Micropropagation of Chayote (Sechium edule L.) var. virens levis in RITA®.

Chayote (Sechium edule) belongs to the Cucurbitaceae family, an important family at the nutritional and medicinal levels, that has been covering international markets. Having vigorous and healthy plants is important for producers, who are very interested in cultivating chayote plants obtained from in vitro tissue culture in their orchards. Bioreactors have become an alternative with high potential for plant propagation, showing significant advantages over micropropagation in semisolid medium, by generating more plant material, larger, and more vigorous. In this chapter, a micropropagation protocol of S. edule in RITA® bioreactors is reported.

First Report of Leaf Blight Caused by Colletotrichum karsti on Peppermint (Mentha x piperitavar. citrata) in Mexico.

Spearmint (Mentha x piperita var. citrata (Ehrh.) Briq.: Lamiaceae) is an aromatic herb widely cultivated owing to its industrial properties. In June 2020, symptoms of leaf blight were observed on 1,500 peppermint plants in a commercial nursery located in Cuautla (18°52'18"N 98°57'58"W), Morelos, Mexico. The incidence of the disease was 89%. Symptoms were initially observed as irregular, small black necrotic spots, that grew rapidly until the leaves were blighted. Fungal isolation was done using diseased leaf tissue on potato dextrose agar (PDA) as described by Ayvar-Serna et al. (2020) and Colletotrichum-like colonies were obtained. Six isolates were purified by single spore culture and only a single morphotype was obtained. One isolate was used for pathogenicity tests, morphological characterization, and multilocus phylogenetic analysis. The isolate (accession no. UACH449) was deposited in the Culture Collection of Phytopathogenic Fungi of the Department of Agricultural Parasitology at the Chapingo Autonomous University. Colonies in PDA grow at a rate of 7.0-10.0 mm/d. After 14 days, the colony was white to orange, and conidia (n =100) were hyaline, cylindrical, and straight with rounded ends, measuring 15.0-17.0 × 4.5-6.5 µm. Appressoria were brown and bullet-shaped. In 28-day-old colonies, the formation of perithecia was observed. Asci were hyaline, unitunicate, 8-spored, fasciculate, and cylindrical to clavate. Ascospores (n =100) were hyaline, unicellular, allantoid, inaequilateral, often straight on the inner side, apices rounded, arranged biseriately within the asci, and measured 14-19 × 4.0-7.5 µm. Morphological features of the isolate placed it tentatively within the Colletotrichm boninense species complex (Damm et al. 2012). For molecular identification, genomic DNA was extracted, and the internal transcribed spacer (ITS) region (White et al. 1990), partial sequences of calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and actin (ACT) (Damm et al. 2012) genes were amplified and sequenced. A phylogenetic tree including published ITS, CAL, GAPDH, and ACT data for Colletotrichum species was constructed and the isolate UACH449 was grouped in the clade of Colletotrichum karsti. Sequences were deposited in GenBank under the accession numbers: ITS, OL825605; CAL, OL855890; GAPDH, OL855891 and ACT, OL855889. Pathogenicity was tested by spraying a suspension of 1 × 10^5 conidia/ml, onto eight healthy peppermint plants 30-days-old var. citrata, while eight control plants were sprayed using sterile distilled water. All plants were kept at 25 +/- 2°C and 70% RH. The characteristic symptoms of the disease were observed seven days after inoculation, while control plants remained symptomless. The pathogenicity test was repeated twice. The fungus was consistently reisolated from the eight inoculated plants and was morphologically identical to that originally isolated from diseased leaves, fulfilling Koch's postulates. To date, this pathogen has not been reported on peppermint (Farr and Rossman, 2022). To our knowledge, this is the first report of Colletotrichum karsti causing foliar blight on peppermint worldwide. According to our field observations, this disease is a threat to the production of peppermint plants.

Inorganic Compounds that Aid in Obtaining Somatic Embryos.

Somatic embryogenesis (SE) is a process that allows formation of embryos from somatic cells; this biological process has different stages that first require micropropagation and conditioning of explant, and then induction, multiplication, development, and germination of somatic embryos (SoE), to obtain seedlings that will be acclimatized and grown in a greenhouse to further be cultivated in the field. Inorganic compounds are supplemented by macro- and micronutrients that can conform different culture media, and with other compounds such as a carbon source, vitamins, and plant growth regulators (PGRs), will direct the fate of the plant cells to obtain SoE that will regenerate into plants. The concentration of these inorganic compounds must be optimized, since at very high concentrations they can cause toxicity and at low concentrations they may not induce the desired response. The objective of this chapter is to describe the most significant advances in the use of inorganic elements during the different stages of SE, starting with the description of the most used basal media and later describing the use of the main studied mineral elements during establishment of SE.

