Analysis of genetic diversity of mezcalero agaves from central México

"En el Altiplano Potosino, un ecosistema árido y semiárido, Agave salmiana Otto Salm Dick ssp. crassispina (Trel Gentry) es una especie utilizada para la recolección de insectos comestibles y para producción de mezcal, bebida alcohólica tradicional con denominación de origen. El aprovechamiento para producción de mezcal de las poblaciones silvestres de Agave salmiana ssp. crassispina carece de un sistema de explotación basado en el conocimiento biológico de la especie. Esto tiene efectos demográficos, y a su vez podría causar el deterioro de sus recursos fitogenéticos y colocar a la especie en situación de riesgo. Este trabajo analizó diversidad genética y estructura poblacional de tres poblaciones silvestres de Agave salmiana ssp. crassispina en San Luis Potosí, México, mediante polimorfismos de longitud de los fragmentos amplificados, AFLP. La evidencia mostró un alto nivel de diversidad genética dentro de las poblaciones analizadas y un bajo nivel de diferenciación entre ellas, probablemente debido a una fragmentación del hábitat producida por actividades antropogénicas. Postulamos que la diversidad genética en las poblaciones analizadas, a pesar de la constante explotación, se origina por el sinergismo entre la polinización cruzada y la actividad de elementos genéticos transponibles. El alto grado de diversidad genética encontrado en las poblaciones silvestres analizadas en San Luis Potosí demuestra la importancia agroecológica de Agave salmiana ssp. crassispina en el Altiplano Mexicano, una región geográfica que abarca casi la mitad del territorio mexicano. Sin embargo, uso descontrolado y manejo inapropiado de las magueyeras silvestres podría arriesgar los recursos fitogenéticos de Agave salmiana ssp. crassispina.""The Potosino Upland Plateau is an arid and semiarid ecosystem. Agave salmiana Otto Salm Dick ssp. crassispina (Trel Gentry), which grows in this environment, is a species used for gathering of edible insects and for production of mezcal. Mezcal is a Mexican alcoholic beverage with denomination of origin. Production of mezcal with wild populations of Agave salmiana ssp. crassispina lacks an exploitation system based on biological knowledge of the species. The lack of organized use has demographic effects, which leads to degradation of genetic resources and risks to this species. This study analyzed genetic diversity and population structure of three wild populations of Agave salmiana ssp. crassispina from San Luis Potosí, Mexico by AFLP's (amplified fragment length polymorphism). The evidence showed a high level of genetic diversity within populations and low level of genetic differentiation among populations, probably as a result of habitat fragmentation produced by anthropogenic activities. We postulate that the genetic diversity in the wild populations analyzed, despite their constant exploitation, is originated by the synergism between cross-pollination and the activity of transposable genetic elements. The high level of genetic diversity found in the wild populations in San Luis Potosí shows agro-ecological relevance of Agave salmiana ssp. crassispina in the Mexican upland plateau. However, uncontrolled use and inappropriate management of wild populations could risk the genetic resources of Agave salmiana ssp. crassispina.

Características morfológicas y genéticas permiten la identificación de cultivares de ajo en las regiones Norte y Central de México

"The objective of this study was to compare the yield and genetic relationships between two Perla garlic selections, obtained by individual selection of cloves, and commercial varieties and cultivars adapted to diverse regions of Mexico (which did not have a selection process). Varieties with fewer cloves showed higher yields. Coreano and California produced 20 ton/ha (fresh weight) and Perla selections between 17-1.9 ton/ha (fresh weight). Six primers of the series OPB were tested for frenetic characterization and OPB-17 was selected. With the. amplified DIVA fragments, a binary matrix was generated; afterwards, a dissimilarity matrix and dendrogram were developed. High genetic diversity was found among all varieties, which were separated into two groups through differential analysis. Varieties of smaller yield were included in one group. The other group was constituted by the best production varieties with fewer cloves per plant (Perla, California, Coreano and Chino). Dissimilarity was found among Perla varieties. The selection method used to obtain plants with better yield characteristics can be applied to the genetic improvements of garlic.

