SNP markers identification by genome wide association study for chemical quality traits of coffee (Coffea spp.) Germplasm.

BACKGROUND: Coffee quality is an important selection criterion for coffee breeding. Metabolite profiling and Genome-Wide Association Studies (GWAS) effectively dissect the genetic background of complex traits such as metabolites content (caffeine, trigonelline, and 5-caffeoylquinic acid (5-CQA)) in coffee that affect quality. Therefore, it is important to determine the metabolic profiles of Coffea spp. genotypes. This study aimed to identify Single Nucleotide Polymorphisms (SNPs) within Coffea spp. genotypes through GWAS and associate these significant SNPs to the metabolic profiles of the different genotypes. METHODS AND RESULTS: A total of 1,739 SNP markers were obtained from 80 genotypes using the DArTseq™ method. Caffeine, trigonelline, and 5-CQA content were determined in coffee leaves using Ultra-Performance Liquid Chromatography/tandem mass spectrometry (UPLC-MS/MS) analyses. The GWAS was carried out using the Genome Association and Prediction Integrated Tool (GAPIT) software and a compressed mixed linear model. Finally, a total of three significant SNP markers out of ten were identified. One SNP, located in the coffee chromosome (Chr) 8, was significantly associated with caffeine. The two remaining SNPs, located in Chr 4 and 5, were significantly associated with trigonelline and six SNPs markers were associated with 5-CQA in Chr 1, 5 and 10, but these six markers were not significant. CONCLUSIONS: These significant SNP sequences were associated with protein ubiquitination, assimilation, and wall receptor kinases. Therefore, these SNPs might be useful hits in subsequent quality coffee breeding programs.

Evaluation of in vitro shoot multiplication and ISSR marker based assessment of somaclonal variants at different subcultures of vanilla (Vanilla planifolia Jacks).

The effect of subculture cycles on somaclonal variation of V. planifolia using intersimple sequence repeat (ISSR) markers was analyzed. Nodal segments of 2 cm in length were established in vitro and multiplied by 10 subculture cycles in Murashige and Skoog (MS) medium supplemented with 8.86 µM BAP (benzylaminopurine). After 45 days in each culture, the length and number of shoots per explant were evaluated. For ISSR markers, ten shoots per each subculture and the mother plant were used. Ten ISSR primers were used and a total of 118 bands were obtained. The polymorphism (%) was calculated and a dendrogram based on Jaccard's genetic distance between the subcultures and the donor plant was obtained. These results show that the multiplication rate tends to increase until subculture five, whereas shoot length decreases as the number of subcultures increases. The ISSR markers revealed an increase in the polymorphism percentage after the fifth culture cycle. The dendrogram showed the formation of two groups. The first group, with less genetic variability, is the donor plant and subcultures 1-5; the second group has greater genetic distance and is formed by subcultures 6-10. The results revealed that the number of subcultures with 8.86 µM BAP is a factor that affects the somaclonal variation during in vitro regeneration of V. planifolia. In conclusion, the subculture number affects somaclonal variation and in vitro development of V. planifolia.

Temporary Immersion Bioreactors for Sugarcane Multiplication and Rooting.

Sugarcane is used to produce sugar, ethanol, and other by-products, so it is considered one of the most important crops worldwide. Using temporary immersion systems for sugarcane micropropagation represents an alternative to reduce the labor force, increase plant development, and improve plant quality. Temporary immersion systems are semi-automated bioreactors designed for the large-scale propagation of tissues, embryos, and organs. These are temporarily exposed in a liquid culture medium under a specific time and immersion frequency. Using this protocol and a temporary immersion bioreactor, it is possible to achieve multiplication and rooting. The use of temporary immersion bioreactors improves the multiplication rate and the rooting of sugarcane, reducing the culture time, labor force, and reagents needed while maintaining high survival rates during acclimatization.

In Vitro Multiplication of Agave (A. marmorata and A. potatorum) by Temporary Immersion in SETIS™ Bioreactor.

In Mexico, wild agaves are important for the production of alcoholic beverages known as mezcal and pulque. However, the propagation of agave seeds and pups is not enough to satisfy the national demand. Temporary immersion systems represent an agave micropropagation alternative that reduces the labor force, increases development, and improves the quality of seedlings. The use of the SETIS™ bioreactor in A. marmorata and A. potatorum improves the multiplication rate and allows rooting. Additionally, this bioreactor reduces the culture time, labor force, and reagents needed while maintaining high survival rates during the acclimatization phase.