Comparison of Different Semi-Automated Bioreactors for In Vitro Propagation of Taro (Colocasia esculenta L. Schott).

Taro is important for its nutritional content, medicinal use, and bioethanol production. The aim of the present study was to compare different semi-automated bioreactors (SABs) during in vitro multiplication of C. esculenta. The SABs used were temporary immersion bioreactors (TIBs), SETIS™ bioreactors and ebb-and-flow bioreactors; semi-solid culture medium was used as a control treatment. At 30 d of culture, different developmental variables, determination of chlorophyll, stomatal content, and survival percentage during acclimatization were evaluated. SABs increased the shoot multiplication rate relative to the semi-solid medium; however, the SETIS™ bioreactor showed the highest shoot production, with 36 shoots per explant, and the highest chlorophyll content. The stomatal index was higher in the semi-solid medium compared to the SABs, while the percentage of closed stomata was higher in the SABs than in the semi-solid culture medium. The survival rate during acclimatization showed no differences among the culture systems assessed, obtaining survival rates higher than 99%. In conclusion, the SETIS™ bioreactor showed the highest multiplication rate; however, other bioreactor alternatives are available for semi-automation and cost reduction for micropropagation of C. esculenta.

Osmo- and cryotherapy of sugarcane (Saccharum spp. L.) shoot-tips infected with sugarcane mosaic virus (SCMV).

In this work, we investigated the effect of different osmoprotective treatments and of cryopreservation using a droplet-vitrification (D-V) protocol to eliminate sugarcane mosaic virus (SCMV) of shoot-tips excised from in vitro propagated infected plantlets. Shoot-tips of sugarcane (Saccharum spp. L.) were precultured on semisolid MS medium supplemented with 0.3 M sucrose for 1 day, loaded in solution with 0.4 M sucrose and 2 M glycerol for 30 min and exposed to plant vitrification solution 2 for 15 min at room temperature prior to ultra-rapid cooling in liquid nitrogen. Virus indexing was performed by the DAS-ELISA immunoenzymatic test. The presence of SCMV was confirmed in the donor-plantlets derived of infected field material. No virus was detected in the regenerated plantlets from shoot-tips subjected to cryopreservation protocol. The progressive decrease in absorbances occurred from the first preculture treatment and no significant differences (P ≤ 0.05) were found with respect to following steps of D-V protocol. These results indicate that the osmotic dehydration treatments (osmotherapy) and cryopreservation (cryotherapy) may be potentially effective strategies to remove the SCMV from infected plants.

Micropropagation of Guarianthe skinneri (Bateman) Dressler et W. E. Higging in Temporary Immersion Systems.

This study's aim was to establish a protocol for the micropropagation of G. skinneri using temporary immersion system (TIS). Different concentrations of 6-Benzylaminopurine (0, 1, 2, and 3 mg L-1), three different systems of cultivate semi-solid (SS) and liquid media under partial (PI) and temporary immersion systems (TIS), different compositions of the inorganic salts, and the number of subcultures were evaluated. The results showed a maximum of 16.56 shoots per explant obtained through TIS, adjusting all the parameters evaluated in our study. One higher number of shoots per explant was observed in the micropropagation of G. skinneri TIS compared to SS and PI. While the use of 3 mg L-1 of BAP + MS (Murashige and Skoog) media was better than 3 mg L-1 of BAP VW (Vacint and Went) for the generation of a greater number of shoots per explant, 6.33 and 2.72, respectively. The immersion frequency of 2 min every 4 h allowed the production to be scaled to 8.54 shoots per explant. While it was necessary to perform three subcultures every 30 days, to obtain 16.56 shoots per explant, a rooting phase was not required due to the generation of adventitious roots during the different subcultures. However, a phase of elongation of the regenerated plants with ½ MS + GA3 (gibberellic acid) was needed to guarantee 100% survival in the process of acclimatization. In conclusion, this plant production system can be applied for the commercial micropropagation of this species for ornamental purposes, as well as for its reintroduction in protected natural areas.

