Glutamate dehydrogenase activity in normal and vitrified plants of Agave tequilana Weber propagated in vitro

Agave tequilana stem explants were used to produce adventitious shoots under a set of different water potentials induced by different concentrations of gelrite in the medium. At high water potentials all shoots were vitrified; as the medium water potential became more negative the degree of vitrification decreased but the number of shoots per explant also diminished. The enzymes NADH and NAD-GDH (EC. 1.4.1.2) were measured along the water potential gradient. GDH activity was high in the non-vitrified tissues and decreased significantly in the vitrified ones.Abbreviations GDH glutamate dehydrogenase - MS Murashige and Skoog medium - MSO methionine sulfoximine - PVP polyvinylpolypyrrolidone - GS glutamine synthetase - GOGAT glutamine: oxoglutarate amino transferase     

Somatic embryogenesis in Agave tequilana Weber cultivar azul

Somatic embryogenesis was achieved from leaves of Agave tequilana Weber cultivar azul utilizing MS medium supplemented with L2 vitamins and the addition of cytokinins: 6-benzylaminopurine (BA), 1-phenyl-3(1,2,3-thiadiazol-5-yl)urea (TDZ), 6-(γ-γ-dimethylamino)purine (2ip) and 6-furfurylaminopurine (KIN), combined with the auxin 2,4-dichlorophenoxyacetic acid (2,4-D). Differences among the six genotypes studied with regard to their embryogenic response in culture were found. Embryos produced by genotype S3 under a hormone regime of high cytokinin (44.4 to 66.6 μM BA) compared to auxin (4.5 μM 2,4-D) contained chlorophyll, whereas those produced when auxin was high compared to cytokinin (9.0 and 13.6 μM 2,4-D and 1.3 and 4.0 μM BA, respectively) were whitish and morphologically similar to their zygotic counterparts. Somatic embryos matured and germinated after transferring the embryogenic calli to maturation and germination medium without growth regulators and enriched with organic nitrogen. Microscopic observations demonstrated a unicellular origin for production of indirect somatic embryos.     

Aberrant meiotic behavior in Agave tequilana Weber var. azul

Background  

Agave tequilana Weber var. azul, is the only one variety permitted by federal law in México to be used for tequila production which is the most popular contemporary alcoholic beverage made from agave and recognized worldwide. Despite the economic, genetic, and ornamental value of the plant, it has not been subjected to detailed cytogenetic research, which could lead to a better understanding of its reproduction for future genetic improvement.     

Stomatal physiology of a micropropagated CAM plant; Agave tequilana (Weber)

Experiments were designed to assess the capacity of an in vitro cultured CAM plant to control water loss and to examine the response of their stomata to various factors. Detached leaves of micropropagated Agave tequilana plants lost water at similar rates as did field-grown plantlets when dehydrated in air. This was consistent with the fact that stomata from micropropagated plants show similar morphology than field-grown plantlets. In addition, stomata from micropropagated plants responded to various factors in a manner similar to those from field-grown plantlets. It appears that in vitro culture does not affect the capacity of leaves to control water loss nor does it alters the nocturnal stomatal opening of this CAM plant.     

In vitro induction of a trisomic of Agave tequilana Weber var. Azul (Agavaceae) by para-fluorophenylalanine treatment

The effect of para-fluorophenylalanine (PFP) on the production of trisomic plants of Agave tequilana Weber var. Azul produced through somatic embryogenesis was investigated. Normal diploid plants with 2n = 2x = 60 were obtained in the control treatment and with 4 mg L⁻¹ PFP exposure, while use of 8 and 12 mg L⁻¹ PFP led to production of trisomics with 2n = 2x = 61. Normal diploid plants showed a bimodal karyotype with five pairs of large chromosomes and 25 pairs of small chromosomes. Trisomic plants also had a bimodal karyotype with a group of three chromosomes in position five of the chromosome set. More than 13 homologous chromosome pairs exhibited structural changes. Differences in chromosome arm ratio (long arm/short arm) were also found in eight chromosome pairs; all these aberrations in the chromosome complement of trisomic plants were probably caused by inversions, deletions, and/or duplications produced by high concentrations of PFP. The gross chromosome structural changes and the presence of a single extra chromosome could have been induced by the effect of PFP on the mitotic spindle by inducing nondisjunction of sister chromatids, resulting in hyperploids (2n + x) and hypoploids (2n − x). Flow cytometric analysis of nuclear DNA content was performed using nuclei isolated from young leaves of normal and trisomic plants. The 2C DNA content of 8.635 pg (1Cx = 4,223 Mbp of trisomic plants was different (p < 0.001) than that of normal plants (2C DNA = 8.389 pg (1Cx = 4,102 Mbp). The difference in genome size was correlated with the large structural changes in the trisomic plant genomes.     

