Lab Report for AGRO/BIOTC460
LAB REPORT 5
NAME: M. Azrul Akmal Saharudin
SUBMISSION DATE: May 3rd , 2011
Transgene Detection by Phenotypic Screening and Molecular Diagnosis Test.
The introduction of Bar gene into maize and using phenotypic screening and molecular diagnosis test to detect the expression of the gene are one of commonly use method for transgene detection. We were using maize plant that was transformed with Bar gene using gene gun. For phenotypic screening, BASTA(Liberty) herbicide was applied to the plant to detect whether the plant show the phenotype of BASTA resistant or not. For molecular diagnosis test, we have used polymerase chain reaction(PCR) and ...view middle of the document...
This is possible due to the outcome of functional genomics research.
In plant transformation, a selectable marker called the Bar gene is commonly used. This selectable marker is very reliable and essential for the recovery of transgenic plants. When a plant express the Bar gene, they will become resistant to BASTA, a non-selective herbicide which can be toxic to most non-transgenic plants2.
Having a very unique mode of action, BASTA is applied on the leaves of the plant. Nitrate is absorbed by the plant and broken down to ammonium ions during the processes of plant metabolism3. When the ammonium ions combine with the glutamate in the cells, glutamine is formed or produced3. This reaction is crucial for photosynthesis. The presence of glutamine synthetase transforms ammonium to glutamate and glutamine. The action of glutamine synthetase is disrupted by the glufosinate-ammonium which will prevent the reaction for photosynthesis. Therefore, yellow color produced due to build up levels of ammonia and the slow production of glutamine3.
This experiment shows that the Bar gene is co-transformed in the maize plants and the phenotype is observed by the phenotype screening procedure. The molecular diagnosis is conducted by using the PCR and gel electrophoresis. Part of plant that showed symptom of dried, brown color or dead after BASTA is applied is considered susceptible and not resistant to the herbicide. Therefore it is called a negative transgenic plant. On the other hand, part of plant that shows no symptom when BASTA is applied is resistant to herbicide and called a positive transgenic plant.
MATERIALS AND METHOD.
Analysis of the putative transformants using herbicide (BASTA) test.
Weâ€™ve been assigned with six transgenic plants and six control plant in this experiment. Each plant was numbered from 1-6. Using a marker pen, small area on each plant was marked and that area was painted with 1% BASTA. This process is called the BASTA treatment. The phenotype was observed after 7 days.
After seven days, all plants that were treated with BASTA were obtained and observed for any development of symptoms. Then, the symptoms on the transgenic plants were compared with those on the negative control (B73 plants). From this we can determined which of the plants were resistant and sensitive to BASTA herbicide. The phenotype of each plant was recorded on worksheet. Two healthy BASTA resistant plants were selected for further analysis such as DNA extraction and PCR amplification of the bar gene.
Genomic DNA isolation using DNAzol direct.
The two labeled stakes provided by TA were placed beside the plants that were selected for analysis. A hole-punch was used to remove two disks of tissue from each plant and the disks of tissue from each plant were placed in the appropriately labeled 1.5 ml tubes. The tubes were labeled with the number on the stake and student name. The tubes were placed on the rack and the tissue...