CRISPR-Cas Technology in Plant Science

CRISPR-Cas technology has raised considerable interest among plant scientists, both in basic science and in plant breeding. Presently, the generation of random mutations at a predetermined site of the genome is well mastered, just like the targeted insertion of transgenes, although both remain restricted to species or genotypes amenable for plant transformation. On the other hand, true genome editing, i.e. the deliberate replacement of one or several nucleotides of the genome in a predetermined fashion, is limited to some rather particular examples that generally concern genes allowing positive selection, for example tolerance to herbicides. Therefore, further technological developments are necessary to fully exploit the potential of genome editing in enlarging the gene pool beyond the natural variability available in a given species. In principle, the technology can be applied to any quality related, agronomical or ecological trait, under the condition of upstream knowledge on the genes to be targeted and the precise modifications necessary to improve alleles. Published proof of concepts concern a wide range of agronomical traits, the most frequent being disease resistance, herbicide tolerance and the biochemical composition of harvested products. The regulatory status of the plants obtained by CRISPR-Cas technology raises numerous questions, in particular with regard to the plants that carry in their genomes the punctual modifications caused by the presence of the Cas9 nuclease but not the nuclease itself. Without clarification by the competent authorities, CRISPR-Cas technology would continue to be a powerful tool in functional genomics, but its potential in plant breeding would remain untapped.     

Breeding Differently: Participatory Selection and Scaling Up Innovations in Colombia

Global challenges like sustainable development, climate change, malnutrition and gender inequality can be tackled from an agricultural perspective. Micronutrient deficiency is a major problem for the health and food security of the population. In Colombia, iron deficiencies affect 32% of the children under the age of five; and 43% of the Colombian population have zinc and vitamin A deficiencies. We designed and implemented a research-for-development programme that links agriculture and nutrition through a two-stage project. In the first stage, we selected three new yellow potato cultivars with better nutritional contents, higher yield and better resistance to late blight than the traditional yellow cultivars. We characterized the Colombian germplasm, Solanum tuberosum Group Phureja, for its nutritional attributes. During this stage, studies of the social and nutritional status of the communities were also conducted. In the second stage, we focused on scaling up new potato cultivars to reach potato producers and consumers by promoting the production and marketing of good quality seed potatoes, by establishing four Rural Entrepreneurs Nuclei. The programme promotes gender equity, good dietary habits, the consumption of more nutritious potatoes and empowers local leadership to strengthen governance. Field work was conducted in southern and central regions of Colombia by employing social strategies such as the Family Farming Community Schools. In 50% of the cultivated area, we are replacing the originally used traditional yellow potatoes by the new, more nutritious, potatoes to reach 6 million consumers. A good quality seed system for small-scale potato growers has been implemented. These achievements have been reached through a comprehensive approach in a period of two years.     

Silage and total mixed ration hygienic quality on commercial farms: implications for animal production

Implications of silage hygienic quality for animal production were investigated on forty‐five dairy farms in South West England. Samples of grass and maize silages and of total mixed rations (TMR) were obtained together with information on silage technology, herd size and animal production. Samples were analysed for mycotoxins, bacteria, yeasts, moulds and chemical composition. Thirteen mycotoxins were assayed, but none were detected in the samples of grass silage. However, mycotoxins were found in 0·9 of all maize and other silage samples, with deoxynivalenol and zearalenone predominating. There was no relationship between total mycotoxin concentration and mean lactation milk yield per cow. Enterobacteria counts tended to be higher in maize silage than in grass silage and higher still in TMR – a cause for concern. There were no relationships between mould counts and mycotoxin concentrations in silages, implying that mycotoxins may have been produced in the field pre‐ensiling.     

Evaluation of Particle Size Influence on Proximate Composition,Physicochemical, Techno-Functional and Physio-Functional Properties of Flours Obtained from Persimmon (<Emphasis Type="Italic">Diospyros kaki</Emphasis> Trumb.) Coproducts

