3. RESULTS:
3.1 Wheat transformation:
3.1.1 Over Expression HarChit and HarCho Genes in Wheat:
3.1.1.13 rt-PCR analysis for the expression of pVst-HarChit and pVst-HarCho
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Figure: 3.15a shows the expression of HarCho gene in T0 and T1 plants of plants ―I.A-18‖ and ―I.A-17‖. ―–ve‖ control, and
―I.A-2‖ were expected to show no expression because ―–ve‖ was non transgenic control plant and ―I.A-2‖ was proved negative by Southern Blot analysis.
Figure 3.15 shows the expression of HarCho. I.A-18 and I.A-17 are expressing the gene in T0
as well as in T1 generation while there is no expression in I.A-2 and I.A-5. I.A-2 did not show the integration of pUbi-HarCho during Southern Blot analysis but this construct was integrated in I.A-5.
The conclusion of molecular analysis of pUbi-Harchit and pUbi-Harcho integration and expression is that six transgenic plants were recovered with the co-integration of pUbi-HarChit and pUbi-HarCho genes into wheat genome. One plant had only pUbi-pUbi-HarChit and one had only pUbi-HarCho. The successful transfer of transgenes was also proved to the progeny of primary transforments.
The expression of transgenes was not possible in all the transgenic plants and progeny. One plant that had got both the genes and marker gene pat showed only the expression of marker gene but did not show the expression of both the genes of interest. The other plant which contained both genes showed the expression of HarChiy only.
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Figure: 3.16a. rt-PCR of the T0 pVst-HarChit positive plants.
Positive control showed the band of the expected size as did all the positive plants while the negative controls never showed the presence of any transcript from HarChit. PCR reaction showed the presence of HarChit transcript after Vst1 promoter induction with wounding stress.
M= Marker, C1= reagents control with H2O as template, C2 = negative control with template from non transgenic plant. C3 = positive control with template from pVst-Chit plasmid.
Figure: 3.16b rt-PCR of the T1 pVst-HarChit positive plants from T0 progenies
Positive control showed the band of the expected size as did all the positive plants while the negative controls never showed the presence of any transcript from HarChit. PCR reaction showed the presence of HarChit transcript after Vst promoter induction with wounding stress in the progenies of pVst-HarChit positive plants.
M= Marker, C1= reagents control with H2O as template, C2 = negative control with template from non transgenic plant. C3 = positive control with template from pVst-HarChit plasmid.
Figures 3.16a and 3.16b shows the presence of Harchit transcription on wound induction in all the T0 plants as well as in the T1 plants. The positive and negative controls gave the expected results. The positive control with a template of plasmid DNA gave a band exactly like the transgenic plants gave. PCR negative control with H2O as template did not give any band and same did when the negative control for transgenics where c-DNA from non transgenic plant was taken as template.
Induction of HarCho:
PCR reactions were done with the synthesized c-DNA using HarCho specific primers. A band of around 400bp was expected on the transgenic plants with induced Vst promoter as well as on pVst-HarCho plasmid DNA. PCR product from all the six pVst-HarCho Southern Blot analysis positive plants and non transgenic control was compared with negative H2O template control.
M C1 C2
I.A-7 I.A-9 I.A-10 I.A-11 I.A-12 C3
T0-Vst-HarChit
400bp
I.A-11 I.A-12 I.A-7 I.A-9 C3
C1 C2 M
T1-Vst-HarChit
400bp
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Figure: 3.17a. rt-PCR of the T0 pVst-HarCho positive plants.
Positive control showed the band of the expected size as did all the positive plants while the negative controls never showed the presence of any transcript from HarChit. PCR reaction showed the presence of HarCho transcript after Vst promoter induction with wounding stress. The PCR products with I.A-7 and I.A-9 showed two bands which may be due to incomplete integration of a copy of gene construct.
M= Marker, C1= reagents control with H2O as template, C2 = negative control with template from non transgenic plant. C3 = positive control with template from pVst-HarCho plasmid.
Figure: 3.17b. rt-PCR of the T1 pVst-HarCho positive plants from T0 progenies
Positive control showed the band of the expected size as did all the positive plants while the negative controls never showed the presence of any transcript from HarChit. PCR reaction showed the presence of HarCho transcript after Vst promoter induction with wounding stress in the progenies of pVst-HarChit positive plants.
The trend of double bands continued even in the T1 generation as is shown in the example above.
M= Marker, C1= reagents control with H2O as template, C2 = negative control with template from non transgenic plant. C3 = positive control with template from pVst-HarCho plasmid.
Figures 3.17a and 3.17b showed the presence of HarCho transcription on wound induction in all the T0 plants as well as in the T1 plants. The positive and negative controls gave the expected results. The positive control with a template of plasmid DNA gave a band exactly like the transgenic plants gave. PCR negative control with H2O as template did not give any band and same did the negative control for transgenics where c-DNA from non transgenic plant was taken as template. I.A-9 continued the trend of double band in the T1 and T2 generation. This can be due to additional incomplete integeration of the gene cassette as these plants were having multicopy integration of pVst-HarCho.
In summery, it was possible to transform wheat with pVst-HarChit and pVst-HarCho and induce both the genes on wound induction. Out of the six transgenic plants five were having the co-integration and expression of pVst-HarChit and pVst-HarCho and one was having only pVst-HarCho.
M C1 C2 I.A-11 I.A-12 I.A-7 I.A-9 C3
400bp
T1-Vst-HarCho
400 bp
M C1 C2 I.A-7 I.A-9 I.A-8 I.A-10 I.A-11 I.A-12 C3
T0-Vst-HarCho
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Table: 3.3 Summery of the Gene Integration and Expression among HarChit and HarCho Transgenics.
No. Lines Southern Blot analysis Northern/rtPCR
HarChit HarCho HarChit HarCho
Transgenic Plants with Constitutive Promoter
1 I.A-1 + + - +
2 I.A-2 + - + -
3 I.A-3 + + + +
4 I.A-4 + + + +
5 I.A-5 + + - -
6 I.A-6 + + + +
7 I.A-17 - + - +
8 I.A-18 + + + +
Transgenic plants with stress inducible promoter
9 I.A-7 + + + +
10 I.A-8 - + - +
11 I.A-9 + + + +
12 I.A-10 + + + +
13 I.A-11 + + + +
14 I.A-12 + + + +
Table 3.3 gives and over view of the HarChit and HarCho transformation and expression in wheat. In total 14 transgenic plants were found with the gene/genes of interest, eight under the control of constitutive promoter and six under stress and disease inducible promoter. One line showed the integration of both HarChit and HarCho under Ubiquitin promoter but there was no expression, another plant showed the integration of both HarChit and HarCho but expression was found only on HarCho. Four plants showed the co-intergration and expression of HarChit and HarCho under Ubiquitin constitutive promoter. One line showed intergration and expression of HarChit only and two lines showed the integration and expression of HarCho only.
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The case was not a lot different when disease and stress inducible promoter was used. A total of six plants were found and five of them showed co-integration and expression of both the genes. One line showed the integration and expression of HarCho only.