-LRR) stress in species of Agave with different levels of ploidy, and how these factors contribute to major response of Agave against environmental stresses, we analyzed 16 morphological trials on five accessions of three species (Agave tequilana Weber, Agave angustifolia Haw. and Agave fourcroydes Lem.) with different ploidy levels (2n=2x=60 2n=3x=90, 2n=5x=150, 2n=6x=180) and evaluated the expression of NBS-LRR and LEA genes regulated by biotic and abiotic stress. It was possible to associate some morphological traits (spines, nuclei, and stomata) to ploidy level. The genetic characterization of stress-related genes NBS-LRR induced by pathogenic infection and LEA by heat or saline stresses indicated that amino acid sequence analysis in these genes showed more substitutions in higher ploidy level accessions of A. fourcroydes Lem. 'Sac Ki' (2n=5x=150) and A. angustifolia Haw. 'Chelem Ki' (2n=6x=180), and a higher LEA and NBS-LRR representativeness when compared to their diploid and triploid counterparts. In all studied Agave accessions expression of LEA and NBS-LRR genes was induced by saline or heat stresses or by infection with Erwinia carotovora, respectively. The transcriptional activation was also higher in A. angustifolia Haw. 'Chelem Ki' (2n=6x=180) and A. fourcroydes 'Sac Ki' (2n=5x=150) than in their diploid and triploid counterparts, which suggests higher adaptation to stress. Finally, the diploid accession A. tequilana Weber 'Azul' showed a differentiated genetic profile relative to other Agave accessions. The differences include similar or higher genetic representativeness and transcript accumulation of LEA and NBS-LRR genes than in polyploid (2n=5x=150 and 2n=6x=180) Agave accessions, thus suggesting a differentiated selection pressure for overcoming the lower ploidy level of the diploid A. tequilana Weber 'Azul'.

Bacterial consortia constructed for the decomposition of Agave biomass.

Research has shown that a greater variety of enzymes, as well as variety of microorganisms producing enzymes, can have an overall synergistic effect on the decomposition of lignocellulosic biomass for the production of value-added bio-products. Here, 8 cellulase-degrading bacterial isolates were selected to develop co-, tri-, and tetra-cultures for the decomposition of lignocellulosic biomass. Glucose and xylose equivalents released from imitation biomass media containing 0.5% (w/v) beechwood xylan and 0.5% (w/v) Avicel was measured using di-nitrosalicylic acid for all consortia, along with cell growth and survival. Thereafter, 6 co- and 2 tri-cultures with greatest decomposition were examined for ability to degrade Agave americana fiber. Interestingly, when strains were paired up in co-culture, four pairs: G+5, G+A, C+A1, and G+A1 produced high reducing sugars in 24 h: 6 µM, 8 µM, 8 µM, and finally, 6 µM, respectively. From 4 co-cultures with highest reducing sugar equivalents, tri- and tetra-cultures were produced. The bacterial consortia which had the highest reducing sugars detected were 2 tri-cultures: G + A1 + A4 and G + A1 + 5, displaying levels as high as 9 µM and 5 µM in day 1, respectively. All co- and tri-cultures maintained high cell survival for 14 days with 0.5 g ground Agave. Upon evaluating Agave dry weight after treatment, it was evident that almost half the biomass could be decomposed in 14 days. Scanning electron microscopy of treated Agave supported decomposition when compared with the control. These bacterial consortia have potential for further study of value-added by-product production during metabolism of lignocellulosic biomasses.