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Creating transgenic plants carrying extra IPT genes
Author
Loh, Chay Boon
Supervisor
Yong, J. W. H. (Jean W. H.)
Tan, Swee Ngin
Fu, Jianlin
Abstract
The isopentenyltransferase (ipt) gene is closely associated with the de novo biosynthesis of cytokinins in plants. Cytokinins, one of the main classes of plant hormones, are found ubiquitously in all plants, the pivotal function being the involvement in the cell cycle. Presently, knowledge of the gene involvement in cytokinin biosynthesis is still unclear. Thus, a detailed study of the cytokinin biosynthesis gene, ipt, not only can enhance our present understanding of cytokinins, it can also provide insights into the general mode of action of plant hormones at the molecular level.
In today's context, creating transgenic plants is fast becoming mandatory in most plant research, especially for investigation at the genomic level. Thus, the objective of this study is to first, establish a sturdy methodology to create transgenic plants that can be used even by inexperienced operators, followed by an irrevocable methodology to identify mutants accurately at its molecular level.
In creating transgenic plants, the ideal transformation method should be a simple and highly efficient process with a short regeneration time. The transgenic plant should essentially have the gene mutated in the desired sector only. In this project, the flower-dip cum vacuum infiltration method was chosen as it fulfilled these prerequisites. Moreover, when using this rapid transformation method where the seeds are the transformed entity, the probability of unintended mutation is greatly reduced.
In this study, two lines of Arabidopsis thaliana mutant have been successfully created using the standard flower-dip cum vacuum infiltration transformation method. The first mutant line, hereby known as pCAM16, was transformed with the empty cloning vector pCAMBIA1301. It served as the assay control. The second mutant, C3, transformed with the CMV-ipt-GUS plasmid, carried an extra ipt gene driven by the 35S-CMV promoter. Transformation efficiency for pCAM16 and C3 mutants was 45.9% and 12.5% respectively. The xintegrity and functionality of the mutants were later confirmed wholly by molecular methods, using only 1 leaf or 2 leaves from each plant. PCR detection and sequencing analysis were employed to check the integrity of the mutants. RT-PCR technique was employed to confirm the functionality and expression level of the incorporated ipt. The ipt expression level of C3 mutant was found to be at least 4-fold higher than the wild-type. Phenotypically, C3 was also observed to be more robust and bushy than the wild-type. However this mutant was unable to produce viable seeds as the flowers shriveled up prematurely. This mutant could only be maintained via tissue culture methods.
In today's context, creating transgenic plants is fast becoming mandatory in most plant research, especially for investigation at the genomic level. Thus, the objective of this study is to first, establish a sturdy methodology to create transgenic plants that can be used even by inexperienced operators, followed by an irrevocable methodology to identify mutants accurately at its molecular level.
In creating transgenic plants, the ideal transformation method should be a simple and highly efficient process with a short regeneration time. The transgenic plant should essentially have the gene mutated in the desired sector only. In this project, the flower-dip cum vacuum infiltration method was chosen as it fulfilled these prerequisites. Moreover, when using this rapid transformation method where the seeds are the transformed entity, the probability of unintended mutation is greatly reduced.
In this study, two lines of Arabidopsis thaliana mutant have been successfully created using the standard flower-dip cum vacuum infiltration transformation method. The first mutant line, hereby known as pCAM16, was transformed with the empty cloning vector pCAMBIA1301. It served as the assay control. The second mutant, C3, transformed with the CMV-ipt-GUS plasmid, carried an extra ipt gene driven by the 35S-CMV promoter. Transformation efficiency for pCAM16 and C3 mutants was 45.9% and 12.5% respectively. The xintegrity and functionality of the mutants were later confirmed wholly by molecular methods, using only 1 leaf or 2 leaves from each plant. PCR detection and sequencing analysis were employed to check the integrity of the mutants. RT-PCR technique was employed to confirm the functionality and expression level of the incorporated ipt. The ipt expression level of C3 mutant was found to be at least 4-fold higher than the wild-type. Phenotypically, C3 was also observed to be more robust and bushy than the wild-type. However this mutant was unable to produce viable seeds as the flowers shriveled up prematurely. This mutant could only be maintained via tissue culture methods.
Date Issued
2008
Call Number
SB123.57 Loh
Date Submitted
2008