Technological and Structural Properties of Oat Cookies Incorporated with Fructans (Agave tequilana Weber)

Food Biophysics - The beneficial effect of agave fructans on health has been demonstrated gaining popularity as a new prebiotic and functional food ingredient, however, their role as an ingredient...     

Active telomerase during leaf growth and increase of age in plants from Agave tequilana var. Azul

In plants, previous studies show that telomerase activity contributes to the maintenance of telomeric length for the proper development of organs and tissues. In this work, we investigated telomerase activity in A. tequilana during several years of cultivation. We found that during growth of the leaf there are two crucial phases: (1) the onset of cell elongation in 3 years and (2) differentiation of vascular bundles in 6 years. This coincides with the ages where the highest telomerase activity is seen. Therefore indicates that telomerase is associated with cellular activities such as; elongation, division, and cell differentiation. Likewise, we detected high activity during the period of vegetative growth, indicating that telomerase also contributes to telomeric maintenance on the leaf in A. tequilana.     

Bioprospecting for fungi with potential pathogenic activity on leaves of Agave tequilana Weber var. Azul

Thirty different fungal strains were isolated from A. tequilana leaves showing disease symptoms such as wilt and curled leaves, black, red and chlorotic spots. Ten genera were identified and confirmed by using the LSU D1/D2 rDNA and ITS1‐5.8S‐ITS2 regions, mainly of the Ascomycota phylum, where the Lasiodiploidia and Neoscytalidium genera were the more (46.6%) abundant. The other genera identified were Cladosporium, Cytospora, Epicoccum, Flavodon, Lasiodiplodia, Myrmaecium, Neoscytalidium, Penicillium, Peniophora, Purpureocillium, Trametes and Fusarium. Five strains of Lasiodiplodia and one of Fusarium were selected based on their representativeness and pathogenic potential on Agaves. Pathogenic potential was analysed by both, an infection assay, evidenced as necrosis, and by pectinolytic activity. Specifically, necrosis infection assay was conducted by puncture (wounded) infection and by direct mycelium contact. In general, Lasiodiplodia strains exhibited different pathogenic profiles according to their necrosis percentages, regardless of the infection method used. Fusarium strain analysed also showed a high necrosis infection (> 99%). Pectinolytic activity used as an indirect measurement of pathogenesis presented a high Fusarium extract activity (peaking at 23.9 U). Lasiodiplodia strains exhibited up 6 times more enzymatic activity (peaking at 143.5) than Fusarium strain analysed. In addition, Agave leaf extracts used totally or partially as carbon source during fungal induction culture may induce different pathogenic activities in these strains. In general, the two pathogenicity assays implemented evidenced differences in the pathogenicity profile of these analysed strains.     

Heterologous expression of Fusarium oxysporum tomatinase in Saccharomyces cerevisiae increases its resistance to saponins and improves ethanol production during the fermentation of Agave tequilana Weber var. azul and Agave salmiana must

