Improvements in Agrobacterium-mediated transformation of cassava (Manihot esculenta Crantz) for large-scale production of transgenic plants
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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 OD600 0.05, compared to 15 plants at the more commonly used OD600 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.
KeywordsCassava Friable embryogenic callus Genetic transformation Moxalactam Oko-iyawo TME 204
This work was supported by the Bill and Melinda Gates Foundation, the United States Agency for International Development from the American people, and the Monsanto Fund. PureMLB® technology was kindly donated by Japan Tobacco Inc. We thank Dr. Edgar Cahoon, Director of the Center for Plant Science Innovation, University of Nebraska-Lincoln, for providing expression cassettes of crtB and DXS. We also thank Tira Jones, Jacquelyn Leise, Amita Rai, Jackson Gehan, Theodore Moll and Danielle Posey at the Donald Danforth Plant Science Center for technical assistance.
Conflict of interest
The authors declare that they do not have any conflict of interest.
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