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Some Physiological Studies on Modern Biotechnology of Wheat Plant
Wheat is occupied a position among global crops because wheat is a staple food for most of the world’s population. The demand for wheat is increased with the increasing of the world’s population. Wheat influenced by many factors, such as fungal infection, that reduce productivity and quality. The most important fungal diseases that infect wheat was ”blight” which caused by the Fusarium graminearum.
Improving the productivity of wheat by using traditional ways was unsatisfactory because it was taken a long time and the results may be unacceptable, therefore, gene transfer technique was used to improve resistance in susceptible varieties to fungal diseases using particle bombardment method.
The aim of this study is the production of Egyptian wheat cultivars resistant to fungal diseases such as Fusarium head blight (FHB). Firstly, two wheat bread cultivars (Giza 164 and Sids 1) and one durum wheat cultivar (Bani Suef 6) were used to study the effect of different concentrations of FCF on regenerated plants from the three wheat cultivars by using tissue culture technique. Secondly, particle bombardment method was used to gene transfer into the three Egyptian wheat cultivars to become resistant to fungal diseases. Finally, transgenic lines of cv. Giza 164 plants were used to the comparative physiological studies between non-inoculated and
inoculated transgenic lines with 20% of FCF for 15 days.
This study was divided into three experiments that interrelated to each other:
Experiment I: Effect of different concentrations of Fusarium graminearum culture filtrate on some physiological parameters of regenerated wheat (Triticium aestivium L.) cv. Giza164, cv. Sids1and (Triticium durum L.) cv. BaniSuef 6:
In this part of the study, Fusarium graminearum grew on solid Czapek’s medium, then the fungus grew on liquid medium to prepare fungal culture filtrate (FCF). Finally, different concentrations of the FCF (5, 10, 20 and 40%) were prepared.
- Immature embryos of the three Egyptian wheat cultivars (Giza 164, Sids 1 and Bani Suef 6) were used, then were grown on callus induction medium supplemented with 2, 4-D. Induced calli were transferred into regeneration medium containing TDZ.
- Rooting media were prepared with different concentrations of FCF, regenerated shoots were transferred into rooting
media. Finally samples were collected at intervals 5, 10 and 15 days after inoculation.
- Estimation of total soluble protein; some antioxidant enzymes (CAT, POX and APX) and PAL enzyme; proline
content, total phenol, flavonoids and protein patterns were carried out.
-The level of total soluble protein decreased at all concentrations of FCF except at 20% in cv. Giza 164. While the amount of the soluble protein increased in cv. Sids 1 and cv. Bani Suef 6 inoulated with different concentrations of FCF reaching the maximum value (2.938 mg g-1 FW) in cv. BaniSuef 6 and (4.061 mg g-1 FW) at 20%.
- Activities of CAT, APX and PAL increased in inoculated plants of the three wheat cultivars compared to non-inoculated plants. The highest increment in their activities was observed at 20% of FCF after 15 days compared with control ones reaching 132.86% in cv. Sids 1 in CAT enzyme, 278.85% in cv. Giza 164 in APX enzyme and 227.05% in cv. Sids 1 in PAL enzyme.
-The three wheat cultivars showed different behavior in the activity of the enzyme POX during inoculation with different concentrations of FCF. The highest increase was (97.06%) for cv. Sids 1 at 20% of FCF.
- The highest level of proline was observed at 20% of FCF in the three wheat cultivars reaching 4.071, 5.271 and 7.704 µg g-1 FW in Giza 164, Sids 1 and Bani Suef 6 compared with their controls, respectively.
- Total phenol content in inoculated of the three wheat cultivars was decreased at 5th day of inoculation but increased at 10th and 15th days of inoculation and reached its maximum value at 20% of FCF which was 2.466, 1.259 and 1.126 mg g-1 FW in Giza 164, Sids 1 and Bani Suef 6, respectively.
- The three wheat cultivars showed fluctuations in flavonoid content when treated with different concentrations of FCF. The highest value of flavonoid content was observed at 20% reaching 75.89%, 127.93% nad 165.56% for cv. Giza 164, Sids 1 and Bani Suef 6, respectively above the control.
- There were no changes in number of proteins after inoculation, new protein appeared and other disappeared, compared with control in cv. Giza 164 and Bani Suef 6. While cv. Sids 1 showed increase in the induced number of proteins at different concentrations of FCF compared with the control.
Experiment II: Co-transformation of immature embryo derived calli of bead wheat (cv.Giza 164 and cv.Sids 1) and durum wheat (cv. Bani Suef 6) with pAHCht-2 harboring rice chitinase gene and pAB6 harboring bar and gus genes:
To improve and increase wheat production, quality and resistance to fungal diseases, transfer three genes (cht-2, bar and gus) into immature embryos derived calli of the three wheat cultivars (Giza 164, Sids 1 and Bani Suef 6) were carried out. Gold particles (1.0µm) coated with pAHCht-2 harboring rice chitinase gene and pAB6 harboring bar and gus genes.Calli were co-bombarded with two plasmids at 1100 psi.
Histochemical gus assay was confirmed the gus expression, transgenic calli were given blue color when treated with gus buffer assay compared to non-transgenic calli that were not changed in color. After bombardment, calli were transferred into selection medium supplemented with PPT for four weeks to selecte transgenic calli from non-transgenic ones.
Selected calli were transferred into regeneration medium containing TDZ for four weeks, then transferred into rooting medium free hormones and acclimatized. The plants were transplanted into pots containing mixture of soil at specific
conditions (temperature, humidity and light) in the growth chamber.
