Genetic Diversity of <em>Coffea arabica</em> L.: A Genomic Approach

Coffea arabica L. produces a high-quality beverage, with pleasant aroma and flavor, but diseases, pests and abiotic stresses often affect its yield. Therefore, improving important agronomic traits of this commercial specie remains a target for most coffee improvement programs. With advances in genomic and sequencing technology, it is feasible to understand the coffee genome and the molecular inheritance underlying coffee traits, thereby helping improve the efficiency of breeding programs. Thanks to the rapid development of genomic resources and the publication of the C. canephora reference genome, third-generation markers based on single-nucleotide polymorphisms (SNPs) have gradually been identified and assayed in Coffea, particularly in C. arabica. However, high-throughput genotyping assays are still needed in order to rapidly characterize the coffee genetic diversity and to evaluate the introgression of different cultivars in a cost-effective way. The DArTseq™ platform, developed by Diversity Arrays Technology, is one of these approaches that has experienced an increasing interest worldwide since it is able to generate thousands of high quality SNPs in a timely and cost-effective manner. These validated SNP markers will be useful to molecular genetics and for innovative approaches in coffee breeding

Gamma Radiation (60Co) Induces Mutation during In Vitro Multiplication of Vanilla (Vanilla planifolia Jacks. ex Andrews)

In vitro mutagenesis is an alternative to induce genetic variation in vanilla (Vanilla planifolia Jacks. ex Andrews), which is characterized by low genetic diversity. The objective of this study was to induce somaclonal variation in V. planifolia by gamma radiation and detect it using inter-simple sequence repeat (ISSR) molecular markers. Shoots previously established in vitro were multiplied in Murashige and Skoog culture medium supplemented with 2 mg&middot;L&minus;1 BAP (6-benzylaminopurine). Explants were irradiated with different doses (0, 20, 40, 60, 80 and 100 Gy) of 60Co gamma rays. Survival percentage, number of shoots per explant, shoot length, number of leaves per shoot, and lethal dose (LD50) were recorded after 60 d of culture. For molecular analysis, ten shoots were used for each dose and the donor plant as a control. Eight ISSR primers were selected, and 43 fragments were obtained. The percentage of polymorphism (% P) was estimated. A dendrogram based on Jaccard&rsquo;s coefficient and the neighbor joining clustering method was obtained. Results showed a hormetic effect on the explants, promoting development at low dose (20 Gy) and inhibition and death at high doses (60&ndash;100 Gy). The LD50 was observed at the 60 Gy. Primers UBC-808, UBC-836 and UBC-840 showed the highest % P, with 42.6%, 34.7% and 28.7%, respectively. Genetic distance analysis showed that treatments without irradiation and with irradiation presented somaclonal variation. The use of gamma rays during in vitro culture is an alternative to broaden genetic diversity for vanilla breeding

In Vitro Conservation and Regeneration of Potato (<i>Solanum tuberosum</i> L.): Role of Paclobutrazol and Silver Nanoparticles

In vitro conservation and regeneration of potato germplasm is important in breeding programs. The objective of this study was to assess the combined effect of paclobutrazol (PAC) and silver nanoparticles (AgNPs) during reduced-growth conservation and their in vitro ability to regenerate S. tuberosum after conservation treatments. For the conservation system, apices were used as explants in Murashige and Skoog culture media with different combinations of PAC (0, 0.5, 1 and 2 mg L−1) and AgNPs (0, 50, 100 and 200 mg L−1). At six months of culture, plant length, number of lateral branches, number of leaves and roots, root length, stomatal density and total chlorophyll content were assessed. For regeneration, explants were internodes cultured in medium with only 2 mg L−1 benzyladenine (BA). At 60 d of culture, the response percentage, number of shoots per explant and shoot length were assessed. For in vitro conservation, the combination of 2 mg L−1 PAC and 50 mg L−1 AgNPs was the best treatment, whereas for in vitro regeneration, the highest number of shoots was in explants that were treated with 1 or 2 mg L−1 PAC with 50 mg L−1 AgNPs. In conclusion, PAC and AgNPs are alternatives for in vitro S. tuberosum conservation, and their evaluation in other species recalcitrant to the effect of ethylene is recommended

Gamma Radiation (⁶⁰Co) Induces Mutation during In Vitro Multiplication of Vanilla (<i>Vanilla planifolia</i> Jacks. ex Andrews)

In vitro mutagenesis is an alternative to induce genetic variation in vanilla (Vanilla planifolia Jacks. ex Andrews), which is characterized by low genetic diversity. The objective of this study was to induce somaclonal variation in V. planifolia by gamma radiation and detect it using inter-simple sequence repeat (ISSR) molecular markers. Shoots previously established in vitro were multiplied in Murashige and Skoog culture medium supplemented with 2 mg·L−1 BAP (6-benzylaminopurine). Explants were irradiated with different doses (0, 20, 40, 60, 80 and 100 Gy) of 60Co gamma rays. Survival percentage, number of shoots per explant, shoot length, number of leaves per shoot, and lethal dose (LD50) were recorded after 60 d of culture. For molecular analysis, ten shoots were used for each dose and the donor plant as a control. Eight ISSR primers were selected, and 43 fragments were obtained. The percentage of polymorphism (% P) was estimated. A dendrogram based on Jaccard’s coefficient and the neighbor joining clustering method was obtained. Results showed a hormetic effect on the explants, promoting development at low dose (20 Gy) and inhibition and death at high doses (60–100 Gy). The LD50 was observed at the 60 Gy. Primers UBC-808, UBC-836 and UBC-840 showed the highest % P, with 42.6%, 34.7% and 28.7%, respectively. Genetic distance analysis showed that treatments without irradiation and with irradiation presented somaclonal variation. The use of gamma rays during in vitro culture is an alternative to broaden genetic diversity for vanilla breeding

Plant Extracts Control In Vitro Growth of Disease-Causing Fungi in Chayote

The use of agrochemicals has caused environmental problems and toxicity to humans, so natural alternatives for disease control during harvest and postharvest have been evaluated. The aim of this study was to evaluate cinnamon essential oil, neem oil, and black sapote fruit extract for in vitro inhibition of fungi isolated from chayote fruit. The extracts were applied at 300, 350, and 400 ppm in Petri dishes and the mycelial growth of Fusarium oxysporum, Fusarium solani, Goetrichum sp., and Phytophthora capsici was evaluated for 7 days, and the percentage of mycelial growth inhibition per day was calculated. Cinnamon oil showed a fungicidal effect at all concentrations. Neem oil at 400 ppm showed a 42.3% reduction in the growth of F. solani and 27.8% reduction in the growth of F. oxysporum, while at 350 ppm it inhibited the mycelial growth of Phytophthora capsici by 53.3% and of Goetrichum sp. by 20.9%; finally, the black sapote extract at 400 ppm inhibited 21.9–28.6% of the growth of all fungi. The growth of postharvest fungi on chayote fruit could be prevented or reduced by applying the plant extracts evaluated at adequate concentrations

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&ndash;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

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