Genetic transformation is essential for introducing new traits into cassava. However, theudcurrent protocols for cassava transformation are inefficient. In this study, the aims were touddevelop a protocol for Agrobacterium tumefaciens-mediated genetic transformation ofudcassava axillary buds and direct regeneration thereof, to screen selected cultivars forudsomatic embryogenesis (SE) potential and tobacco leaf discs were transformed with aud221bp Rep transgene derived from South African cassava mosaic virus (SACMV) touddetermine the efficiency of the antisense transgene to silence SACMV. Various explantudpre-treatments were tested prior to transformation, followed by Agrobacterium-infiltration.udCo-cultivation at different temperatures (22 and 25ºC), photoperiod (16h light 8h dark, andudcomplete darkness) as well as co-cultivation time periods, were evaluated. GUS assaysudshowed that putative transgenic plants had not been transformed. The most widely usedudexplants for cassava transformation are friable embryogenic callus (FEC) and somaticudcotyledons. In this study, 9 cassava cultivars were tested for SE and FEC induction. Mediaudcontaining various plant growth regulators and various explants (40 explants perudexperiment) were used for the production of SE. The optimal media and explants for SEudwere shown to be axillary buds on MS2 containing 50μM picloram, except for cultivarudAR9-18 which showed increased SE production using immature leaf lobes on MS2udcontaining 50μM picloram. The cultivars with the highest SE efficiency were cultivarsudTMS60444 (model cultivar), T200, AR 9-18, MTAI16, CR25-4 and CM523-7. Low SE efficiency was found in BRA 1183, MCOL2261 and SM707-17 cultivars. Cultivars withudlow SE efficiency produced mostly globular stage embryos and friable embryogenicudcallus. Tobacco leaf discs were transformed to test the viral-silencing efficiency of theud221bp Rep construct against SACMV. Results showed that regenerated transgenic tobaccoudlines infected with SACMV showed reduced symptom development compared withuduntransformed infected plants, and statistical analysis from RT-PCR results showed thatudthere was a significant decrease in the amount of virus present in four of the fiveudtransgenic lines compared with non-transgenic controls.
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