Biogeografía del tomate Solanum lycopersicum var. cerasiforme (Solanaceae) en su centro de origen (sur de América) y de domesticación (México)

El tomate (Solanum lycopersicum) es una de las principales hortalizas en el mundo debido a las importantes ganancias económicas que genera su comercialización; sin embargo, a pesar de que la especie es ampliamente cultivada en el mundo, su diversidad genética se considera restringida. Esto hace que su resistencia a plagas y enfermedades en las variedades actualmente cultivadas sea baja. Existe la forma silvestre S. l. var ceraciforme que se distribuye desde Suramérica (centro de origen) hasta México (área de domesticación), específicamente en la vertiente del Golfo de México en Veracruz. Como objetivo, se quiso identificar patrones de diversificación ambiental con los que se pudiesen inferir procesos de adaptación de las poblaciones en el área de domesticación con respecto a su centro de origen. Asimismo, se planteó proponer medidas de conservación y rescate del tomate S. l. var. cerasiforme. Se realizaron modelos de nicho ecológico (MNE) con Maxent versión 3.4.1 (evaluados con AUC y ROC-parcial) para determinar la idoneidad de las condiciones ambientales, incluyendo proyecciones del centro de domesticación al área de origen y viceversa. Esto se realizó con base en ocho variables de temperatura y precipitación. Los registros de tomate fueron tomados de fuentes bibliográficas y trabajo de campo. Seguidamente, se realizó la prueba de comparación de nichos (equivalencia y similitud), propuesta por Broennimann et al. (2012), para evaluar la similitud de condiciones ambientales en ambas regiones. Posteriormente, se realizó un análisis de varianza seguido de una prueba de comparación de medias (Tukey, P ≤ 0.05) con las ocho variables ambientales utilizadas. Los valores de AUC (0.93 y 0.80) y ROC-parcial obtenidos (1.86 y 1.71; P = 0.0001) de los MNE muestran que fueron buenos modelos predictores. Se observó que en el centro de México existen condiciones ambientales similares a las del centro de origen, a diferencia de la vertiente del Golfo de México donde son diferentes. La prueba de equivalencia mostró que la comparación ambiental entre ambas regiones es menos equivalente que lo esperado por azar (P = 0.003). Mientras tanto, la otra prueba indica que la similitud que existe entre ambas regiones también se puede obtener por azar (P = 0.683). También, se encontraron diferencias significativas en tres variables de temperatura y precipitación. En conclusión, el centro de origen y de domesticación de S. l. var. cerasiforme tienen características ambientales en común a pesar de la distancia geográfica, pero existen zonas geográficas (vertiente del Golfo de México en Veracruz) en el área de domesticación con condiciones ambientales diferentes a las de su centro de origen y con un potencial importante como bancos de germoplasma.

The tomato (Solanum lycopersicum) is one of the main horticultural crops in the world because of the important economic benefits that its commercialization generates. Even though the species is widely cultivated in the world, it is susceptible to pests, diseases, and environmental stresses due to the loss of its genetic diversity. There is a wild form called S. l. var ceraciforme that is distributed from South America (its center of origin) to Mexico (its area of domestication), specifically on the slope of the Gulf of Mexico in Veracruz. Due to the large genetic diversity of these wild tomatoes, it is important to improve its crop. The objective of this investigation was to identify patterns of environmental diversification of the tomato, infer the processes of adaptation of the populations in the area of domestication with respect to their center of origin and propose measures of conservation and variation of S. l. var. cerasiforme. We generated two ecological niche models (MNE) with Maxent version 3.4.1 (evaluated with AUC and partial-ROC) to determine the suitability of environmental conditions including their respective projections from the domestication center to the area of origin and vice versa. We used eight variables of temperature and precipitation. Additionally, we included tomato records from bibliographical sources and fieldwork. We also used the niche comparison test (equivalency and similarity) proposed by Broennimann et al. (2012) to evaluate the similarity of environmental conditions in both regions. Subsequently, we carried out an analysis of variance followed by a mean comparison test (Tukey, P ≤ 0.05) with all environmental variables measured. The values of AUC (0.93 and 0.80) and partial-ROC (1.86 and 1.71, P = 0.0001) of the MNE showed that they were good predictive models. We observed that, in the center of Mexico, there are environmental conditions similar to those of the center of origin, unlike the slope of the Gulf of Mexico where they are different. The equivalency test showed that the environmental comparison between both regions is less similar than expected by chance (P = 0.003). The similarity test indicated that the existing similarity between both regions can also be obtained by chance (P = 0.683). We also found significant differences in three temperature and precipitation variables. In conclusion, we determined that the center of origin and domestication of S. l. var. cerasiforme has similar environmental characteristics despite the geographic distance; nevertheless, there are geographical zones (the Gulf of Mexico in Veracruz) in the area of domestication with different environmental conditions. Those places have the potential to contain valuable germplasms.