Expression analysis of <i>MySP</i> during nymph developmental stages of <i>M. persicae</i>.

<p>(A) Expression profiles of <i>MySP</i> at different developmental stages of <i>M. persicae</i> ranging from early (1–2 day-old new-born nymph), median (3–5 day-old nymph) to late (6–7 day-old apterous adult) was determined by semi-quantitative RT-PCR. cDNA, prepared from 5 µg of total RNA in each sample, was PCR amplified with <i>MySP</i> specific primers (lanes 2–4; upper panel) and 18SrRNA (lanes 2–4; lower panel). The latter was used as endogenous control showing similar RNA concentrations in aphid samples collected at different developmental stages. (B) Integrated density values (IDV) of the PCR products of <i>MySP</i> in the nymphs collected at different development stages as described in (A).</p

Dendrogram illustrating the relatedness of serine protease gene sequence of <i>M. persicae</i> (<i>MySP</i>) with those of different Indian pollinators.

<p>Phylogenetic relationship of <i>MySP</i> and its counterpart from Indian pollinators was conducted using MEGA 4.0.2 which is shown along with branch lengths. DNA sequences were aligned using CLUSTAL W and a tree was constructed by neighbour-joining program from a similarity matrix of pairwise comparisons. The Nucleotide/ p-distance option was selected in substitution model section. Bootstrap values (*) were assessed with 1000 replicates and are shown at the dendrogram nodes. The scale bar represents sequence divergence.</p

Detection of <i>MySP</i>-specific siRNAs in different SP transgenic lines.

<p>Total RNA (50 µg) was extracted from the control and different SP transgenic lines (SP8, SP17, and SP20), run on 15% denaturing acrylamide gel, and hybridized with <i>MySP</i>-specific RNA probes. Northern hybridization detected 21–24 nt siRNAs in transgenic samples (upper panel). Loading of equal amounts of RNA was confirmed by ethidium bromide staining (lower panel).</p

siRNA-mediated suppression of <i>MySP</i> transcript and gut protease activity in aphid nymphs fed on SP transgenic lines.

<p>(A) Aphids were collected from the control and SP-transgenic line at different time points after their release and also those developed parthenogenetically for quantitative Real-time PCR analysis of the relative expression profiles of <i>MySP</i>. 18S rRNA was used as an internal control, and values for the control plants (inoculated nymphs and parthenogenetic progenies) were normalized to 1. Data presented are means of two independent biological replicates with three technical replicates of each ± SD. Asterisks * and ** indicate reductions in the expression of <i>MySP</i> by ≤ 0.5 fold and > 0.5 fold, respectively in the aphids that were fed on the SP-transgenics as compared to those on the control plants. (B) Gut protease activity was estimated by EnzChek protease assay performed with 10 µg of total protein extracted from <i>M. persicae</i> collected from the control and SP transgenics 7 days after the release of the aphids. Protease activity was expressed as equivalent to trypsin (ng/ml). Values are mean ± SE; <i>n = </i>4. Different letters on the histograms indicate that the means differ significantly (<i>P</i> ≤ 0.05).</p

Effect of various concentrations of BA and IBA on shoot induction from nodal explants of <i>P</i>. <i>eriocarpum</i>.

<p>Effect of various concentrations of BA and IBA on shoot induction from nodal explants of <i>P</i>. <i>eriocarpum</i>.</p

Assessment of genetic fidelity of <i>in vitro</i> raised plants of <i>Pittosporum eriocarpum</i>.

<p>(A) SCoT fragments obtained with primer SCoT 35. (B) ISSR fragments obtained from primer number 860. (C)RAPD fragments obtained with primer OPL10. L- 1Kb ladder; MP- mother plant (Control); A1-A9- <i>in vitro</i> raised plants.</p

<i>Pittosporum eriocarpum</i>.

<p>(A) Nodal explant. (B) Shoot induction in MS medium supplemented with BA (mg/l). (C) Root induction in MS medium supplemented with IBA (mg/l). (D) <i>In vitro</i> grown plantlets. (E) <i>In vitro</i> grown plantlets in soil rite. (F) <i>In vitro</i> raised plantlets in natural conditions.</p

Effect of one hour incubation in IBA on number of shoots, leaves and callus diameter per explants.

<p>Effect of one hour incubation in IBA on number of shoots, leaves and callus diameter per explants.</p

Dendrogram illustrating coefficient similarity among <i>in vitro</i> raised plants with mother plant of <i>P</i>. <i>eriocarpum</i> by UPGMA cluster analysis from the RAPD data set showing genetic relationship.

<p>MP- mother plant; A1-A9- <i>in vitro</i> raised plants.</p

ANOVA of the effect of BA and IBA and combined BA and IBA on shoot length.

<p>ANOVA of the effect of BA and IBA and combined BA and IBA on shoot length.</p