The aim of the work was to study the influence of particle size in the composition, physicochemical, techno-functional and physio-functional properties of two flours obtained from persimmon (Diospyros kaki Trumb. cvs. ‘Rojo Brillante’ (RBF) and ‘Triump’ (THF) coproducts. The cultivar (RBF and THF) and particle size significantly affected all parameters under study, although depending on the evaluated property, only one of these effects predominated. Carbohydrates (38.07–46.98 g/100 g) and total dietary fiber (32.07–43.57 g/100 g) were the main components in both flours (RBF and THF). Furthermore, insoluble dietary fiber represented more than 68% of total dietary fiber content. All color properties studied were influenced by cultivar and particle size. For both cultivars, the lower particle size, the higher lightness and hue values. RBF flours showed high values for emulsifying activity (69.33–74.00 mL/mL), while THF presented high values for water holding capacity (WHC: 9.47–12.19 g water/g sample). The bile holding capacity (BHC) and fat/oil binding values were, in general, higher in RBF (19.61–12.19 g bile/g sample and 11.98–9.07, respectively) than THF (16.12–12.40 g bile/g sample and 9.78–7.96, respectively). The effect of particle size was really evident in both WHC and BHC. Due to their dietary fiber content, techno-functional and physio-functional properties, persimmon flours seem to have a good profile to be used as potential functional ingredient.     

<Emphasis Type="Italic">Lupinus albus</Emphasis> Conglutin Gamma Modifies the Gene Expressions of Enzymes Involved in Glucose Hepatic Production <Emphasis Type="Italic">In Vivo</Emphasis>

Lupinus albus seeds contain conglutin gamma (Cγ) protein, which exerts a hypoglycemic effect and positively modifies proteins involved in glucose homeostasis. Cγ could potentially be used to manage patients with impaired glucose metabolism, but there remains a need to evaluate its effects on hepatic glucose production. The present study aimed to analyze G6pc, Fbp1, and Pck1 gene expressions in two experimental animal models of impaired glucose metabolism. We also evaluated hepatic and renal tissue integrity following Cγ treatment. To generate an insulin resistance model, male Wistar rats were provided 30% sucrose solution ad libitum for 20 weeks. To generate a type 2 diabetes model (STZ), five-day-old rats were intraperitoneally injected with streptozotocin (150 mg/kg). Each animal model was randomized into three subgroups that received the following oral treatments daily for one week: 0.9% w/v NaCl (vehicle; IR-Ctrl and STZ-Ctrl); metformin 300 mg/kg (IR-Met and STZ-Met); and Cγ 150 mg/kg (IR-Cγ and STZ-Cγ). Biochemical parameters were assessed pre- and post-treatment using colorimetric or enzymatic methods. We also performed histological analysis of hepatic and renal tissue. G6pc, Fbp1, and Pck1 gene expressions were quantified using real-time PCR. No histological changes were observed in any group. Post-treatment G6pc gene expression was decreased in the IR-Cγ and STZ-Cγ groups. Post-treatment Fbp1 and Pck1 gene expressions were reduced in the IR-Cγ group but increased in STZ-Cγ animals. Overall, these findings suggest that Cγ is involved in reducing hepatic glucose production, mainly through G6pc inhibition in impaired glucose metabolism disorders.     

Digestibility of Quinoa (<Emphasis Type="Italic">Chenopodium quinoa</Emphasis> Willd.) Protein Concentrate and Its Potential to Inhibit Lipid Peroxidation in the Zebrafish Larvae Model

Quinoa protein concentrate (QPC) was extracted and digested under in vitro gastrointestinal conditions. The protein content of QPC was in the range between 52.40 and 65.01% depending on the assay used. Quinoa proteins were almost completely hydrolyzed by pepsin at pH of 1.2, 2.0, and 3.2. At high pH, only partial hydrolysis was observed. During the duodenal phase, no intact proteins were visible, indicating their susceptibility to the in vitro simulated digestive conditions. Zebrafish larvae model was used to evaluate the in vivo ability of gastrointestinal digests to inhibit lipid peroxidation. Gastric digestion at pH 1.2 showed the highest lipid peroxidation inhibition percentage (75.15%). The lipid peroxidation activity increased after the duodenal phase. The digest obtained at the end of the digestive process showed an inhibition percentage of 82.10%, comparable to that showed when using BHT as positive control (87.13%).     

Performance of Granular Starch with Controlled Pore Size during Hydrolysis with Digestive Enzymes

Studies on porous starch have been directed to explore different industrial applications as bio-adsorbents of a variety of compounds. However, the analysis of starch digestibility is essential for food application. The objective of this study was to determine the impact of porous structure on in vitro starch digestibility. Porous starches were obtained using a range of concentrations of amyloglucosidase (AMG), α-amylase (AM), cyclodextrin-glycosyltransferase (CGTase) or branching enzyme (BE). Porous starches exhibited major content of digestible starch (DS) that increased with the intensity of the enzymatic treatment, and very low amount of resistant starch (RS). Porous starches behaved differently during in vitro hydrolysis depending on their enzymatic treatment. AMG was the unique treatment that increased the digestive amylolysis and estimated glycemic index, whereas AM, CGTase and BE reduced them. A significant relationship was found between the pore size and the severity of the amylolysis, suggesting that a specific pore size is required for the accessibility of the digestive amylase. Therefore, pore size in the starch surface was a limiting factor for digestion of starch granules.     

