Browsing by Author "Taylor, Nigel"

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    Efficient CRISPR/Cas9 Genome Editing of Phytoene desaturase in Cassava
    (Frontiers in Plant Science, 2017) Odipio, John; Alicai, Titus; Nusinow, Dmitri; Bart, Rebecca; Ingelbrecht, Ivan; Taylor, Nigel
    CRISPR/Cas9 has become a powerful genome-editing tool for introducing genetic changes into crop species. In order to develop capacity for CRISPR/Cas9 technology in the tropical staple cassava (Manihot esculenta), the Phytoene desaturase (MePDS) gene was targeted in two cultivars using constructs carrying gRNAs targeting two sequences within MePDS exon 13. After Agrobacterium-mediated delivery of CRISPR/Cas9 reagents into cassava cells, both constructs induced visible albino phenotypes within cotyledon-stage somatic embryos regenerating on selection medium and the plants regenerated therefrom. A total of 58 (cv. 60444) and 25 (cv. TME 204) plant lines were recovered, of which 38 plant lines (19 from each cultivar) were analyzed for mutagenesis. The frequency of plant lines showing albino phenotype was high, ranging from 90 to 100% in cv. TME 204. Observed albino phenotypes were comprised of full albinos devoid of green tissue and chimeras containing a mixture of white and green tissues. Sequence analysis revealed that 38/38 (100%) of the plant lines examined carried mutations at the targeted MePDS site, with insertions, deletions, and substitutions recorded. One putatively mono-allelic homozygous line (1/19) was found from cv. 60444, while 1 (1/19) and 4 (4/19) putatively bi-allelic homozygous lines were found in 60444 and TME204, respectively. The remaining plant lines, comprised mostly of the chimeras, were found to be putatively heterozygous. We observed minor (1 bp) nucleotide substitutions and or deletions upstream of the 50 and or downstream of the 30 targeted MePDS region. The data reported demonstrates that CRISPR/Cas9-mediated genome editing of cassava is highly efficient and relatively simple, generating multi-allelic mutations in both cultivars studied. Modification of MePDS described here generates visually detectable mutated events in a relatively short time frame of 6–8 weeks, and does not require sequencing to confirm editing at the target. It therefore provides a valuable platform to facilitate rapid assessment and optimization of CRISPR/Cas9 and other genome-editing technologies in cassava.
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    Production of friable embryogenic callus and regeneration of Ugandan farmer-preferred cassava genotypes
    (African Journal of Biotechnology, 2015) Apio, Hellen B.; Alicai, Titus; Baguma, Yona; Mukasa, Settumba B.; Bua, Anton; Taylor, Nigel
    Generation of embryogenic callus is a key step in genetic engineering of many crop species, including cassava. Protocols for generation of friable embryogenic callus (FEC) have been lacking for Ugandan cassava genotypes, thereby delaying their genetic engineering for agronomic and other desirable traits. The objective of this study was to determine conditions suitable for production and regeneration of FEC in the Ugandan cassava genotypes; Aladu, Bukalasa and Ebwanateraka, and control cultivar 60444. Immature leaf lobe explants were established on Murashige and Skoog (MS) based media for initiation of organized embryogenic callus (OES). To produce FEC, resulting OES were established on Gresshoff and Doy based callus induction media with varying levels of sucrose, maltose, tyrosine, tryptophan, naphthalene acetic acid (NAA) under light and dark conditions. Subsequently, FEC was subcultured to MS-based embryo maturation and embryo regeneration media. All genotypes produced OES. All genotypes produced FEC except Bukalasa. The amino acid tyrosine favoured production of FEC in Aladu and Ebwanatereka, but not in 60444, while 20 g/L of sucrose trigged production of FEC in Aladu and 60444, but 40 g/L of sucrose was superior for Ebwanatereka. Media supplemented with 1 ml/L naphthalene acetic acid NAA facilitated embryo regeneration in Ebwanatereka and 60444, while Aladu responded better to 5 ml/L NAA. Light, tyrosine and sucrose were essential for FEC production in Uganda cultivars while NAA was required for regeneration of somatic embryos. Ability to produce FEC in these genotypes lays a foundation for their improvement through genetic transformation for the desired and agronomic traits.