Less than 10% of the estimated average requirement (EAR) for iron and zinc is provided by consumption of storage roots of the staple crop cassava (Manihot esculenta Crantz) in West African human ...populations. We used genetic engineering to improve mineral micronutrient concentrations in cassava. Overexpression of the Arabidopsis thaliana vacuolar iron transporter VIT1 in cassava accumulated three- to seven-times-higher levels of iron in transgenic storage roots than nontransgenic controls in confined field trials in Puerto Rico. Plants engineered to coexpress a mutated A. thaliana iron transporter (IRT1) and A. thaliana ferritin (FER1) accumulated iron levels 7-18 times higher and zinc levels 3-10 times higher than those in nontransgenic controls in the field. Growth parameters and storage-root yields were unaffected by transgenic fortification in our field data. Measures of retention and bioaccessibility of iron and zinc in processed transgenic cassava indicated that IRT1 + FER1 plants could provide 40-50% of the EAR for iron and 60-70% of the EAR for zinc in 1- to 6-year-old children and nonlactating, nonpregnant West African women.
Summary
Cassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive‐sense ...RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Diseases caused by the family Potyviridae require the interaction of viral genome‐linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms. Cassava encodes five eIF4E proteins: eIF4E, eIF(iso)4E‐1, eIF(iso)4E‐2, novel cap‐binding protein‐1 (nCBP‐1), and nCBP‐2. Protein–protein interaction experiments consistently found that VPg proteins associate with cassava nCBPs. CRISPR/Cas9‐mediated genome editing was employed to generate ncbp‐1, ncbp‐2, and ncbp‐1/ncbp‐2 mutants in cassava cultivar 60444. Challenge with CBSV showed that ncbp‐1/ncbp‐2 mutants displayed delayed and attenuated CBSD aerial symptoms, as well as reduced severity and incidence of storage root necrosis. Suppressed disease symptoms were correlated with reduced virus titre in storage roots relative to wild‐type controls. Our results demonstrate the ability to modify multiple genes simultaneously in cassava to achieve tolerance to CBSD. Future studies will investigate the contribution of remaining eIF4E isoforms on CBSD and translate this knowledge into an optimized strategy for protecting cassava from disease.
DNA methylation is important for the regulation of gene expression and the silencing of transposons in plants. Here we present genome-wide methylation patterns at single-base pair resolution for ...cassava (Manihot esculenta,cultivar TME 7), a crop with a substantial impact in the agriculture of subtropical and tropical regions. On average, DNA methylation levels were higher in all three DNA sequence contexts (CG, CHG, and CHH, where H equals A, T, or C) than those of the most well-studied model plantArabidopsis thaliana.As in other plants, DNA methylation was found both on transposons and in the transcribed regions (bodies) of many genes. Consistent with these patterns, at least one cassava gene copy of all of the known components ofArabidopsisDNA methylation pathways was identified. Methylation of LTR transposons (GYPSYandCOPIA) was found to be unusually high compared with other types of transposons, suggesting that the control of the activity of these two types of transposons may be especially important. Analysis of duplicated gene pairs resulting from wholegenome duplication showed that gene body DNA methylation and gene expression levels have coevolved over short evolutionary time scales, reinforcing the positive relationship between gene body methylation and high levels of gene expression. Duplicated genes with the most divergent gene body methylation and expression patterns were found to have distinct biological functions and may have been under natural or human selection for cassava traits.
Endogenous FLOWERING LOCUS T homolog MeFT1 was transgenically overexpressed under control of a strong constitutive promoter in cassava cultivar 60444 to determine its role in regulation of flowering ...and as a potential tool to accelerate cassava breeding. Early profuse flowering was recorded in-vitro in all ten transgenic plant lines recovered, causing eight lines to die within 21 days of culture. The two surviving transgenic plant lines flowered early and profusely commencing as soon as 14 days after establishment in soil in the greenhouse. Both transgenic lines sustained early flowering across the vegetative propagation cycle, with first flowering recorded 30-50 days after planting stakes compared to 90 days for non-transgenic controls. Transgenic plant lines completed five flowering cycles within 200 days in the greenhouse as opposed to twice flowering event in the controls. Constitutive overexpression of MeFT1 generated fully mature male and female flowers and produced a bushy phenotype due to significantly increased flowering-induced branching. Flower induction by MeFT1 overexpression was not graft-transmissible and negatively affected storage root development. Accelerated flowering in transgenic plants was associated with significantly increased mRNA levels of MeFT1 and the three floral meristem identity genes MeAP1, MeLFY and MeSOC1 in shoot apical tissues. These findings imply that MeFT1 encodes flower induction and triggers flowering by recruiting downstream floral meristem identity genes.
