Cassava tissue culture and transformation : Improvement of culture media and the effect of different antibiotics on cassava

The culture medium most often used for in vitro culture of cassava is based on that developed by Murashige and Skoog. In the published papers on cassava tissue culture this medium is usually modified by optimizing the concentration of its organic component (e.g., growth regulators, sucrose), but not of its mineral elements. In our experiments with shoot tip cultures, we found that raising the concentration of cupric sulfate in the MS-medium from 0.1 uM (original concentration) to 2.0 uM resulted in an increase in shoot length and fresh weight of about 50 percent. Using young leaf lobes for the induction of embryogenesis, 2.0 uM cupric sulfate increased the number of lobes producing embryos in three independent experiments from about 5-25 percent to about 20-70 percent. As selective marker for transformation experiments we had chosen the nptII gene, which confers resistance to aminoglycoside antibiotics. Using kanamycin for the selection of Agrobacterium- treated somatic embryos (see abstract by Chavarriaga et al,) so far we have obtained chimeric embryos. The regeneration of plantlets or the induction of secondary embryogenesis starting from transformed tissues on media with kanamycin was not possible. For that reason we tested alternative antibiotics (glyphosate, phosphinotricin, and hygromycin) for their effects on non-transformed cassava embryo clumps. The results show that both glyphosate and hygromycin suppress embryogenesis at comparatively low concentrations, while permitting callus formation at higher concentrations. In contrast to that, phosphinotricin inhibits embryogenesis and formation of callus to a similar extent and therefore seems to be better suited for the selection of transformed tissues.