Pathological Testing of the transgenic plant with over expression of HarChit

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3.1.1.15 Pathological Testing of the transgenic plant with over expression of HarChit and

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Figure: 3.25 shows the relative number of colonies developed on the leaf disks of the transgenic plants (having HarChit and/or HarCho expressed under stress/disease inducible promoter) compared to the non transgenic and transgenic control plants. Here control-1 is the non transgenic control while control-2 is the transgenic contol with gus and bar constructs. The rest bars represent transgenic plants.

The data collected for the number of colonies developing on the young leaf segments was taken nine days post inoculations and an average number of developing colonies per leaf segment were calculated. The averages of all the individual transgenic lines were then compared with non transgenic control. The results showed that all the lines had different average number of colonies after nine days of inoculations. Control2 (transgenic control) had 1.03 colonies as compared to every colony on control1 (non transgenic control). All the other transgenic lines had less number in comparison to non transgenic control except line I.A-9 who had 1.14 colonies in comparison to every colony found on the non transgenic control.

Sizes of the selected developing colonies were measured three weeks post inoculations in terms of millimetres square and an average was calculated for every individual line. These averages were then compared taking the non transgenic control line colony size as one.

0 0.2 0.4 0.6 0.8 1 1.2 1.4

Control1 I.A-7 I.A-8 I.A-9 I.A-10 I.A-11 I.A-12 Control2 Transgenic lines under inducible promoter

Relative size of colonies

Figure: 3.26 Comparison of Colony size among Transgenics under Inducible Promoter

Figure: 3.26 represent the relative colony size of the transgenic plants (having HarChit and/or HarCho expressed under inducible promoter) compared to the non transgenic and transgenic controls. Here, control-1 is the non transgenic control while control-2 is the transgenic contol with gus and pat constructs. The rest bars represent transgenic plants. The data were taken 21days after inocution with 300-400 conida per cm². The results indicate that some transgenic plants which are relatively more susceptible than the controls while the others are visibley less susceptible than the negative controls.

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The data collected for colony size is presented above. It is evident from here that a detectable reduction in colony size was observed in three out of seven lines under the test. Among these lines I.A-11 showed the most reduction. If non transgenic line produced an average colony size of 1 millimetre I.A-11 colony size was 0.25 millimetre. Lines I.A-7 and I.A-12 also showed good reduction in colony size with 0.74 and 0.64 millimetres in comparison to a colony size of 1 millimetre for non transgenic control. Non transgenic control, line I.A-10 and transgenic control lines produced almost the same size while the colony size was even bigger than the controls in lines I.A-8 and I.A-9.

Figure: 3.27 Experimental sample of PM inoculation on over expression transgenic line I.A-11

4 cm long young leaf segments of all the transgenic plants were cultured separately on anti senescence media in comparison with non transgenic leaf segments and powdery material was blown onto them ensuring 300-400 conidia per cm². Transgenic control (with gus and bar genes) was also compared with non transgenic control. The experiment was kept under ideal conditions for fungal growth mentioned in the section 2.2.6.1 of this manuscript. First data were taken 9dpi for the number of developing colonies per leaf segment compared to the non transgenic control (shown in figure 3.18). The second data were taken 21 dpi on the colony size in terms of mm² and the average size on control and transgenic lines was compared

Figure: 3.27 show the real picture of the experiment 21 dpi. 1-6 are repeats used per experiment. Control is non transgenic line Florida used as negative control. Figure shows the development of E. graminis colonieas on control and transgenic line ‗I.A-6‘ 21 dpi.

1 2 3 4 5 6

control

I.A-11

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Figure: 3.28 Close view of Experimental sample of PM inoculation on over expression transgenic line I.A-11

Figure: 3.28 close view of the experiment presented in figure 3.27 shows clear difference in the transgenic and non transgenic control in terms of resistance to E. graminis. Transgenic has completely controlled the pathogen after 3 weeks but the non transgenic could not.

The figures 3.27 and 3.28 above show real examples of the same experiment that shows not only the reduction in the number of the PM colonies per leaf segment but also the deterioration of the health of the colonies. Control leaf segments showed healthy colonies with a lot of mycelia while transgenic leaf segments had fewer colonies and all the mycelia were also dead. The transgenic leaf segments also seemed to have more plant cell death shown in terms of more yellowness.

FHB (Fusarium graminearum) inoculations:

F. graminearum is a pathogen of the wheat head. Two central wheat spikelets were inoculated with 200 conidia/10µL of water at just before anther maturity stage. The infection process

Control

I.A-11 1 2

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was followed for three weeks. The differences started to become clear 9 days after inoculations.

0 10 20 30 40 50 60 70

Control-1 I.A-8 I.A-11 I.A-12 Control-2

Transgenic lines under inducible promoter

No. of visually affected kernels

Figure: 3.29 Comparisons of Visually Effected Kernals among Transgenics under Inducible Promoter Figure: 3.29 show the percentage of the visually affected kournals 21 dpi in the non transgenic, transgenic controls along with transgenic lines (having HarChit and/or HarCho expressed under stress/disease inducible promoter). The inoculations were made with 200 conidia per spikelet in the middle two spikelets just before fertilization. The experiment was kept at recommended condition for F. graminearum development stated in the section 2.2.6.2 of this manuscript

Three weeks after inoculations the data were taken on the number of affected kernels. Only three transgenic lines were checked for their reaction against F. graminearum. The data were taken and the percentage of the visually effected kernels was calculated on all the individual transgenic lines as well as control lines. The data presented above depicts that the control lines showed highest percentage of the effected kernels with a percentage of 58.25 and 53.75 for non transgenic and transgenic controls respectively. Out of the three lines tested I.A-12 showed the least visually effected kernel with a percentage of 32.75 effected kernels. I.A-8 and I.A-11 also showed a detectable reduction of visually effected kernels with 35.30% and 49.26% of visually effected kernels respectively.

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Figure: 3.30 Visual examples of FBH experiments.

Figure: 3.30A= Comparison of water control, non transgenic control and transgenic line I.A-12, 9 days after inoculation, B= Infection area of the non transgenic control 21 dpi. C=Infection area of transgenic (I.A-12) 21 dpi

Figure 3.30 shows the examples of the general trends in the pathology experiments and reveals how the transgenic lines resist against the pathogen and stop it from spreading to the next spikelets.