Recovery of transgenic plants from “escape” shoots

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Abstract
The problem of escapes is well known to those investigating the regeneration of transgenic shoots from transformed callus. Shoots can pass various tests and assays for transformation, and are then scored as transgenic, but the progeny do not express the transferred trait and do not contain the T-DNA. Explanations for these enigmatic “escapes” include instability of the T-DNA, genomic rearrangements during meiosis, or merely non-rigorous selection or identification assays giving rise to spuriously positive scorings. At least some shoots, however, are likely to simply be chimeric, containing both transformed and non-transformed cell lines. In this case, the transformed cells are responsible for the positive selection and scoring on tests, but either do not contribute to the germ line (resulting in no transgenic progeny) or contribute to only a portion of the germ line (resulting in many fewer positive segregants than expected). We describe two methods which we used to recover fully transgenic plants from apparent escapes. One method involved analyzing more progeny than would normally be necessary (to identify minority transgenic contribution to the cell line). The other method, (to recover transgenic plants from primary selectants with no transgenic contribution to the germ line) involved regenerating new shoots from leaf tissue used in a selection assay to score the initial shoot as a positive transgenic.