ISHS Acta Horticulturae 459: International Symposium Brassica 97, Xth Crucifer Genetics Workshop
NON-DESTRUCTIVE SCREENING OF HAPLOID EMBRYOS FOR GLUFOSINATE AMMONIUM RESISTANCE FOUR WEEKS AFTER MICROSPORE TRANSFORMATION IN BRASSICA |
| Authors: | M. Dormann, N. Datla, A. Hayden, D. Puttick, J. Quandt |
| Keywords: | bromocresol purple, phosphinothricin (PPT), bar, pat, Brassica napus, B. rapa, β-glucuronidase (GUS), phosphinothricin acetyltransferase (PAT) |
| Abstract:
High frequencies of embryogenesis and plant regeneration make embryogenic microspores a desirable system for genetic transformation. We show the recovery of non-chimeric GUS (  -glucuronidase) positive embryos four weeks after microspore transformation, evidence that integrative transformation happened at the single cell stage.
Since microspore-derived embryos green up within a few days under light, an early screening for transgenic embryos carrying the pat or bar (phosphinothricin acetyltransferase) gene should be possible at this time.
The objective of this study was to explore the possibility of discriminating transgenic PAT (phosphinothricin acetyltransferase) positive embryos from wild-type embryos employing an early non-destructive assay. We designed experiments with transgenic and non-transgenic haploid populations with and without the herbicidal compound glufosinate ammonium (syn. phosphinothricin, PPT) and the pH indicator bromocresol purple. Explants sensitive to PPT release ammonium into the medium, resulting in an increased pH, which is visualized by a change of the pH indicator from yellow (pH 5.6) to red-purple (De Block, et al., 1995). Multiwell plates with one embryo per well were used. PPT sensitive embryos change yellow coloured bromocresol purple medium to red-purple two to three days after incubation with PPT. The medium remains yellow if the explants are PPT-resistant. In the absence of PPT, healthy green embryos (PAT positive and wild-type) acidified red coloured bromocresol purple medium (pH 5.8 and higher) to yellow within one day. The optimized bromocresol purple test allowed the reliable identification of transgenic haploid Brassica napus embryos carrying the pat gene in a non-destructive test within two to three days. Prerequisite is healthy, uniform and green embryos as they are obtained from efficient microspore cultures. The efficiency of the non-destructive screening for the detection of new primary transformants carrying the pat gene is currently being tested. | |
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