الفهرس 
MOLECULAR GENETICS STUDIES ON OVEREXPRESSION OF OSMOTIN (Tbosm) GENE TO IMPROVE SALINITY RESISTANCE IN WHEAT (Triticum aestivum) PLANTOnly 14 pages are availabe for public view
 |  | from | 91 |  |  |
| | | from | 91 | | |
Abstract
Wheat is one of the major staple food crops grown worldwide; however, productivity in cereal crops has not kept pace with the world population growth. A significant increase in wheat production is needed simply to keep up with the growing demand. This increase is unlikely to be achieved by conventional plant breeding methods because of the limited gene pool available.
The application of recombinant techniques to improve wheat quality and yield is not only desirable but also has potential to open up new opportunities. Although there has been significant progress in developing gene-transformation technologies for improving these traits, this remains an important challenge for plant biotechnology.
Wheat crop productivity is often low, due to abiotic stresses, such as drought or salinity and to biotic stresses such as pests and diseases. Wheat is classified as a semi tolerant crop to salinity.
The main objective of the present research is to:
Evaluate the most suitable concentration of growth regulators for calli induction and plant regeneration from wheat embryo, Regeneration and high throughput calli for transformation, Transformed of Tbosm gene by particle bombardment to calli samples, screening the genetic transformation of this gene on the end products.
The present study using two wheat (Triticum aestivum L) cultivars; namely ’’Misr-1 and Sakha – 93’’, produced the following results. Seeds of each cultivar were washed under running tap water for 10-15 mint and then surface-sterilized sequentially with 70% ethanol for 3-5 minutes followed by three rinses with sterilized distilled water. The mature seeds were sterilized with Clorox 10% for 20 min followed by three rinses with sterilized distilled water. Sterilized seeds were imbibed in sterile water over night, at room temperature (22 °C ) in complete darkness.
Mature embryos were aseptically isolated and cultivated on Ms Medium with different concentration of regulatory hormones. After three week embryogenic callus was produced.
Results showed that Ms3 induced the best result for callus induction for varieties Misr1 and Sakha -93 were 98, 24% and 99.1% respectively. Variety Sakha 93was considered more efficient than variety Misr 1 in callus production.
Embryogenic calli were collected and transferred to an osmotic medium (Ms medium 30g mannitol and 30g sorbitol) for four hours prior to bombardment using single shouts of gold particles coated with Tbosm gene. After a bombardment, callus were cultured on induction medium for 7-10 days recovery and then transferred to the same culture medium with four concentration of NaCl 50,100,150,200 Mm for selection of tolerant calli. After two cycles of selection, transformed callus were transferred to the regeneration medium.
The number of alive calli decreased in media that contain 200 Mm NaCl for both varieties Misr1and Sakha 93.
The surviving calli were transformed to regeneration medium. Calli with clearly differentiated shoots and roots were scored as regenerating calli. Calli, were subjected to three selection.
After five selections the percentage of dead callus was 6.7% for Misr1 and 12.6% for Sakha93
The percentage of regeneration was 8% for Misr1 and 30 % for Sakha 93
The Data showed more calli regeneration and transformation efficiency in variety Sakha- 93 than Misr-1.
The results confirmed that the Tbosm gene was successfully transformed to both wheat varieties. PCR analysis was conducted on all the plantlets regenerated from mature embryos transformation. To a certain the existence of the primers. Electrophoresis revealed the presence of Os1 at 320bp, Os2 at 134bp and Os3 at 101bp.
In this study we used three specific primers for osmotin gene. The first one Os1 was present in variety Misr-1 using (100,150 mM) NaCl only. While in Sakh-93 it was present using all concentration with high numbers. Os2 was found in both varieties using all concentration of NaCl with increased number in Sakha. In Misr-1, the primer Os3 wasn`t present using the concentration of 50 Mm NaCl .While in Sakha-93 it was present in all concentration.
Indicating that the variety Sakha-93 could tolerate salinity more than the cultivar Misr-1 and it could be used in breeding programs in reclamation lands.