CBN-V Video Archives - S8-25
An Efficient in vitro Viral Replication Assay for African Cassava Mosaic Virus Clones in Cassava Leaves

Zhang P., J. Puonti-Kaerlas and W. Gruissem

Institute for Plant Sciences, ETH-Zentrum/LFW E 17, CH-8092 Zürich, Switzerland ZHANG.PENG@IPW.BIOL.ETHZ.CH

        Like other geminiviruses, African cassava mosaic virus (ACMV) replicates in nucIei of mature plant cells after inducing the accumulation of host DNA replication machinery. Under in vitro conditions, however, viral replication from cloned ACMV in host tissues is difficult to be detected. In order to develop a transient assay for ACMV replication in vitro, cassava leaf discs were infected with ACMV clones pDNA-A and pDNA-B, originating from West Kenya isolate 844, using biolistic inoculation. By using the methylation sensitive DpnI and Mbol, methylated input DNA could be distinguished from de novo synthesized viral DNA that is not methylated. Different media that could be used for pre- and post- culture of the bombarded leaf discs were investigated. Without pre-culture, input DNA is rapidly degraded in all cases. Media combination between pre- and post- culture were optimized and their durations were evaluated. Southern analysis revealed that only with 4-day pre-culture on medium M1 combined with 4-day post-culture on medium M3 that the cloned virus could replicate efficiently. Changing the culture duration resulted in the failure of viral replication even by a difference of 2 days. Optimization of the stringent parameters for successful transient assay of ACMV replication in vitro was achieved by this study.

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