Development of Transgenic Cotton Lines Expressing <i>Allium sativum</i> Agglutinin (ASAL) for Enhanced Resistance against Major Sap-Sucking Pests

<div><p>Mannose-specific <i>Allium sativum</i> leaf agglutinin encoding gene (<i>ASAL</i>) and herbicide tolerance gene (<i>BAR</i>) were introduced into an elite cotton inbred line (NC-601) employing <i>Agrobacterium</i>-mediated genetic transformation. Cotton transformants were produced from the phosphinothricin (PPT)-resistant shoots obtained after co-cultivation of mature embryos with the <i>Agrobacterium</i> strain EHA105 harbouring recombinant binary vector pCAMBIA3300-<i>ASAL</i>-<i>BAR</i>. PCR and Southern blot analysis confirmed the presence and stable integration of <i>ASAL</i> and <i>BAR</i> genes in various transformants of cotton. Basta leaf-dip assay, northern blot, western blot and ELISA analyses disclosed variable expression of <i>BAR</i> and <i>ASAL</i> transgenes in different transformants. Transgenes, <i>ASAL</i> and <i>BAR</i>, were stably inherited and showed co-segregation in T₁ generation in a Mendelian fashion for both PPT tolerance and insect resistance. <i>In planta</i> insect bioassays on T₂ and T₃ homozygous <i>ASAL</i>-transgenic lines revealed potent entomotoxic effects of ASAL on jassid and whitefly insects, as evidenced by significant decreases in the survival, development and fecundity of the insects when compared to the untransformed controls. Furthermore, the transgenic cotton lines conferred higher levels of resistance (1–2 score) with minimal plant damage against these major sucking pests when bioassays were carried out employing standard screening techniques. The developed transgenics could serve as a potential genetic resource in recombination breeding aimed at improving the pest resistance of cotton. This study represents the first report of its kind dealing with the development of transgenic cotton resistant to two major sap-sucking insects.</p></div

Effect of ASAL on the fecundity of jassid and whitefly insects.

<p>(A) Total number of nymphs produced by three pairs of adult jassid insects fed on untransformed control and transgenic plants were counted and plotted on the graph. (B) Total number of nymphs produced by five pairs of adult whitefly insects fed on untransformed control and transgenic plants were counted and plotted on the graph. UC: untransformed control plants. NC_3-1-8, NC_9-1-15, NC_12-1-11 and NC_16-1-6: Different Transgenic cotton lines expressing ASAL. Bioassays were carried out with five replications and were repeated thrice. Differences between control and transgenic plants were significant at p<0.0001. Bars indicate mean ± SE.</p

Jassid and whitefly bioassays on homozygous transgenic plants of cotton.

<p>(A) 45-day old transgenic lines along with untransformed control plant infested with jassid. (B) 45-day old transgenic lines along with untransformed control plant infested with whitefly. UC: untransformed control plants showing susceptibility against jassid and whitefly infestation with complete damage (4 on a 1 to 4 scale). NC_3-1-8, NC_9-1-15, NC_12-1-11 and NC_16-1-6: Transgenic cotton lines expressing ASAL showing significant resistance (1 to 2 on a 1 to 4 scale) against jassid and whitefly infestation with minimal plant damage.</p

Basta treated leaves of cotton transformants showing tolerance to the herbicide.

<p>UC: Untransformed control leaf showing damage to the herbicide. 1–9: Leaves of different cotton transformants showing tolerance to the herbicide.</p

Southern blot analyses of transgenic cotton plants.

<p>(A) Restriction map of T-DNA region of pCAMBIA3300 containing <i>ASAL</i> and <i>BAR</i> expression units. (B) Genomic DNA digested with EcoRI and probed with <i>ASAL</i> coding sequence. (C) Genomic DNA digested with HindIII and probed with <i>BAR</i> coding sequence. Lane UC: DNA from untransformed control plant. Lanes NC3, NC5, NC9, NC10, NC12 and NC16: DNA from different transgenic lines.</p

Northern and western blot analyses for the expression pattern of transgenes in transgenic cotton lines.

<p>(A) RNA probed with <i>ASAL</i> coding sequence. (B) RNA probed with <i>BAR</i> coding sequence. (C) Protein extracts from cotton plants treated with anti-ASAL antibodies. Lane UC: Samples from untransformed control plants. Lanes NC3, NC5, NC9, NC10, NC12 and NC16: Samples from different transgenic lines. Ethidium bromide stained 28S RNA band is shown under northern blots for amount of RNA loading.</p