Effect of cytokinins on the in vitro propagation of Mexican agaves

"La falta de sistemas eficientes de propagación es un factor que limita el aprovechamiento racional de varias especies de Agave, que en muchos casos han tenido una reducción peligrosa de sus poblaciones debido a la sobreexplotación de materiales silvestres. En este estudio se desarrollaron protocolos para la propagación in vitro de Agave cupreata, A. difformis, A. karwinskii, A. obscura y A. potatorum. Como explantes se utilizaron tejidos meristemáticos extraídos de plántulas germinadas in vitro. Se logró la formación de brotes múltiples en explantes basales en medio MS adicionado con 30 g L-1 de sacarosa, 8 g L-1 de agar y varios tratamientos con citocininas [6- bencilaminopurina (BA), 6-γ,γ-dimetilalilaminopurina (2iP), cinetina (Cin), tidiazurón (TDZ) y meta-topolina o N6-(metahidroxibencil) adenina (MT)]. Las eficiencias más altas en producción de brotes en A. cupreata y A. karwinskii se obtuvieron con 1.5 y 1 mg L-1 de BA, donde se generaron 10.5 y 6.1 brotes por explante, respectivamente. En A. difformis y A. obscura las mejores respuestas se obtuvieron con 0.2 mg L-1 TDZ con 8.5 y 11 brotes por explante, respectivamente. En A. potatorum la mejor respuesta ocurrió con 3 mg L-1 Cin, en el que se produjeron 6.9 brotes por explante. El enraizamiento de los brotes generados in vitro se alcanzó en medio MS basal con eficiencias entre 80 y 100 %, y la frecuencia de supervivencia de las plantas una vez transferidas a suelo fue de 72 % en promedio.""The lack of efficient propagation systems is one factor limiting the rational use of several species of Agave, which in many cases have had a dangerous reduction of their populations due to the over-exploitation of wild materials. In this work in vitro propagation protocols were developed for Agave cupreata, A. difformis, A. karivinskii, A. obscura and A. polatorum. Meristematic tissues front in vitro germinated seedlings were used as explants. Multiple shoot formation from basal explants was achieved oil MS medium supplemented with 30 g L(-1) sucrose, 8 g L(-1) agar and various treatments with cytokinins [6-benzylaminopurine (BA) 6-gamma,gamma-dimethylallylaminopurine (2iP), kinetin (Kin), thidiazuron (TDZ) and meta-topolin or N(6)-(meta-hydroxylbenzyl)adenine (M)]. The highest shoot production efficiencies for A. cupreata and A. karivinskii were obtained with 1.5 and 1 mg L(-1) BA, which rendered 10.5 and 6.1 shoots per explant, respectively. In A. difformis and A. obscura the best responses were obtained with 0.2 mg L(-1) TDZ, which yielded 8.5 and 11 shoots per explant, respectively. In A. potatorum the best response occurred with 3 mg L(-1) Kill where 6.9 shoots per explant were produced. Rooting of the in vitro generated shoots was achieved on MS basal media with frequencies ranging from 80 to 100%, and survival of plants transferred to soil was 72%, on the average.

Antitumor and immunostimulatory activities of a genotype V recombinantattenuated veterinary Newcastle disease virus vaccine

"Antitumor conventional treatments including chemo/radiotherapy result in several side effects and non-specificity. Therapies including the use of oncolytic viruses, particularly the Newcastle disease virus (NDV), have emerged as an attractive alternative due to their capacity to kill cancer cells directly or through stimulation of the immune system. In the present study, a commercial vaccine composed of a recombinant attenuated NDV strain P05 (rNDV-P05) was assessed for antitumor and immunostimulatory activity. Firstly, hemagglutination activity was evaluated at different pH and temperature conditions. Then, cancer cell lines and peripheral blood mononuclear cells (PBMC) were co-cultured with or without rNDV-P05 and cytoplasmic nucleosomes were measured by enzyme-linked immunosorbent assay (ELISA) as an apoptosis indicator. Antitumor cytokines produced by PBMC in response to the virus were analyzed by ELISA and reverse transcription quantitative polymerase chain reaction. Characterization ofrNDV-P05 indicates that the virus is slightly sensible to acid and basic pH, and stable at temperatures no greater than 42 degrees C. The majority of cell lines developed apoptosis in co-culture with rNDV-P05 in a dose-time dependent manner. The highest level of HeLa, HCC1954 and HepG2 cell apoptosis was at 48 h/50 hemagglutination units (HU), and HL-60 was 24 h/50 HU.A549 cell line and PBMC did not show sensitivity to apoptosis by the virus. PBMC from healthy donors stimulated with the rNDV-P05 increased significantly the levels of interferon (IFN)-alpha, IFN-gamma, tumor necrosis factor (TNF)-alpha and soluble TNF-related apoptosis-inducing I igand in culture supernatants, as well as their mRNA expression. These results demonstrate that the pro-apoptotic effect ofrNDV-P05 and its magnitude is specific to particular tumor cell lines and is not induced on PBMC; and the virusstimulates the expression of several key antitumor cytokines. This study promotes the use of rNDV-P05 in an alternate application of different viral strains during virotherapy with NDV.

