SOME PHYSIOLOGICAL STUDIES ON MODERN BIOTECHNOLOGY OF WHEAT PLANT

Abstract

Fusarium graminearum leads to severe losses in grain yield and quality of wheat. Therefore, it is very important to improve wheat resistance. In the present study, immature embryos of three Egyptian wheat cultivars (Giza 164, Sids 1 and Bani Suef 6) and plasmid pAHCht-2 harboring rice chitinase gene for pathogen resistance and plasmid pAB6 containing the gus reporter gene and the bar selectable marker gene have been used for studying the effect of different concentrations of Fugal Culture Filtrate (FCF) and genetic transformation. The three wheat cultivars exhibited different responses for inoculation with different concentrations of FCF (5%, 10%, 20% and 40%). However, Inoculation with 20% FCF significantly increased antioxdant enzymes activities and PAL activities in shoots of wheat cultivars compared with uninoculated ones. Also, total soluble protein and secondary metabolites significantly increased throughout experiment period. Protein profiling by SDS-PAGE revealed that there are no changes in the number of protein bands, but new proteins appeared and other disappeared in the two wheat cultivars. cv. Sids 1 synthesized new proteins compared with uninocualted plants. Cht-2, bar and gus genes were introduced into immature embryos derived calli of the three wheat cultivars using particle bombardment. The bombarded calli were transferred to medium supplemented with phosphinothricin (PPT) at 3 mg/L for the selection of the transformed calli. The transgenic calli were confirmed for the expression of GUS by histochemical analysis of β-glucuronidase. The regeneration ix was carried out on MS medium supplemented with 1.5 mg/L thidiazuron (TDZ). Herbicide-resistant putative transformants were selected after leaf painting with 0.2% Liberty. Presence and integration of transgenes were assessed by subjecting the DNA of the transgenic plants to PCR analysis using specific primers for Cht-2, bar and gus genes. Among the three cultivars studied, Giza 164 recorded the highest transformation efficiency 6.01% followed by Sids1 with 5.29% and Bani Suef 6 with 4.67%. The incorporation of the rice chitinase gene in the genome of the transformants was confirmed by the Dot-blot analyses. The results showed that there are variations in the physiological characteristics between transgenic and non-transgenic wheat lines (cv. Giza 164) inoculated with 20% of FCF and between inoculated transgenic lines and non-inoculated transgenic ones. In this respect, the results showed significant decreases in the soluble protein, proline, phenols and flavonoids contents; antioxidant enzyme activities (POX, APX and CAT) and chitinase activity in shoots of transgenic lines compared with non-transgenic plants. On the other hand, inoculation with 20% FCF had increased stotal phenol and flavonoids; antioxidant enzymes and chitinase activity in transgenic lines compared with the non-inoculated transgenic ones. Keywords: Wheat, Fusarium graminearum, particle bombardment, antioxidant enzymes, secondary metabolites, SDS-PAGE, rice chitinase, bar, gus, PCR, Dot blot, Fungal culture Filtrate, protein profile.