Reverse Genetics Approach to Identify Genes Required for Glandular Trichome Development in Medicago truncatula

Abstract

Trichome development has been used as an important model system to study plant development. The traditional method for doing this has involved inducing mutations in simple plants such as Arabidopsis thaliana and screening for mutant phenotypes. Once these phenotypes are identified several laboratory techniques can be used to determine in which genes the mutations occurred. We used a reverse approach to identifying these genes by starting with a list of the genes most commonly expressed only in the glandular trichomes of Medicago truncatula. We used PCR amplification to isolate these DNA fragments. We then cloned the fragments into the pCR8 and then pHG8 vectors. When agrobacteria are transformed with the pHG8 vector and grown on a Medicago truncatula tissue culture a double-stranded RNA copy of the vector becomes present in the plant cell. The machinery of the cell recognizes the double-stranded RNA construct and inhibits the expression of any regions of the genome that have the same nucleotide sequence. This effectively knocks out the gene we inserted into the plasmid. The tissue cultures can then be screened for a mutant phenotype in their trichome development. If a mutant phenotype is observed it suggests that the gene we started with has a role in the development of glandular trichomes in Medicago truncatula. By finding these genes we can then compare them to the many known genes that are responsible for trichome development in Arabidopsis thaliana; we can address the question of whether trichome development is controlled by the same genetic pathways throughout the plant kingdom.