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Title: Overexpression of Aspartic Acid in Cotton Against Insects
Authors: Gul, Ambreen
Keywords: Molecular Biology
Issue Date: 2019
Publisher: University of the Punjab , Lahore
Abstract: Whitefly is a major threat to cotton production causing more than 60% yield losses to cotton crop. Owing to the limitations of whitefly control strategies through chemical insecticides, and lack of long term control offered by application of different strategies in the field of molecular biology instigates the establishment of new biochemical pathways pertaining to the whitefly resistance of the cotton plant. Current research work entails the enhancement of free aspartic acid in cotton by utilizing two different genes; OsAAT2 and ZmASN encodes aspartate and asparaginase proteins respectively. Both OsAAT2 and ZmASN genes were separately cloned in pCAMBIA-1301 vector under the control of CaMV35S promoter. Cotton variety CIM-482 was transformed through Agrobacterium tumefaciens (LBA4404) with a PCR-based transformation efficiency of 2.5%. Relative quantification of OsAAT2 and ZmASN transcripts by qRT-PCR depicted an increase of 31% and 48% respectively. UPLCbased quantification of amino acids in the transgenic cotton plants was assessed in the seeds of T0 and phloem sap of T1 progeny portraying an increase in amino acid concentrations. Aspartic acid concentration in the seeds of OsAAT2 and ZmASN transgenic cotton and non-transgenic control line was found to be 9.6mg/100g, 9.96mg/100g and 1.43mg/100g respectively. Similarly, an increase in the glutamic acid and asparagine concentration of 9.29mg/100g and 76mg/100g respectively in OsAAT2 transgenic line and 11.8mg/100g and 37mg/100g respectively in ZmASN transgenic line was observed in contrast to the non-transgenic control (3. 7mg/100g and 10.67 mg/100g). Free aspartic acid detected in the phloem sap of both OsAAT2 and ZmASN transgenic cotton lines revealed an increase of 3.48 x 10-2 mg and 2.19 x 10-2 mg respectively from the non-transgenic control cotton plant (0.845 x 10-2 mg). Different agronomic characters like height, number of bolls and yield of OsAAT2 and ZmASN transgenic cotton plants were analysed and exhibited an improvement in the aforementioned traits when compared with non-transgenic control plant. Agronomic characters of transgenic cotton like height, number of bolls and yield of transgenic cotton plants of OsAAT2 and ZmASN and non-transgenic control were also analysed and the results exhibited an improvement in the these traits when compared with non-transgenic control cotton plant. An improvement in height, number of bolls and yield was observed in the transgenic plants of OsAAT2 (192cm, 152 and 265g respectively) and ZmASN (149cm, 130 and 218g respectively) than the nontransgenic control (59cm, 23 and 50g). Cotton yield is determined in terms of quantity as well as quality. The quality of cotton fiber was determined by subjecting the cotton fiber to scanning electron microscope analysis for determination of fiber surface. Scanning electron microscopic analysis showed a fine surface of the transgenic cotton lines of both genes than the non-transgenic control cotton plants. The altered amino acid content has its direct influence on determination of plant physiology as was evident in case of transgenic cotton lines. The maximum increase in photosynthesis and stomatal conductance in transgenic lines of OsAAT2 was found to be (17.33 µmol m-2s- 1 and 322 mmol m-2s-1) and ZmASN (22 µmol m-2s-1 and 492 mmol m-2s-1) compared to non-transgenic control cotton line where it was found to be 9.56 and 37.66 respectively. More than 90% whitefly adult mortality was achieved in the transgenic cotton lines of both genes (OsAAT2 and ZmASN) as compared to the non-transgenic control cotton lines. Fluorescence in situ hybridization analysis of the advanced generation selected plant revealed that both genes were integrated as single copy gene in the plant genome at chromosome number 12 and 1 for OsAAT2 and ZmASN respectively.
Gov't Doc #: 19585
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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