Temporary immersion systems induce photomixotrophism during in vitro propagation of agave Tobalá.

This study aimed to determine the photomixotrophic and physiological responses using different temporary immersion systems (TIS) during in vitro multiplication of agave Tobalá. The culture systems were SETIS™ bioreactor, Temporary Immersion Bioreactor (TIB), Monobloc Advance Temporary Immersion System, and semisolid culture medium as the control. At six weeks of culture, different physiological variables were evaluated: chlorophyll content, stomatal index, percentage of closed stomata, Phosphoenolpyruvate (PEP) and Rubisco during the multiplication stage, and survival percentage in the acclimatization stage. TIS increased multiplication rate (41%), stomatal index (44%), percentage of closed stomata (11%) and chlorophyll content (45%) with respect to the semisolid culture medium system. The highest PEP content (> 35%) was observed in temporary immersion (TI), whereas, Rubisco content, showed no differences among the culture systems evaluated. Regarding survival percentage during acclimatization, the highest shoot survival was obtained in TI, and all regenerate shoots were rooted ex vitro. This study shows that in vitro photomixotrophism was induced in TIS during the multiplication stage. In conclusion, SETIS™ bioreactor and TIB systems are an alternative for mass multiplication of A. potatorum; however, all TIS evaluated guarantee a high survival rate during acclimatization.

Arbuscular Mycorrhizal Symbiosis Improves Ex Vitro Acclimatization of Sugarcane Plantlets (Saccharum spp.) under Drought Stress Conditions.

The symbiotic associations between arbuscular mycorrhizal fungi (AMF) and plants can induce drought stress tolerance. In this study, we evaluated the effect of Glomus intraradices, a mycorrhizal fungus, on the ex vitro development and survival of sugarcane plantlets subjected to drought stress during the acclimatization stage of micropropagation. In vitro obtained sugarcane plantlets (Saccharum spp. cv Mex 69-290) were inoculated with different doses of G. intraradices (0, 100, and 200 spores per plantlet) during greenhouse acclimatization. Sixty days after inoculation, plantlets were temporarily subjected to drought stress. We evaluated the survival rate, total chlorophyll, total protein, carotenoids, proline, betaine glycine, soluble phenolic content, and antioxidant capacity every 3 days for 12 days. Symbiotic interaction was characterized by microscopy. Our results showed that the survival rate of inoculated plants was higher in 45% than the treatment without mycorrhizae. Total chlorophyll, protein, proline, betaine glycine content, and antioxidant capacity were increased in AMF inoculated plants. The soluble phenolic content was higher in non-inoculated plants than the treatment with mycorrhizae during the drought stress period. Microscopy showed the symbiotic relationship between plant and AMF. The early inoculation of 100 spores of G. intraradices per sugarcane plantlet during the acclimatization stage could represent a preconditioning advantage before transplanting into the field and establishing basic seedbeds.

In Vitro Stress-Mediated Somatic Embryogenesis in Plants.

Somatic embryogenesis (SE) serves as a key biological model for studying cell totipotency and the ontogenic processes of zygotic embryogenesis in plants. The SE process, under in vitro conditions, can be induced from different sources of explant cultivated in a culture medium with plant growth regulators (PGR) or by subjecting tissues to abiotic stress treatments. Somatic embryogenesis, in plant tissue culture (PTC), is a multifactorial event. The use of PGR, particularly auxins, is an important factor during induction. However, in vitro abiotic stress treatments are physiologically, biochemically, and genetically relevant and should be further studied.

Arbuscular Mycorrhizal Fungi Induce Tolerance to Salinity Stress in Taro Plantlets (Colocasia esculenta L. Schott) during Acclimatization.

