CBN-V Video Archives - S7-20
Influence of Explant Source and Light on Efficiency of Agrobacterium-mediated Transformation of Cassava

Msikita W., R.T. Sayre, V. White and J. Marks

Department of Plant Biology, Ohio State University, 1735 Neil Avenue, Columbus, OH 43210 sayre.2@osu.edu

        Apical leaves, undifferentiated calli pieces, and somatic embryos for cassava cultivar Mcol 2215 were inoculated with Agrobacterium-plasmids coding the genes for phosphomannose isomerase (pmi), and neomycin phosphotransferase II (nptII). The pmi gene confers ability to convert mannose-6-phosphate to fructose-6-phosphate (and hence only transformed cells are capable of utilizing mannose as a carbon source), while the npt II gene confers resistance to the antibiotic kanamycin. Following inoculation, explants were incubated in the dark (1, 2, or 3 weeks) or transferred directly to a 12-hour light regime, and maintained on selection media (0.3% mannose for pmi gene or 75 mg/l paromomycin for npt II gene) for 10-12 weeks. Recovery of putative transgenic plants for both gene types was influenced by the source of explant and duration of dark incubation. Germinating somatic embryos incubated in the dark for one week gave the highest number of putative transgenic plants. Overall, more putative plants carrying the npt II gene were recovered than for the pmi gene. Putative transgenic plants were rooted on half strength Murashige and Skoog medium supplemented with 10 mg/L -napthlaneacetic acid (NAA). Integration of the genes into the plants was verified by PCR analysis, and ranged between 2% (for apical leaves) and 5% (for germinating somatic embryos). The results demonstrate the importance of choice of explant type, and treatment of explant in transformation efficiency of cassava.

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