This paper describes the effect of the heterologous expression of tomatinase from Fusarium oxysporum f. sp lycopersici in Saccharomyces cerevisiae. The gene FoTom1 under the control of the S. cerevisiae phosphoglycerate kinase (PGK1) promoter was cloned into pYES2. S. cerevisiae strain Y45 was transformed with this vector and URA3 transformant strains were selected for resistance to α-tomatine. Two transformants were randomly selected for further study (designated Y45-1 and Y45-2). Control strain Y45 was inhibited at 50 μM α-tomatine, in contrast, transformants Y45-1 and Y45-2 did not show inhibition at 200 μM. Tomatinase activity was detected by HPLC monitoring tomatine disappearance and tomatidine appearance in the supernatants of culture medium. Maximum tomatinase activity was observed in the transformants after 6 h, remaining constant during the following 24 h. No tomatinase activity was detected in the parental strain. Moreover, the transformants were able to grow and produce ethanol in a mix of Agave tequilana Weber var. azul and Agave salmiana must, contrary to the Y45 strain which was unable to grow and ferment under these conditions.     

In vitro propagation of Agave arizonica Gentry & Weber

Agave arizonica Gentry & Weber, an extremely rare and endangered species native to Arizona, was successfully propagated in vitro using modified Murashige and Skoog media. Adventitious shoots developed from callus which formed on bulbil explants grown in a medium supplemented with 1.4 μM 2,4-dichlorophenoxyacetic acid. These shoots proliferated by subculture in media supplemented with 44.4 μM 6-benzylaminopurine, and either 0.5 or 5.4 μM naphthaleneacetic acid. Rooting occurred on shoots transferred to a growth regulator free medium. Rooted plants transferred to potting soil could be established under greenhouse conditions following gradual acclimatization indoors.     

The occurrence of dicotyledonar embryos in Agave tequilana

Agave tequilana Weber is a monocot plant species member of the Asparagaceae family. One of the characteristics of monocot species is that their embryos show only one cotyledon. In this work, the occurrence of embryos with two cotyledons and fused cotyledons in A. tequilana is reported for the first time. The occurrence of dicotyledonar embryos in a species that by definition should have only one cotyledon could bring an opportunity to elucidate the mechanisms that have given the origin to the only one cotyledon present in monocots. Syncotyly is considered in this work as the possible mechanism that gave rise to the only cotyledon mostly present in this species.     

Utilization of by-products from the tequila industry. Part 2: Potential value of Agave tequilana Weber azul leaves

The leaves of the agave plant are left in the field after harvesting the heads for tequila production. Different types of agave leaves were isolated, classified, and their content in the total plant determined. The usable fractions were collected and their properties determined. Of the total wet weight of the agave plant, 54% corresponds to the agave head, 32% corresponds to materials which could be usable for sugar and fiber production which leaves 14% of the wet plant without apparent utility. The fractions with higher total reducing sugars (TRS) content were the fresh fraction of partially dry leaves stuck to the head and the leaf bases with a TRS content of 16.1% and 13.1%, respectively. The highest TRS concentration (16-28%) is in the agave head which is used for tequila production. The leaves are 90-120 cm long and 8-12 cm wide and contain fiber bundles that are 23-52 cm long and 0.6-13 mm wide. The ultimate fiber length is approximately 1.6 mm with an average width of 25 microns. There are several types of leaf fibers that can be utilized depending on what part of the plant they come from and what product is desired. Agave leaf fibers were pulped using a soda pulping process and the pulp was hand formed into test sheets. Test sheets made from pulped agave leaf fibers had a breaking length comparable to paper made from both pine and eucalyptus fibers, but the tear index and burst index were lower than the other two papers.     

Spatial and temporal dynamics of blue agave (Agave tequilana Weber var. azul) wilt in Jalisco Mexico