Evaluation of a SUMO E2 conjugating enzyme involved in resistance to Clavibacter michiganensis subsp. michiganensis in Solanum peruvianum, through a tomato mottle virus VIGS assay

"Clavibacter michiganensis subsp. michiganensis (Cmm) causes bacterial wilt and canker of tomato. Currently, no Solanum lycopersicum resistant varieties are commercially available, but some degree of Cmm resistance has been identified in Solanum peruvianurn. Previous research showed up regulation of a SUMO E2 conjugating enzyme (SCEI) transcript in S. peruvianum compared to S. lycopersicum following infection with Cmm. In order to test the role of SCEI in resistance to Cmm, a fragment of SCEI from S. peruvianum was cloned into a novel virus induced gene silencing (VIGS) vector based on the geminivirus, Tomato Mottle Virus (ToMoV). Using biolistic inoculation, the ToMoV-based VIGS vector was shown to be effective in S. peruvianum by silencing the magnesium chelatase gene, resulting in leaf bleaching. VIGS with the ToMoV_SCEI construct resulted in 61% silencing of SCEI in leaves of S. peruvianum as determined by quantitative RT-PCR. The SCEI -silenced plants showed unilateral wilting (15 dpi) and subsequent death (20 dpi) of the entire plant after Cmm inoculation, whereas the empty vector-treated plants only showed wilting in the Cmm-inoculated leaf. The SCEI-silenced plants showed higher Cmm colonization and an average of 4.5 times more damaged tissue compared to the empty vector control plants. SCEI appears to play an important role in the innate immunity of S. peruvianum against Cmm, perhaps through the regulation of transcription factors, leading to expression of proteins involved in salicylic acid dependent defense responses.

Ripening-related cDNAs in guava fruit (Psidium guajava L.). Characterization and expression analysis

"En este estudio se presentan los análisis bioinformáticos y de la expresión de cuatro clonas de ADNc que codifican para una poligalacturonasa (PG), para ácido 1-aminociclopropano-1-carboxílico oxidasa (ACCo), y para dos α-expansinas (a-Exp) en guayaba (Psidium guajava L.). Mediante RT-PCR se obtuvo un fragmento parcial de 301 pb (PgPGl) correspondiente a una PG que se expresa a partir de fruto maduro, uno de 320 pb (PgACOl) para una ACCo de fruto sobremaduro, y dos para α-expansinas: uno de 466 pb (PgEXP2) de fruto sobremaduro y otro de 362 pb (PgEXP3) de pedúnculo. El análisis bioinformático de los ADNc mostró que codifican para proteínas putativas con una alta homología con proteínas relacionadas. La secuencia de aminoácidos de la proteína parcial PgPG1 contiene regiones características y conservadas de las PGs en plantas superiores y está relacionada con la maduración de frutos; PgACO1 mostró características presentes en todas las ACCo y está relacionada con la maduración; PgEXP2 y PgEXP3 contienen parte de los dos dominios presentes de las expansinas, y están agrupadas filogenéticamente con las α-expansinas. Los estudios de expresión mediante Dot Blot mostraron que el gen PgPGl fue visible en todos los estadios de maduración del fruto, con mayor intensidad durante el estadio maduro; el gen PgACO1 fue visible en los cinco estadios de maduración del fruto y presentó su expresión más alta durante el estadio de transición, cuando comienza el cambio de color verde al amarillo (estos dos genes muestran comportamientos similares a los reportados en frutos climatéricos); en PgEXP2 la expresión génica se detectó en todos los tejidos, con un incremento a partir del estadio verde 2 al sobremaduro, similar al comportamiento reportado en frutos no climatéricos; para PgEXP3 la expresión fue visible en cuatro estadios de maduración del fruto y en pedúnculo, con mayor intensidad en el estadio maduro que en todos los demás.""Bioinformatics analysis and gene expression studies of four clones of guava cDNA encoding for a polygalacturonase (PG), an acid 1-aminocyclopropane-1-carboxylate oxidase (ACCo), and two α-expansins (a-Exp) in guava (Psidium guajava L.), is presented here. Using RT-PCR, a partial cDNA fragment of 301 bp (PgPGl) was associated to a PG from mature fruit, one of 320 bp (PgACOl) for an ACCo in overriped fruit, and two fragments for α-expansins: a 466 bp (PgEXP2) of overripe fruit and a 362 bp of peduncle (PgEXP3). cDNA analysis showed that these fragments encode putative proteins with high homology with related proteins. The aminoacid sequence of PgPG1 contains particular features and conserved regions of the PGs in higher plants and is related to fruit ripening; PgACO1 showed features present in all the ACCo and is related to fruit maturation; PgEXP2 and PgEXP3 partially contain two domains present in expansins; and they are phylogenetically grouped with α-expansins. Dot Blot expression studies showed that gene PgPGl was visible in all stages of fruit ripening, with higher intensity during mature stage. Gene PgACOl was visible in the five stages of fruit ripening, and was highest during the transition stage (these two genes displayed behaviors similar to those reported in climacteric fruits); PgEXP2 gene expression was detected in all tissues, with an increase from the Green Stage 2 to Overripe Stage 1, as it has been reported in non-climacteric fruits; for PgEXP3 the expression was visible in four stages of fruit ripening and at peduncle, with highest intensity at the mature stage.

