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| Optimization of Agrobacterium-mediated Transient Gene Expression System and Its Utilization in RNAi Based Gene Silencing of Rose(Rosa hybrida) Petals |
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Abstract The gene functional identification in roses is limited by the lack of efficient stable transformation protocols. As an alternative, Agrobacterium-mediated transient gene expression and RNAi-based gene silencing can be used conveniently and effectively in plants. To optimize a transient gene expression system in rose petals, we investigated the effects of rose cultivars, flowering stages, petal positions, Agrobacterium strains, bacterial density and infiltration solution on transient gene expression in rose(Rosa hybrida) petals. The agro-infiltration method was used and GUS was selected as a marker gene in this study. We found that the cultivar Honey could have strongest GUS expression among all the 5 cultivars tested, while the other 4 cultivars Hollywood, Samantha, Honeymoon and Xiushanhong showed weak or no GUS expression. Petals of the middle whirl at flowering stage 3 displayed the highest expression level of GUS in Honey, suggesting that vigorous and fully developed petals were more suitable for transient gene expression. Compared to Agrobacterium strains of C58C1 and EHA105, GV3101 was a more effective laboratory strain and the optimal bacterial density was OD600=0.9. The infiltration solution is better to be supplemented with 10 mmol/L MgCl2 and 10 mmol/L 2-(N-Morpholino) ethanesulfonic acid (MES). The optimized transient system could result in around 100% strong GUS expression. By using the optimized transient expression system, we transiently expressed ihpRNA constructs to silence the reporter gene GUS and RhSAG. Compared to control, GUS-silencing showed less strongly stained petals and a lower average staining density, while RhSAG-silenced petals wilted more slowly, indicating senescence delay. In addition, both silencing treatments led to decreased gene transcripts. Together, the results suggested the system could be used in gene silencing and provide an effective tool for gene function analysis in rose petals.
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Received: 02 May 2013
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