Cassava axillary bud transformation and production of somatic embryos of selected cassava cultivars

• Main Author: Rossin, Claudia
• Format: Others
• Language: en
• Published: 2009
• Online Access: http://hdl.handle.net/10539/6725

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.