Ivan Ingelbrecht

Efficient CRISPR/Cas9 Genome Editing of Phytoene desaturase in Cassava

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 5′ and or downstream of the 3′ 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.

Primary Somatic Embryos from Axillary Meristems and Immature Leaf Lobes of Selected African Cassava Varieties

The study evaluated high-value African cassava varieties for primary somatic embryogenesis usingaxillarymeristems (AM) and immature leaf lobes (LL) on picloram based medium. The study was conducted at the Central Biotech Lab, International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria between 2006 and 2009. Completely randomized design with four replicates was used for the study. Using LL explants, there were significan t (P=.05) differences in percent responding leaf lobes, percent explant with pre -embryogenic structure, PSEF and PSEE among cassava varieties. The PSEF of the only three varieties that produced mature somatic embryo were 93.6, 88.5 and 85.7% for TME 12, Ki

Influence of Type and Age of Primary Somatic Embryo on Secondary and Cyclic Somatic Embryogenesis of Cassava ( Manihot esculenta Crantz)

This study investigated the influence of age of the cotyledons, cut from primary somatic embryos (PSE) developed from shoot meristems (SM) or immature leaf lobes (LL), on secondary somatic (SSE) and cyclic (CSE) embryogenesis of two cassava cultivars at th e Central Biotech Laboratory, International Institute of Tropical Agriculture (IITA), Ibadan, between 2006 and 2010. A completely randomized design with three replicates was used for the study. Only PSE at the age of 4 weeks recorded significant (P<0.05) d ifferences in SSE frequency and efficiency between the SM and LL sources. CSE production was highest using 0 to 4 weeks old SSE cotyledons, and significant (P<0.05) differences were only recorded between the SM and LL sources when the age of the SSE cotyle dons was older than 6 weeks. The CSE frequencies from the SM source were significantly greater than that from the LL source when 8 and 10 week -old SSE cotyledons were used. The OriginalResearch Article