Soil salinity is a problem that affects soil fertility and threatens agri-food crop production worldwide. Biotechnology, through plant micropropagation and the use of biofertilizers such as arbuscular mycorrhizal fungi (AMF), is an alternative to increase productivity and induce tolerance to salinity stress in different crops. This study aimed to evaluate the effect of different doses of the fungus Glomus intraradices on the ex vitro development of taro (Colocasia esculenta L. Schott cv. Criolla) plantlets under salinity stress during the acclimatization stage. In vitro-obtained C. esculenta plantlets were inoculated at different doses (0, 100, and 200 spores per plantlet) of G. intraradices during acclimatization. At 60 d of acclimatization in the greenhouse, plantlets were exposed to 100 mM NaCl salinity stress for 10 d. After the stress period, plantlet development, colonization percentage, and biomass were evaluated. In addition, the content of chlorophyll, carotenoids, proteins, proline, glycine-betaine, soluble phenols, and antioxidant capacity were quantified. The results showed differences in the developmental, physiological, and biochemical variables evaluated; however, no changes in total protein content were observed. Spore colonization showed that the symbiotic association has positive effects on the development of plantlets with or without salinity stress. This symbiotic interaction contributes to salinity stress tolerance in C. esculenta plantlets. The early application of AMF in in vitro-obtained taro plantlets is an alternative to increase or maintain the productivity of this crop in saline soils.

A temporary immersion system for mass micropropagation of pitahaya (Hylocereus undatus).

Scaling-up techniques in temporary immersion systems are an alternative for commercial micropropagation. In vitro propagation of pitahaya (Hylocereus undatus) using temporary immersion with liquid culture medium improves micropropagation efficiency compared to the conventional method in semisolid medium. The objective of this work was to evaluate the efficiency of traditional culture and temporary immersion during micropropagation of pitahaya to facilitate the rapid establishment of new commercial plantations of high genetic and phytosanitary quality. Semisolid culture, liquid media in partial immersion and temporary immersion in an Ebb-and-Flow bioreactor were evaluated. Also, in temporary immersion, different immersion frequencies (every 4, 8, 12, and 16 h) and culture densities (5, 10, 15 and 20 explants per bioreactor) were evaluated. For the multiplication stage, new shoots and length were recorded per explant at 45 d of in vitro culture and in the acclimatization stage, the survival percentage was determined at 30 d of greenhouse cultivation. A temporary immersion of 2 min every 4 h and 15 explants per bioreactor was the best culture system, obtaining on average 10.7 shoots per explant with a length of 1.9 cm. No significant differences were observed among treatments during acclimatization, obtaining survival percentages of 98%-100%. This study reports for the first time a protocol for scaling-up techniques in temporary immersion for commercial micropropagation of pitahaya (and for any species of the Cactaceae family) and its establishment in a productive plantation.

In vitro response of vanilla (Vanilla planifolia Jacks. ex Andrews) to PEG-induced osmotic stress.

Drought-induced water stress affects the productivity of the Vanilla planifolia Jacks. ex Andrews crop. In vitro culture technique is an effective tool for the study of water stress tolerance mechanisms. This study aimed to evaluate the morphological, physiological and biochemical response of V. planifolia under in vitro water stress conditions induced with polyethylene glycol (PEG). In vitro regenerated shoots of 2 cm in length were subjected to different concentrations of PEG 6000 (0, 1, 2 and 3% w/v) using Murashige and Skoog semi-solid culture medium. At 60 days of culture, different growth variables, dry matter (DM) content, chlorophyll (Chl), soluble proteins (SP), proline (Pro), glycine betaine (GB), stomatal index (SI) and open stomata (%) were evaluated. Results showed a reduction in growth, Chl content, SP, SI and open stomata (%) with increasing PEG concentration, whereas DM, Pro and GB contents rose with increasing PEG concentration. In conclusion, PEG-induced osmotic stress allowed describing physiological and biochemical mechanisms of response to water stress. Furthermore, the determination of compatible Pro and GB osmolytes can be used as biochemical markers in future breeding programs for the early selection of water stress tolerant genotypes.

Multi-Walled Carbon Nanotubes Improved Development during In Vitro Multiplication of Sugarcane (Saccharum spp.) in a Semi-Automated Bioreactor.

