Development of transgenic cotton lines expressing Allium sativum agglutinin (ASAL) for enhanced resistance against major sap-sucking pests

Abstract

Mannose-specific Allium sativum leaf agglutinin encoding gene (ASAL) and herbicide tolerance gene (BAR) were introduced into an elite cotton inbred line (NC-601) employing Agrobacterium-mediated genetic transformation. Cotton transformants were produced from the phosphinothricin (PPT)-resistant shoots obtained after co-cultivation of mature embryos with the Agrobacterium strain EHA105 harbouring recombinant binary vector pCAMBIA3300-ASAL-BAR. PCR and Southern blot analysis confirmed the presence and stable integration of ASAL and BAR genes in various transformants of cotton. Basta leaf-dip assay, northern blot, western blot and ELISA analyses disclosed variable expression of BAR and ASAL transgenes in different transformants. Transgenes, ASAL and BAR, were stably inherited and showed co-segregation in T1 generation in a Mendelian fashion for both PPT tolerance and insect resistance. In planta insect bioassays on T2 and T3 homozygous ASAL-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.

Figure 1. Basta treated leaves of cotton transformants showing tolerance to the herbicide.

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

Figure 2. Southern blot analyses of transgenic cotton plants.

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

Figure 3. Northern and western blot analyses for the expression pattern of transgenes in transgenic cotton lines.

(A) RNA probed with ASAL coding sequence. (B) RNA probed with BAR 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.

Figure 4. Jassid and whitefly bioassays on homozygous transgenic plants of cotton.

(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.

Figure 5. Survival of jassid and whitefly insects on transgenic cotton lines expressing ASAL.

(A) Twenty jassid nymphs were released onto each single leaf enclosure and nymphal survival was recorded at every 3-day interval upto 21 days. (B) One adult whitefly pair (1male:1female) was released onto each single leaf enclosure and nymphal survival was recorded from eighth day onwards for every 4-day interval upto 32 days. Transgenic lines NC_3-1-8, NC_9-1-15, NC_12-1-11 and NC_16-1-6 are depicted by square, triangle, rectangle and circle, respectively. Untransformed control (UC) plants are depicted by diamond. 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.

Figure 6. Effect of ASAL on the development of jassid and whitefly insects.

(A) Twenty jassid nymphs were released onto each single leaf enclosure. After 21 days, the number of nymphs reached adult stage and the number of nymphs remained immature were plotted on the graph. (B) One adult whitefly pair was released onto each single leaf enclosure and after 32 days the number of nymphs reached adult stage and the number of nymphs remained immature were 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.

Figure 7. Effect of ASAL on the fecundity of jassid and whitefly insects.

(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.