The present investigation was carried out with an objective of establishing a model system in tobacco for assessing the efficiency of different PTGS vectors developed for reducing ricin and RCA in castor. The strategy involved developing transgenic tobacco expressing full-length preproricin gene that could serve as an excellent model system to assess the efficiency of the PTGS constructs for silencing ricin and RCA.
Towards this end, tobacco was transformed, through Agrobacterium-mediated leaf-disc transformation, with a gene cassette containing full-length ricin gene under CaMV 35S promoter. The transgenicity of the putative transgenics was confirmed subjecting them to gene specific PCRs. Different combinations of primers for amplifying various component sequences were used to ascertain that the presence of the entire gene cassette so that the introduced ricin gene expressed and produced the transcript. The expression of ricin gene in these transgenics was confirmed by RT-PCR studies. Again, by using different primer combinations it was confirmed that the entire transcript of ricin was produced.
The confirmed tobacco transgenics were then re-transformed with the developed PTGS constructs and the putative transgenic shoots for each of the PTGS constructs were obtained. However, as the shoots were small and were in clusters, the molecular analysis could not be completed with these shoots.
Parallel to this, transgenic tobacco plants were developed independently with the five PTGS constructs. These plants were developed with the objective of crossing them with the plants expressing ricin gene so that in the progeny the efficiency of each of the PTGS vectors in silencing of the ricin gene. Transgenic tobacco harbouring PTGS constructs was confirmed by PCR.
Analysis of the expression pattern and levels of transgenic tobacco expressing individual PTGS constructs and confirmation of transgenic tobacco re-transformed with individual PTGS constructs would be the future line of work. |
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