•AtVIT1 overexpression showed 3–4 times increase in cassava storage root iron concentration.•AtVIT1 transgenic cassava plants localize iron in and near the vascular tissues.•AtVIT1 transgenic cassava ...plants have altered expression of genes involved in iron homeostasis.•AtVIT1 overexpression indicates a potential strategy for iron biofortification in food crops.
Iron is extremely abundant in the soil, but its uptake in plants is limited due to low solubility in neutral or alkaline soils. Plants can rely on rhizosphere acidification to increase iron solubility. AtVIT1 was previously found to be involved in mediating vacuolar sequestration of iron, which indicates a potential application for iron biofortification in crop plants. Here, we have overexpressed AtVIT1 in the starchy root crop cassava using a patatin promoter. Under greenhouse conditions, iron levels in mature cassava storage roots showed 3–4 times higher values when compared with wild-type plants. Significantly, the expression of AtVIT1 showed a positive correlation with the increase in iron concentration of storage roots. Conversely, young leaves of AtVIT1 transgenic plants exhibit characteristics of iron deficiency such as interveinal chlorosis of leaves (yellowing) and lower iron concentration when compared with the wild type plants. Interestingly, the AtVIT1 transgenic plants showed 4 and 16 times higher values of iron concentration in the young stem and stem base tissues, respectively. AtVIT1 transgenic plants also showed 2–4 times higher values of iron content when compared with wild-type plants, with altered partitioning of iron between source and sink tissues. These results demonstrate vacuolar iron sequestration as a viable transgenic strategy to biofortify crops and to help eliminate micronutrient malnutrition in at-risk human populations.
Morphogenic culture systems are central to crop improvement programs that utilize transgenic and genome editing technologies. We previously reported that CMD2-type cassava (Manihot esculenta) ...cultivars lose resistance to cassava mosaic disease (CMD) when passed through somatic embryogenesis. As a result, these plants cannot be developed as products for deployment where CMD is endemic such as sub-Saharan Africa or the Indian sub-continent.
In order to increase understanding of this phenomenon, 21 African cassava cultivars were screened for resistance to CMD after regeneration through somatic embryogenesis. Fifteen cultivars were shown to retain resistance to CMD through somatic embryogenesis, confirming that the existing transformation and gene editing systems can be employed in these genetic backgrounds without compromising resistance to geminivirus infection. CMD2-type cultivars were also subjected to plant regeneration via caulogenesis and meristem tip culture, resulting in 25-36% and 5-10% of regenerated plant lines losing resistance to CMD respectively.
This study provides clear evidence that multiple morphogenic systems can result in loss of resistance to CMD, and that somatic embryogenesis per se is not the underlying cause of this phenomenon. The information described here is critical for interpreting genomic, transcriptomic and epigenomic datasets aimed at understanding CMD resistance mechanisms in cassava.
Cassava (
Manihot esculenta
Crantz) is a major staple food crop of the humid tropics. As a heterozygous, vegetatively propagated crop, robust transformation protocols must be developed for elite ...cultivars that allow predictable production of large numbers of independent transgenic plant lines. A high throughput
Agrobacterium
-mediated transformation system was developed for the elite East African farmer-preferred cassava cultivar TME 204 using the GFP visual marker gene. Inclusion of the antibiotic moxalactam in culture medium used to produce embryogenic target tissues prior to inoculation with
Agrobacterium
increased recovery of independent GFP-expressing transgenic callus lines by up to 113-fold compared to the control. Enhanced transformation was also observed when TME 204 tissues were pretreated with other cephalosporins, namely cefoperazone, cefoxitin, cefmetazole and cefotaxime. Similar but less dramatic increases in transformation efficiencies were seen for the West African cultivars Oko-iyawo and 60444 when pre-treated with moxalactam. Dilution of
Agrobacterium
suspensions used for co-culture was found to increase transformation efficiencies, resulting in regeneration at an average of 33 GFP-expressing TME 204 plants per cc settled cell volume at OD
600
0.05, compared to 15 plants at the more commonly used OD
600
0.5. The optimized transformation systems were successfully utilized for the integration of genetic constructs for disease resistance and nutritional enhancement into more than 750 plants of TME 204.