Transgenic plants expressing a novel polypeptide which is encoded by a synthetic gene that contains immunoprotective sequences from corinebacterium diphteriae, bordetella pertussis and clostridium tetani exotoxins

"La presente invención se refiere a una vacuna producida en plantas contra difteria tos ferina y tétanos conocida como vacuna triple DPT. La vacuna incluye un complejo inmunogénico a partir de la transformación de jitomate con un gen quimérico optimizado para expresarse en plantas, que codifica un nuevo polipéptido recombinante el cual no existe en la naturaleza y que contiene los principales epítopos inmunoprotectores de difteria, tos ferina y tétanos. También se describen los métodos para construir el vector de transformación genética y los métodos de regeneración mediante cultivo de tejidos vegetales. Asimismo se presentan los resultados de la producción de anticuerpos en pruebas realizadas en ratones los cuales pueden ser útiles para la inmunización de mamíferos.""Provided is a plant produced vaccine acting against diphtheria, pertussis and tetanus, which is best known as DPT vaccine. The vaccine includes an immunogenic complex resulting from the transformation of tomatoe with a chimeric gene optimised to be expressed in plants, which encodes a novel recombinant polypeptide that is not available in nature and which contains the main immunoprotective epitopes of diphtheria, pertussis and tetanus. In addition, methods for building the genetic transformation vector are described, as well as regeneration methods based on the culture of vegetable tissues. The results of the antibody production in tests performed to mice are also shown, which may be useful for the mammals immunisation.

Identificación y relaciones evolutivas de secuencias parciales de genes del grupo dehidrina en tres especies de cactáceas

