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Figure 6 | BMC Plant Biology

Figure 6

From: Conjugated polymer nanoparticles for effective siRNA delivery to tobacco BY-2 protoplasts

Figure 6

CPN delivery of CesA-1 siRNAs suppress cell wall regeneration. (A) Protoplasts were harvested and immediately stained with calcofluor white (T = 0 h) to verify complete digestion and elimination of cell walls. Image shows no calcofluor fluorescence. (B) Protoplasts at 72 h treated with 10 μM CPNs and 200 nM NtCesA-1 siRNAs show few faint patches of blue fluorescence at the plasma membrane. (C) Untreated protoplasts at 72 h show significant deposition of cellulose at the cell surface. Experiments were repeated five times. Scale bars represent 10 μm. (D) The average percent of protoplasts from two experiments that showed calcofluor staining at 0, 24, 48, and 72 h following treatment with CPNs and NtCesA-1 siRNAs. (E) Propidium iodide was used to determine the percent viable protoplasts at 0, 24, 48, and 72 h following treatment with CPNs and NtCesA-1 siRNAs. Averages were determined for three replicate experiments. (F) Ethidium bromide- stained 1% agarose gels containing semi quantitative RT-PCR products detecting NtCesA-1 or ubiquitin (Ubi) gene expression. The treatments with siRNAs and CPNs are indicated above each panel and the numbers of PCR cycles from 30-45 are indicated below each lane. Lane "L" indicates DNA ladder at the bottom of the gel and size (bp) markers are indicated on the left. As a control, semi-quantitative PCR shows ubiquitin gene expression.

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