DNA was extracted from acclimatized plants and PCR analysis was used to confirm the presence of transgenes (cht-2, bar and gus) using specific primers under suitable conditions.
- The results showed that cv. Sids 1 was recorded the highest percentage of selection rate in the first and second selection phases using PPT reaching 69.31% and 30.1%, respectively.
- In regeneration stage, results showed that cv. Giza 164 was recorded the highest percentage of regeneration rate on regeneration medium supplemented with TDZ reaching 65.21%.
- In acclimatization stage, results showed percentage reached to 16.02%, 16.54% and 14.28% in cv. Giza 164, Sids 1 and Bani Suef 6, respectively.
- Results of PCR analysisshowed that in the case of cht-2 (1100bp) there were 31, 15 and 11 plants that gave positive results in cv. Giza 164, Sids 1 and Bani Suef 6, respectively.
- In the case of bar gene, there were 45, 20 and 17 plants that showed positive results (443bp) in cv. Giza 164, Sids 1 and Bani Suef 6, respectively.
- In the case of gus gene, there were 40, 19 and 18 plants that exhibited positive results (1050bp) in Giza 164, Sids 1 and Bani Suef 6, respectively.
- To confirm bar gene expression, leaf painting was used, herbicide Liberty at 0.2% concentration. Results were identical to the results that obtained from PCR analysis. While expression of cht-2 gene confirmed by using Dot Blot technique. Results showed that plants which gave positive results with the PCR analysis gave positive results with Dot Blot.
- The results showed that cv. Giza 164 scored the highest efficiency of transformation which was 6.01%, followed by Sids1which was 5.29% and cv. Bani Suef 6 recorded the lowest efficient transformation which was 4.67%.
Experiment III: Comparative study between non- inoculated and inoculated transgenic wheat plants cv. Giza 164 with F. graminearum culture filtrate:
On the basis of the results obtained from the first and second experiment, the concentration 20% of FCF and cv.Giza 164, that recorded the highest regeneration rate, were chosen.
- Seven transgenic lines of cv.Giza 164 were used. PCR analysis carried out to confirm the presence of transgenes (cht-2, bar and gus).
- Transgenic lines were divided into two groups:
- group (non-inoculated): transgenic lines were transferred into rooting medium free hormones.
- group (inoculated): transgenic lines were transferred into rooting medium supplemented with 20% of FCF.
- Plants were analyzed in both groups after 15 days and physiological comparative studies were done between the two groups.
- The amount of soluble protein in transgenic lines was lower than that in non-transgenic plants, in the case of inoculation with 20% of FCF for 15 days, the soluble protein content was increased in both transgenic lines and non-transgenic plants reaching maximum value (58.57%) in line B-3 as compared with non-inoculated transgenic line.
- The results showed a marked change in CAT and POX activities between transgenic lines and non-transgenic plants. Inoculation with 20% of FCF increased CAT and POX activities in non-transgenic and transgenic lines reaching 31.86% and 21.73% compared with non-inoculted non-transgenic plants, respectively. The maximum CAT activity was (71.36%) in line E-7 while the maximum POX activity was (157.39%) in line D-6 as compared to non-ioculated ones.
- The results showed different behavior in the APX and PAL activities in transgenic lines compared to non-transgenic plants. Inoculation with 20% of FCF increased the enzyme activities in both non-transgenic and transgenic lines reached the maximum value (56.88%) in C-4 line in case of APX and (39.57%) in B-3 line in case of PAL enzyme as compared with non-inoculated transgenic lines.
- The results showed significant increase in chitinase activity in transgenic lines as compared with non-transgenic plants. Inoculation with 20% of FCF increased chitinase activity reaching 37.14% in non-transgenic plants and 80.75% in D-6 line as compared with non-inoculated ones.
- The results showed slight change in proline content in transgenic lines as compared with non-transgenic plants. Inoculation with 20% of FCF increased proline content in non-transgenic plants reaching 35.65% as compared with non-inoculated non-transgenic plants. Inoculated transgenic lines showed fluctuations in proline content on response to inoculation.
- The results showed different behavior in the total phenol content in transgenic lines compared to non-transgenic plants.
Inoculation with 20% of FCF significantly increased in phenol content in non-transgenic plants reaching 43.35% as compared with non-inoculated ones. The phenol content was decreased in most non-inoculated transgenic lines reaching
20.45% in D-6 line except two lines (B-3 and C-4) phenol content was increased reaching to 36.56% and 23.37%, respectively compared to non-inoculated transgenic lines.
- Flavonoid content decreased in most transgenic lines compared to non-transgenic plants. Inoculation with 20% of FCF markedly increased in flavonoid content in non-transgenic plants reached to 19.77% compared to non-inoculated non-transgenic plants. Inoculated transgenic lines exhibited significant decrease in flavonoid content in some lines which reached to 28.32% in A-2 line and increasing in flavonoid in other lines which reached to 69.44% in H-1 line.
- The results exhibited new six induced proteins induced in transgenic lines compared to non-transgenic plants which have molecular weights (53.57, 50.72, 38.34, 31.19, 28.33 and 27.37 KDas).
- Inoculation with 20% of FCF of F. graminearum induced new nine proteins in transgenic lines compared with non-transgenic plants, their molecular weights are 66.81, 62.8, 50.41, 41.62, 36.96, 29.74, 28.27 and 27.24 KDas.