"Dehydrins or Group 2 Late Embryogenesis Abundant (LEA) proteins play an important role in the response and adaptation to different types of abiotic stresses such as droughts, high salinity and low temperatures. Using PCR techniques, we identified three gene fragments that encoded dehydrin-like proteins in three cacti species Opuntia ficus-indica (OpfiDHN-like), Leuchtenbergia principis (LepDHN-like) and Mammillaria bombycina (MabDHN-like). Bioinformatic sequence analysis showed an identity between 96 and 97% with the Opuntia streptacantha dehydrin 1 (OpsDHN1) gene, demonstrating that the amplified fragments corresponded to dehydrin- like gene sequences, and that the designed oligonucleotides were effective for similar gene amplification in different cacti genera. Multiple OpfiDHN-like, LepDHN-like and MabDHN-like alignments showed that they possessed three repetitions of the conserved K segment. Also, a histidine rich motif was found, which is believed to facilitate the binding of these proteins with metal ions that probably evolved differently in the Opuntioidea and Cactoidea subfamilies of the Cactaceae family. Bioinformatic tools demonstrated that each of the three partial amino acid sequences corresponded to acidic, highly hydrophilic, and disordered protein fragments, which are characteristics of dehydrin proteins. Phylogenetic analysis using maximum parsimony, indicated that cacti dehydrins-like proteins were monophyletic, as well as those of other families.""Las dehidrinas o proteínas abundantes de la embriogénesis tardía (LEA) del grupo 2 juegan un rol importante en la respuesta y adaptación a diferentes tipos de estrés abiótico como deshidratación, alta salinidad y bajas temperaturas. Usando técnicas de PCR, se identificaron tres fragmentos de genes que codifican para proteínas tipo dehidrina de tres especies de cactus: Opuntia ficusindica (OpfiDHN-like), Leuchtenbergia principis (LepDHN-like) y Mammillaria bombycina (MabDHN-like). El análisis bioinformático de las secuencias mostró que poseen una identidad entre el 96 y 97% con la secuencia del gen dehidrina 1 (OpsDHN1) de Opuntia streptacantha, demostrando que los fragmentos amplificados corresponden a secuencias de genes tipo dehidrina, y que los oligonucleótidos diseñados fueron efectivos para la amplificación de genes similares en diferentes géneros de cactáceas. El alineamiento múltiple de OpfiDHN- like, LepDHN-like y MabDHN-like mostró que poseen tres repeticiones del segmento K conservado. También se encontró un motivo rico en histidinas, el cual se cree que facilita la unión de estas proteínas con iones metálicos que probablemente evolucionaron de manera diferente en las subfamilias Opuntioidea y Cactoidea de la familia Cactaceae. Se demostró mediante técnicas bioinformáticas que cada una de las tres secuencias de aminoácidos parciales son ácidas, altamente hidrofílicas y desordenadas, las cuales son características de las proteínas tipo dehidrina. El análisis filogenético usando máxima parsimonia demuestra que las proteínas tipo dehidrina de las cactáceas son monofiléticas, así como las de otras familias de plantas.

Graphene–Cu Nanocomposites Induce Tolerance against <i>Fusarium oxysporum</i>, Increase Antioxidant Activity, and Decrease Stress in Tomato Plants

The tomato crop is susceptible to various types of stress, both biotic and abiotic, which affect the morphology, physiology, biochemistry, and genetic regulation of plants. Among the biotic factors, is the phytopathogen Fusarium oxysporum f. sp. lycopersici (Fol), which can cause losses of up to 100%. Graphene–Cu nanocomposites have emerged as a potential alternative for pathogen control, thanks to their antimicrobial activity and their ability to induce the activation of the antioxidant defense system in plants. In the present study, the effect of the Graphene–Cu nanocomposites and the functionalization of graphene in the tomato crop inoculated with Fol was evaluated, analyzing their impacts on the antioxidant defense system, the foliar water potential (Ψh), and the efficiency of photosystem II (PSII). The results demonstrated multiple positive effects; in particular, the Graphene–Cu nanocomposite managed to delay the incidence of the “vascular wilt” disease and reduce the severity by 29.0%. This translated into an increase in the content of photosynthetic pigments and an increase in fruit production compared with Fol. In addition, the antioxidant system of the plants was improved, increasing the content of glutathione, flavonoids, and anthocyanins, and the activity of the GPX, PAL, and CAT enzymes. Regarding the impact on the water potential and the efficiency of the PSII, the plants inoculated with Fol and treated with the Graphene–Cu nanocomposite responded better to biotic stress compared with Fol, reducing water potential by up to 31.7% and Fv/Fm levels by 32.0%

Bacterial wilt and canker of tomato: fundamentals of a complex biological system

"Tomato (Solanum lycopersicum) is well-known as a model for study of plant–pathogen interactions, since it is a crop of global relevance and susceptible to multiple bacterial, fungal, viral and nematode pathogens. Among bacterial phytopathogens, the actinomycete Clavibacter michiganensis subsp. michiganensis (Cmm) is the causal agent of bacterial wilt and canker of tomato, considered a quarantine disease at international level. The tomato–Cmm interaction has been studied to decipher the pathogenicity mechanisms in Cmm, susceptibility mechanisms in tomato, molecular basis of resistance to Cmm in wild species relative to domesticated tomato, and the level of genetic variability in Cmm. The objective of this review is to discuss recent advances in tomato–Cmm compatible interaction, which can be integrated for application in early diagnosis and biological control of bacterial wilt and canker of tomato. Further study of plant–microorganism interactions is a promising field for improvements in tomato pathogen resistance.