Tequila is a Mexican alcoholic beverage made from the fermentation and distillation of the blue agave (Agave tequilana Weber var. azul) stem. This crop is affected by a wilt associated mainly with Fusarium oxysporum. This disease can produce considerable yield losses. Little is known about the spatial and temporal behaviour of blue agave wilt. In this work, the spatial and temporal dynamics of the disease in commercial blue agave plantations in the state of Jalisco, Mexico, were analysed. Four fields of approximately 1 ha were selected in the municipalities of Arandas and Magdalena, in which disease assessments were carried out over a year of evaluation. Each plant was categorized based on a scale with four severity classes (healthy plant, severity class 1, severity class 2 and severity class 3). Maps of disease distribution were made. The spatial pattern was analysed by means of four indicators of spatial variation for binomial data; the spatial and temporal variation was analysed by means of transition matrices. An aggregate spatial pattern was observed in all fields. The transitions in severity classes were not completely unidirectional; some plants showed symptom remission between the date of first and second disease assessment, while others remained at their original severity. Severity class 1 occurred most frequently in Arandas fields (from 12.9% to 40.3%). There was a notable increase in wilt severity to class 2 in the Magdalena fields (from 4% to 50.6%). The rates of disease development towards severity class 3 are low and do not suppose a significant loss for the crop; nevertheless, the rates of disease development towards the wilt severity class 2 do put in risk the health of the crop and the availability of the raw material for the making of tequila.     

Cellular and molecular changes associated with somatic embryogenesis induction in Agave tequilana

In spite of the importance of somatic embryogenesis for basic research in plant embryology as well as for crop improvement and plant propagation, it is still unclear which mechanisms and cell signals are involved in acquiring embryogenic competence by a somatic cell. The aim of this work was to study cellular and molecular changes involved in the induction stage in calli of Agave tequilana Weber cultivar azul in order to gain more information on the initial stages of somatic embryogenesis in this species. Cytochemical and immunocytochemical techniques were used to identify differences between embryogenic and non-embryogenic cells from several genotypes. Presence of granular structures was detected after somatic embryogenesis induction in embryogenic cells; composition of these structures as well as changes in protein and polysaccharide distribution was studied using Coomassie brilliant blue and Periodic Acid-Schiff stains. Distribution of arabinogalactan proteins (AGPs) and pectins was investigated in embryogenic and non-embryogenic cells by immunolabelling using anti-AGP monoclonal antibodies (JIM4, JIM8 and JIM13) as well as an anti-methyl-esterified pectin-antibody (JIM7), in order to evaluate major modifications in cell wall composition in the initial stages of somatic embryogenesis. Our observations pointed out that induction of somatic embryogenesis produced accumulation of proteins and polysaccharides in embryogenic cells. Presence of JIM8, JIM13 and JIM7 epitopes were detected exclusively in embryogenic cells, which supports the idea that specific changes in cell wall are involved in the acquisition of embryogenic competence of A. tequilana.     

Electrophysiological and behavioural responses of Scyphophorus acupunctatus (Col., Curculionidae) to Agave tequilana volatiles

Abstract:  The weevil Scyphophorus acupunctatus Gyllenhal (Col., Curculionidae) is the most important insect pest of cultivated agaves in Mexico. The objective of this study was to search for potential attractants for this weevil species from the freshly cut fleshy leaves of healthy Agave tequilana Weber var. Blue plants. Combined gas chromatography-electroantennography (GC-EAD) analysis of A. tequilana volatile extracts showed that seven peaks elicited antennal responses from males and females. Five of these peaks were identified by gas chromatography-mass spectrometry (GC-MS) as o -xylene, α -pinene, 3-carene, γ -terpinene and linalool. Electroantennogram (EAG) records of both sexes to 1-, 10- and 100- μ g-stimulus load of these five compounds showed that there was a dose effect on the EAG responses for linalool, 3-carene and α -pinene, but not for γ -terpinene and o -xylene. There were no sexual differences between male and female EAG responses to any of the compounds tested. Y-tube olfactometer bioassays of the compounds showed that males and females were attracted to α -pinene, 3-carene, γ -terpinene and/or linalool at doses of 1 and 10  μ g, while weevils were repelled by linalool at a dose of 100  μ g.     

Telomere length constancy during aging of Saccharomyces cerevisiae.

It has been proposed that a decrease in the length of telomeres with the successive rounds of DNA replication that accompany mitotic division could play a causal role in the aging process. To investigate this possibility, telomeres from cells of the budding yeast Saccharomyces cerevisiae that varied in replicative age were examined. No change in the lengths of the telomeres was observed in cells that had completed up to 83% of the mean life span. We conclude that the length of the telomeres is not a contributing factor in the natural aging process in individual yeast cells.