Carbon nanotubes play an important role in plant biotechnology due to their effects on the growth and differentiation of cells, tissues, organs, and whole plants. This study aimed to evaluate the effect of multi-walled carbon nanotubes (MWCNTs) during in vitro multiplication of sugarcane (Saccharum spp.) using a temporary immersion system. Morphological characterization of MWCNTs was carried out under a transmission electron microscope. Different concentrations (0, 50, 100, 200 mg L-1) of MWCNTs were added to Murashige and Skoog liquid culture medium in the multiplication stage. At 30 d of culture, number of shoots per explant, shoot length, number of leaves per shoot, total chlorophyll, dry matter percentage, carbon percentage, and macro- and micronutrient content were evaluated. Results showed an increase in the development of sugarcane shoots at concentrations of 100 and 200 mg L-1 MWCNT. Total chlorophyll content increased at concentrations of 50 and 100 mg L-1 MWCNT, whereas macro- and micronutrient content was variable at the different MWCNT concentrations. Results suggest a hormetic effect, characterized by stimulation at low concentrations. In conclusion, the use of low concentrations of MWCNTs had positive effects on development, total chlorophyll, carbon percentage, and macro- and micronutrient (N, Ca, S, Fe, Cu, Zn and Na) contents during in vitro multiplication of sugarcane and may have a potential use in other species of agricultural interest.

Silicon induces hormetic dose-response effects on growth and concentrations of chlorophylls, amino acids and sugars in pepper plants during the early developmental stage.

BACKGROUND: Silicon (Si) is a beneficial element that has been proven to influence plant responses including growth, development and metabolism in a hormetic manner. METHODS: In the present study, we evaluated the effect of Si on the growth and concentrations of chlorophylls, total amino acids, and total sugars of pepper plants (Capsicum annuum L.) during the early developmental stage in a hydroponic system under conventional (unstressed) conditions. We tested four Si concentrations (applied as calcium silicate): 0, 60, 125 and 250 mg L-1, and growth variables were measured 7, 14, 21 and 28 days after treatment (dat), while biochemical variables were recorded at the end of the experiment, 28 dat. RESULTS: The application of 125 mg L-1 Si improved leaf area, fresh and dry biomass weight in leaves and stems, total soluble sugars, and concentrations of chlorophylls a and b in both leaves and stems. The amino acids concentration in leaves and roots, as well as the stem diameter were the highest in plants treated with 60 mg L-1 Si. Nevertheless, Si applications reduced root length, stem diameter and total free amino acids in leaves and stems, especially when applied at the highest concentration (i.e., 250 mg L-1 Si). CONCLUSION: The application of Si has positive effects on pepper plants during the early developmental stage, including stimulation of growth, as well as increased concentrations of chlorophylls, total free amino acids and total soluble sugars. In general, most benefits from Si applications were observed in the range of 60-125 mg L-1 Si, while some negative effects were observed at the highest concentration applied (i.e., 250 mg L-1 Si). Therefore, pepper is a good candidate crop to benefit from Si application during the early developmental stage under unstressed conditions.

Cytotoxic, Genotoxic, and Polymorphism Effects on Vanilla planifolia Jacks ex Andrews after Long-Term Exposure to Argovit® Silver Nanoparticles.

Worldwide demands of Vanilla planifolia lead to finding new options to produce large-scale and contaminant-free crops. Particularly, the Mexican Government has classified Vanilla planifolia at risk and it subject to protection programs since wild species are in danger of extinction and no more than 30 clones have been found. Nanotechnology could help to solve both demands and genetic variability, but toxicological concerns must be solved. In this work, we present the first study of the cytotoxic and genotoxic effects promoted by AgNPs in Vanilla planifolia plantlets after a very long exposure time of six weeks. Our results show that Vanilla planifolia plantlets growth with doses of 25 and 50 mg/L is favored with a small decrease in the mitotic index. A dose-dependency in the frequency of cells with chromosomal aberrations and micronuclei was found. However, genotoxic effects could be considered as minimum due to with the highest concentration employed (200 mg/L), the total percentage of chromatic aberrations is lower than 5% with only three micronuclei in 3000 cells, despite the long-time exposure to AgNP. Therefore, 25 and 50 mg/L (1.5 and 3 mg/L of metallic silver) were identified as safe concentrations for Vanilla planifolia growth on in vitro conditions. Exposure of plantlets to AgNPs increase the polymorphism registered by inter-simple sequence repeat method (ISSR), which could be useful to promote the genetic variability of this species.