Identifying and exploring significant genomic regions associated with soybean yield,seed fatty acids,protein and oil

Soybean [Glycine max (L.) Merrill] yield and seed fatty acids, protein, and oil content are important traits for which an improved understanding of significant genomic regions would be useful. To accomplish this, a soybean population consisting of 203 F5 derived recombinant inbred lines (RILs) was developed and genotyped with 11,633 polymorphic single nucleotide polymorphisms (SNPs). Each RIL was grown in a single plot at Knoxville, TN in 2010; followed by replicated, multi-location field trials in 2013 and 2014. The data from 2010, 2013, and 2014 were analyzed together in order to detect quantitative trait loci (QTL) for these traits, and 30 total QTLs were detected. Five QTLs are candidates for confirmed status and one QTL is a candidate for positional confirmation. Many of the genes with mutations in close proximity to the fatty acid QTLs are involved in biological processes for fatty acids and/or lipids and could be considered possible candidate genes. Similarly, genes with mutations in genomic regions near yield, protein, and oil QTLs were plentiful and may contribute to the variation observed in these traits. Except for yield and stearic acid, each trait displayed pleiotropic effects with other traits in this study. Notable are the pleiotropic effects for oleic and linolenic acid on chromosomes 9, 13, and 19. Overall, the findings from this research contribute new information to the genetic understanding of soybean yield and seed fatty acids, protein and oil content. This understanding will be useful in making trait improvements.     

Genotype by environment interactions (GEIs) for barley grain yield under salt stress condition

Changes in the relative genetic performance of genotypes across environments are referred to as genotype × environment interactions (GEIs). GEIs can affect barley breeding improvement for salt tolerance because it often complicates the evaluation and selection of superior genotypes. The present study evaluated the GEIs over 60 barley genotypes for yield components and grain yield in six salinity environments in North Delta, Egypt. Data were analyzed using the additive main effects and multiplicative interaction (AMMI) and Tai’s stability parameters. GEIs effects on yield explained 20.3, 20.1, 14.6, and 33.0% of the total variation besides, the first two principal components account for 67.3, 56.3, 64.3, and 83.7% of the explained variance in the four sets, respectively. Six genotypes namely G-4, G-7, G-20, G-34, G-36, and G-39 were found to be most stable and high yielding across environments (GY >2.00 t ha^-1), and located close to zero projection onto the AEC ordinate. Tai’s stability parameters demonstrated that these genotypes were more responsive to the environmental changes. The genotypes G-50 and G-53 showed perfect/static stability (α = -0.95, -0.91, respectively). In contrast, the genotype; G-36 had α = 0 and λ = 1.10, indicating parallel with the environmental effects followed by G-44. Overall, we found that GEIs for grain yield are highly significant in all sets, suggesting that responded differently across environments. This interaction may be a result of changes in genotypes’ relative performance across environments, due to their differential responses to various abiotic factors.     

Evaluation of carbon sources,gelling agents,growth hormones and additives for efficient callus induction and plant regeneration in Indian wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) genotypes using mature embryos

Various factors affecting in vitro regeneration like different carbon sources, different gelling agents, and growth additives were assessed comprehensively for callus induction and plant regeneration for five Indian wheat cultivars using mature embryos as the explants for the first time. The tissue culture responses of cultivars WH-1105, HD-2967, and PBW-343 have not been reported earlier. Besides, the effect of different concentrations of the cytokinin, zeatin has also been optimized. Using the optimized factors, the efficiency of five different varieties, i.e., HD 2967, C 306, RAJ 3765, WH 1105, and PBW 343 was evaluated for regeneration. Modified MS basal medium containing dicamba reduced precocious germination of the embryo and induced embryogenic callus more efficiently. Removal of embryogenic calli from non-regenerable structures during early callus phase improved plant regeneration. These calli on zeatin (1.0 mgl^-1) and dicamba (0.1 mgl^-1) containing medium showed the highest regeneration frequency (98%) with a maximum of 8-9 shoots per calli. Maltose had the maximum callusing and regeneration percentage than other carbon sources. Various gelling agents did not have any significant difference on the regeneration. Of all the varieties, C-306 and HD-2967 were found to be more regenerative and can be used in transformation experiments.