ISHS Acta Horticulturae 688: IV International Symposium on Edible Alliaceae
TRANSGENIC ONIONS WITH REDUCED ALLIINASE ACTIVITY: BIOCHEMICAL AND MOLECULAR ASSESSMENT |
| Authors: | C.C. Eady, L. Trueman, J. McCallum, M. Shaw, M. Pither-Joyce, S. Davis, J. Reader |
| Keywords: | RNA interference, antisense, sulfur, carbohydrate, transformation, onion |
| Abstract:
Transgenic onion plants with an antisense version of the bulb alliinase gene under CaMV35s promotional control have been produced. Biochemical pyruvate analysis showed that some transgenic plants, containing this gene construct, had significantly reduced alliinase activity. More detailed analysis of individual clones indicated that the reduced alliinase activity was only apparent in some plants suggesting that either chimeric plants were produced; escapes were regenerated along with transformants; differential gene silencing occurred; or there was a huge environmental variation in alliinase expression. The biochemical data was supported by multiplex RT-PCR that indicated a reduction in alliinase transcript in some lines. In at least one line, an approximate 10-fold reduction in alliinase transcript was detected. This corresponded to bulb material that was shown to have no measurable alliinase activity. In one further test, SNuPE analysis of the alliinase transcript present in the RNA was performed to determine whether a particular alliinase gene member had been silenced. The results indicated that all alliinase transcripts were equally silenced. Molecular fingerprinting techniques are now being developed to match the plants sacrificed for this analysis to their ‘real’ clones and offspring so that phenotypic assessment of reduced alliinase plants can be undertaken. | |
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