Development of Transgenic Egyptian Cotton Varieties Tolerant To Some Abiotic Stress Conditions Using Levan Sucrase Gene

Abstract

Fructans are polyfructose molecules that function as non­ structural storage carbohydrates in several plant species. Owing to their solubility, fructans may help cotton survive periods of osmotic stress induced by drought or by cold. We developed transgenic Egyptian cotton plants (Gossypium barbadense), from long staple variety, Giza 85, and extra long staple variety, Giza 88 by introducing the microbial sacB gene of Bacillus subtilis coding for levan sucrase enzyme fused to the vacuolar targeting sequence of the yeast carboxypeptidase Y (cpy) gene and the selectable marker gene bar encoding phosphoinothricin acetyl transferase gene which detoxifies basta herbicide. The transformation was achieved by particle bombardment using shoot meristematic apices as target. R0 plants acclimatized in the greenhouses were submitted to preliminary screening for chimeric transformants by painting the plant leaves with basta herbicide (200 mgs/L) to confirm bar gene integration. Basta painting experiment showed that the bar gene was incorporated into about 30% of transformed cotton plants. Further analyses on the positive- basta putatively transformed cotton plants were performed using Southern blot analysis using the labeled cpy-sacB chimeric gene as a probe following polymerase chain reaction (PCR) which revealed the presence of the intact cpy­ sacB chimeric gene in eight and seven cotton plants from both Giza 85 and Giza 88, respectively. Genomic Southern blot analyses confirmed the integration of cpy-sacB gene into the plant genome. cpy-sacB gene was found to be integrated into the genome of only two plants out of one thousand of each cotton variety. Polyfructose molecules were detected in plant cotton tissue by HPLC and the increase of dry weight in fructose level was determined in putatively transgenic plants transformed with the sacB gene compared to non­ transgenic ones. Reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out to permit the detection of RNA transcripts in the cotton plant genome. A 1.7 Kb eDNA fragment was detected using RT-PCR, which confirms the transcription of the chimeric cpy­ sacB gene