Amelioration of Gene Silencing,Polyadenylation Signal,Homozygotes

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	Amelioration of Gene Silencing - Stable transgene expression can be obtained by screening a large number of transformants and by a selection for stable expression in a breeding programme based on transgenic plants. In addition, several steps may be taken to minimise the likelihood of transgene silencing; some of these are briefly outlined below. 1. The transformation vector should not have duplicated sequences. 2. Each gene construct in the vector should have a different promoter and polyadenylation signal.
3. All the gene constructs in a vector should have the same orientation and should not be located adjacent to each other. 4. The AT composition of transgene should be similar to that of the host chromosome. In addition, the transgene sequence should be optimal for codon usage in plants and should not contain sequences that interfere with mRNA function/stability. 5. The transgene should be integrated in a single copy, and away from chromosomal locations having hypermethylation. 6. Stability of transgene expression in homozygotes may be tested in tissue culture by exposing transgenic tissues to propionic or butyric acid. This test would eliminate plants in which silencing could occur due to environmental stress.