Cassava mosaic disease (CMD) suppresses cassava yields across the tropics. The dominant CMD2 locus confers resistance to cassava mosaic geminiviruses. It has been reported that CMD2-type landraces ...lose resistance after regeneration through de novo morphogenesis. As full genome bisulfite sequencing failed to uncover an epigenetic mechanism for this loss of resistance, whole genome sequencing and genetic variant analysis was performed and the CMD2 locus was fine-mapped to a 190 kilobase interval. Collectively, these data indicate that CMD2-type resistance is caused by a nonsynonymous, single nucleotide polymorphism in DNA polymerase δ subunit 1 (MePOLD1) located within this region. Virus-induced gene silencing of MePOLD1 in a CMD-susceptible cassava variety produced a recovery phenotype typical of CMD2-type resistance. Analysis of other CMD2-type cassava varieties identified additional candidate resistance alleles within MePOLD1. Genetic variation of MePOLD1, therefore, could represent an important genetic resource for resistance breeding and/or genome editing, and elucidating mechanisms of resistance to geminiviruses.
Tef (Eragrostis tef) is an underutilized food crop rich in minerals, vitamins, and amino acids. However, mineral profiling of diverse tef accessions, and estimation of bioavailable iron from tef has ...been lacking. In this study, we analyzed the mineral content of 41 tef accessions along with major cereals. Our analysis revealed that tef seeds contain significantly more minerals than maize, rice, and the wheat varieties used in this study. A significant variation in mineral content was also observed across the tef accessions. We also performed a relative estimation of Fe bioavailability from selected tef accessions and reference crops using an established Caco-2 cell bioassay. This bioassay measures human intestinal cell Fe uptake via intracellular ferritin formation, a storage protein that is a validated marker of Fe uptake. Higher levels of Fe uptake were observed in the PI-494307, PI-494425, and PI-195937 accessions, than those recorded in cells fed wheat, rice, or tef accessions PI-329681, PI-494408 and PI-494293. There was no marked difference in phytic acid (PA) content between tef and wheat, while the PA level in rice was lower than tef and wheat. Enhanced Fe uptake evident in tef accession PI494425 could not be explained by seed Fe content. The Fe content of PI-494425 was lower than the other tef accessions, suggesting that other factors control the amount of bioavailable Fe from tef. Considerable variation in mineral content and bioavailable Fe between tef and other cereals indicate a potential for improving mineral nutrition from this vital food crop.
Tef (
(Zucc.) Trotter) is a staple food crop for 70% of the Ethiopian population and is currently cultivated in several countries for grain and forage production. It is one of the most nutritious ...grains, and is also more resilient to marginal soil and climate conditions than major cereals such as maize, wheat and rice. However, tef is an extremely low-yielding crop, mainly due to lodging, which is when stalks fall on the ground irreversibly, and prolonged drought during the growing season. Climate change is triggering several biotic and abiotic stresses which are expected to cause severe food shortages in the foreseeable future. This has necessitated an alternative and robust approach in order to improve resilience to diverse types of stresses and increase crop yields. Traditional breeding has been extensively implemented to develop crop varieties with traits of interest, although the technique has several limitations. Currently, genome editing technologies are receiving increased interest among plant biologists as a means of improving key agronomic traits. In this review, the potential application of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (CRISPR-Cas) technology in improving stress resilience in tef is discussed. Several putative abiotic stress-resilient genes of the related monocot plant species have been discussed and proposed as target genes for editing in tef through the CRISPR-Cas system. This is expected to improve stress resilience and boost productivity, thereby ensuring food and nutrition security in the region where it is needed the most.
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