Figure 1.
Amplification of PME genes and cloning in plant expression vector.
(A) Amplification of pme genes, AnPME; PME from A. niger, M; 500bp DNA ladder, AtPME; PME from A. thaliana. (B) Schematic representation of the plant expression vector (pAnPME and pAtPME). (C) Restriction analysis of pAnPME ; M, 500bp DNA Ladder ; 1, Digestion with Xba I and EcoRI; 2, Digestion with Xba I and Sac I; 3, Digestion with Xba I and XhoI. D, Restriction analysis of pAtPME; M, 500bp DNA Ladder; 1, Digestion with Xba I and Sac I; 2, Digestion with Xba I and EcoRI.
Figure 2.
Molecular analyses of putative transgenic lines.
Genomic DNA PCR (A) with primer pairs AnF and AnR. (B) with primer pairs nptF and nptR, M; 500bp DNA ladder, An 1-5; different transgenic lines of AnPME, -ve; wild type (NTPH), +ve; positive control (pAnPME). (C) with primer pairs AtF and AtR . (D) with primer pairs nptF and nptR, M; 500bp DNA ladder, At 1-5; different transgenic lines of AtPME, -ve; control plant (wild type), +ve; positive control (pAtPME). (E) cDNA PCR of AnPME transgenic lines, An 1-5; different transgenic lines of AnPME. (F) cDNA PCR of AtPME transgenic lines, At 1-5; different transgenic lines of AtPME. Actin was taken as an internal control.
Figure 3.
Assay of transgenic plants for PME activity.
(A) Percent enrichment of PME activity in different transgenic plants over wild type (WT). (B) & (C) In-gel PME activity analysis of different transgenic lines expressing AnPME (An 1-5) & AtPME (At 1-5). Arrow head shows desired PME bands in transgenic plants.
Figure 4.
Estimation of methanol in transgenic plants.
(A) Quantification of methanol content in control (NTPH) and selected transgenic lines by 1H NMR (inset image shows NMR spectra), Transgenic plants showed up to 16 fold higher methanol content compared to control plants. (B) Quantification of methanol emission in transpirated water through the stomata on leaves surface, Leaves of transgenic plant was also emitted more methanol (~12 fold) than control plant.
Figure 5.
Evaluation of transgenic plants against neonate larvae of Helicoverpa armigera and Spodoptera litura.
(A) Larval mortality on different transgenic lines. (B) Reduction in weight larvae on fed on different transgenic lines. Feeding damage and larval size of H. armigera (C) and S. litura (D) on wild type and transgenic leaves.
Figure 6.
Evaluation of transgenic plants against aphids and whiteflies on different transgenic lines.
(A) In-planta Bioassay of different transgenic lines with aphids and whiteflies. (B) Bioassay with aphids and whiteflies using detached leaves. Transgenic plant show complete absence of aphids (C) and whiteflies (D).
Figure 7.
Confocal microscopic images of transgenic and control plants.
(A) Propidium iodide stained transverse section of transgenic and control plants showing well integrated cell network. (B) Structure of stomata, circumference of stomata of transgenic plant (113.48±6.7) was similar to WT (114.96±4.6). (C) Structure of trichomes (1) and stomatal density of transgenic and WT plants (2) were also similar to control plant. (D) Pollen germination of transgenic and WT (after 3 hrs). (E) FDA stained single pollen showing equal pollen tube length for transgenic and WT (1). FDA stained (2) DIC and (3) merge of FDA and DIC.
Figure 8.
Transcript level of important cell wall enzyme in transgenic plant.
Transcript level of all analysed genes [endo-1,4-ß-glucanases (Cel2, Cel4, Cel5, Cel7, Cel8), Cellulose synthase (celsyn), endo-xyloglucan transferase (Xytr), Expansin (NtExp1) and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)] was up regulated in AtPME transgenic lines (At-5) over WT (NTPH) while AnPME transgenic plants (An-4) also showed expression pattern similar to AtPME except Cel4. Transcript level of housekeeping genes like GADPH was almost unaltered in transgenic plants.