A novel protocol for de novo shoot organogenesis from cassava has been developed utilizing meta-topolin to stimulate shoot regeneration from leaf, petiole and stem internode explants. While use of ...meta-topolin alone was capable of inducing shoot regeneration, a two-stage system combining meta-topolin with 2,4-D in a first stage medium, followed by subculture onto elevated levels of meta-topolin, was superior for inducing multiple shoot regeneration events in more than 35% of explants in cultivar TME 7. Caulogenesis was achieved in eleven additional cultivars. Meta-topolin was also found to be beneficial for stimulating shoot regeneration from somatic embryos and cotyledon explants. The shoot organogenesis techniques described enhance the capacity of existing embryogenic systems and present previously unavailable morphogenic pathways for developing genetic transformation and gene editing technologies in cassava.
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.
Summary
Effective weed control can protect yields of cassava (Manihot esculenta) storage roots. Farmers could benefit from using herbicide with a tolerant cultivar. We applied traditional ...transgenesis and gene editing to generate robust glyphosate tolerance in cassava. By comparing promoters regulating expression of transformed 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) genes with various paired amino acid substitutions, we found that strong constitutive expression is required to achieve glyphosate tolerance during in vitro selection and in whole cassava plants. Using strategies that exploit homologous recombination (HR) and nonhomologous end‐joining (NHEJ) DNA repair pathways, we precisely introduced the best‐performing allele into the cassava genome, simultaneously creating a promoter swap and dual amino acid substitutions at the endogenous EPSPS locus. Primary EPSPS‐edited plants were phenotypically normal, tolerant to high doses of glyphosate, with some free of detectable T‐DNA integrations. Our methods demonstrate an editing strategy for creating glyphosate tolerance in crop plants and demonstrate the potential of gene editing for further improvement of cassava.
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
Delivering the benefits of agricultural biotechnology to smallholder farmers requires that resources be directed towards staple food crops. To achieve effect at scale, beneficial traits must ...be integrated into multiple, elite farmer‐preferred varieties with relevance across geographical regions. The staple root crop cassava (Manihot esculenta) is consumed for dietary calories by more than 800 million people, but its tuberous roots provide insufficient iron and zinc to meet nutritional needs. In Africa, cassava yields are furthermore limited by the virus diseases, cassava mosaic disease (CMD) and cassava brown streak disease (CBSD). In this study, we strove to develop cassava displaying high‐level resistance to CBSD and CMD to attain food and economic security for cassava farmers, along with biofortified levels of iron and zinc to enhance consumer health. RNAi‐mediated technology was used to achieve resistance to CBSD in two East African and one Nigerian farmer‐preferred cultivars that harboured resistance to CMD. The Nigerian cvs. TMS 95/0505 and TMS 91/02324 were modified with T‐DNA imparting resistance to CBSD, along with AtIRT1 (major iron transporter) and AtFER1 (ferritin) transgenes to achieve nutritionally significant levels of iron and zinc in cassava storage roots (145 and 40 µg/g dry weight, respectively). The inherent resistance to CMD was maintained in all four disease resistant and mineral enhanced cassava cultivars described here, demonstrating that this technique could be deployed across multiple farmer‐preferred varieties to benefit the food and nutritional security of consumers in Africa.
Cassava (Manihot esculenta) feeds c. 800 million people world-wide. Although this crop displays high productivity under drought and poor soil conditions, it is susceptible to disease, postharvest ...deterioration and the roots contain low nutritional content.
Here, we provide molecular identities for 11 cassava tissue/organ types through RNA-sequencing and develop an open access, web-based interface for further interrogation of the data.
Through this dataset, we consider the physiology of cassava. Specifically, we focus on identification of the transcriptional signatures that define the massive, underground storage roots used as a food source and the favored target tissue for transgene integration and genome editing, friable embryogenic callus (FEC). Further, we identify promoters able to drive strong expression in multiple tissue/organs.
The information gained from this study is of value for both conventional and biotechnological improvement programs.
Summary
Tef is a staple food and a valuable cash crop for millions of people in Ethiopia. Lodging is a major limitation to tef production, and for decades, the development of lodging resistant ...varieties proved difficult with conventional breeding approaches. We used CRISPR/Cas9 to introduce knockout mutations in the tef orthologue of the rice SEMIDWARF‐1 (SD‐1) gene to confer semidwarfism and ultimately lodging resistance. High frequency recovery of transgenic and SD‐1 edited tef lines was achieved in two tef cultivars by Agrobacterium‐mediated delivery into young leaf explants of gene editing reagents along with transformation and regeneration enhancing morphogenic genes, BABY BOOM (BBM) and WUSCHEL2 (WUS2). All of the 23 lines analyzed by next‐generation sequencing had at least two or more alleles of SD‐1 mutated. Of these, 83% had tetra‐allelic frameshift mutations in the SD‐1 gene in primary tef regenerants, which were inherited in subsequent generations. Phenotypic data generated on T1 and T2 generations revealed that the sd‐1 lines have reduced culm and internode lengths with no reduction in either panicle or peduncle lengths. These characteristics are comparable with rice sd‐1 plants. Measurements of lodging, in greenhouse‐grown plants, showed that sd‐1 lines have significantly higher resistance to lodging at the heading stage compared with the controls. This is the first demonstration of the feasibility of high frequency genetic transformation and CRISPR/Cas9‐mediated genome editing in this highly valuable but neglected crop. The findings reported here highlight the potential of genome editing for the improvement of lodging resistance and other important traits in tef.
•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.
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