| Summary: | Genetic transformation is essential for introducing new traits into cassava. However, the
current protocols for cassava transformation are inefficient. In this study, the aims were to
develop a protocol for Agrobacterium tumefaciens-mediated genetic transformation of
cassava axillary buds and direct regeneration thereof, to screen selected cultivars for
somatic embryogenesis (SE) potential and tobacco leaf discs were transformed with a
221bp Rep transgene derived from South African cassava mosaic virus (SACMV) to
determine the efficiency of the antisense transgene to silence SACMV. Various explant
pre-treatments were tested prior to transformation, followed by Agrobacterium-infiltration.
Co-cultivation at different temperatures (22 and 25ºC), photoperiod (16h light 8h dark, and
complete darkness) as well as co-cultivation time periods, were evaluated. GUS assays
showed that putative transgenic plants had not been transformed. The most widely used
explants for cassava transformation are friable embryogenic callus (FEC) and somatic
cotyledons. In this study, 9 cassava cultivars were tested for SE and FEC induction. Media
containing various plant growth regulators and various explants (40 explants per
experiment) were used for the production of SE. The optimal media and explants for SE
were shown to be axillary buds on MS2 containing 50μM picloram, except for cultivar
AR9-18 which showed increased SE production using immature leaf lobes on MS2
containing 50μM picloram. The cultivars with the highest SE efficiency were cultivars
TMS60444 (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 with
low SE efficiency produced mostly globular stage embryos and friable embryogenic
callus. Tobacco leaf discs were transformed to test the viral-silencing efficiency of the
221bp Rep construct against SACMV. Results showed that regenerated transgenic tobacco
lines infected with SACMV showed reduced symptom development compared with
untransformed infected plants, and statistical analysis from RT-PCR results showed that
there was a significant decrease in the amount of virus present in four of the five
transgenic lines compared with non-transgenic controls.
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