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Post-Transcriptional Silencing of Flavonol Synthase mRNA in Tobacco Leads to Fruits with Arrested Seed Set

Figure 6

In vitro and in vivo pollen germination rescue assays of FLS silenced transgenic tobacco.

A, Effect of quercetin on in vitro pollen germination rate of silenced transgenic pollens. Pollens were collected from freshly dehiscent anthers of FLS silenced tobacco plant and suspended in GM containing (a) only DMSO added to final concentration of 1 µM; (b) 10 nM quercetin; (c) 20 nM quercetin and (d) 1 µM quercetin. The pollen germination rate and pollen tube length was increased in GM containing 1 µM quercetin as compared to pollens that were exposed to 10 nM, 20 nM and with no quercetin. B, Different developmental stages of flower bud (stage I, II, III and IV) in tobacco (Nicotiana tabacum cv xanthi). It has been proven experimentally that auxin is mainly accumulated in anthers and stigma during stage II (the stage after bud formation when flower has not yet opened) of floral development [39]. C, Histochemical staining of pollen tubes growth in carpels of floral buds (stage II) from silenced transgenic lines exposed to different concentrations of quercetin i.e. 10 nM, 20 nM and 1 µM through pollen maturation media (PM). After 2 days of pollination, carpels were stained with aniline blue to specifically stain callose present in growing pollen tubes. Pollen tube growth in self pollinated carpels of floral bud of silenced transgenic lines exposed only to PM without any treatment (a1-a5); carpels of floral bud exposed to PM containing 10 nM quercetin (b1-b5); 20 nM (c1-c5) and 1 µM (d1-d6) quercetin. Pollen germination is visible at stigma region (a1, b1, c1 and d1). It was found to be maximum with 1 µM (d1) and least in silenced pollens with no treatment (a1). The pictures a2-a4, b2-b4, c2-c4 and d2-d4 show proliferation of pollen tube growth in the middle of the style. The pollen tubes grew only to nine-tenths of way down the style after 2 days of pollination in case of silenced flora buds exposed to 10 nM (b2-b4), 20 nM (c2-c4) quercetin and with no treatment (a2-a4). Whereas pollen tubes are reaching the base of the style in case of floral buds exposed to 1 µM quercetin (d2-d5). On the other hand, in untreated buds and in buds exposed to 10 nM, 20 nM quercetin, no pollen tubes are seen in carpels reaching the base of style (a5, b5 and c5) within the same time period. All micrographs are of the same magnification and the scale bar in A, and C pictures represent 50 µm. The pictures were taken under UV filter of florescent microscope equipped with Nikon digital camera Dxm 1200.

Figure 6

doi: https://doi.org/10.1371/journal.pone.0028315.g006