The two most common reporters utilized in plant biology, (and the two wewill use in this lab), are the bacterial enzyme -glucuronidase (GUS) and the jellyfishprotein Green Florescent Protein (GFP). A reporter gene can be fused to a gene ofinterest (or just to the gene of interest’s promoter) to aid investigation of that genewe will use a reporter gene to analyze the response of Arabidopsis seedlings to the plant hormone Auxin

Auxins are a class of plant growth hormones that play roles in almost all aspects of plant development and growth

and the most commonly used is the highly stabile 2,4-Dichloro-phenoxyacetic acid (2,4-D).The DR5 promoter is an artificially created promoter that was made by modifying a fewbases in a naturally occurring AuxRE. AuxREs contain the Auxin-responsive sequenceelement TGTCTC, and DR5 contains two of these in a tandem repeat. The DR5promoter has greater activity and Auxin responsiveness than naturally occurringAuxREs, and has been heavily utilized as a marker for the presence of Auxin in planttissues. We will use the DR5 promoter to drive the expression of two differentcommonly used markers in plant research: GUS and GFP-glucuronidase (GUS) is an enzyme from bacteria that catalyzes the breakdown ofCarbohydrates.  β-glucuronidase GFP can be used to label genes by attaching it to a gene or promoter of interest tomonitor gene expression. If the gene is expressed, then the GFP will also be expressedand the organism (or tissue) will glow green in blue light. The expression of GFP canalso be examined by western blottingGFP is visualized by itsgreen florescence under UV or blue light. Some benefits of GFP are that it can bevisualized in living tissue and can remain attached to your protein of interest, revealingthe sub-cellular localization of your protein.

GUS staining in the transition zone (zone 2) in wildtype but not in aux1-T root tips. The minor effects of ethylene treatment on the DR5:GUS maximum in aux1-T root apexes were consistent with DR5:GUS expression in aux1-7 root apexes unaffected by the ethylene biosynthesis precursor 1-aminocyclopropane-1-carboxylic acid (Stepanova et  al., 2007). Unexpectedly, GUS staining was not observed or was extremely weak in aux1rcr1 root apexes, regardless of ethylene treatmentDR5:GUS expression is elevated in the root tip.Of note, ethylene inhibited root elongation, and the region below the mature zone was largely shortened compared with no-ethylene treatmentAs a result, root cells grow faster with lower than with higher auxin concentrations, and differential cell growth is facilitated. The differential root cell growth facilitates a curvature formation that re-orients the root growth towards gravity (GUS and GFP are competing DNA markers used in many studies, these reporter genes can also be used to detect multiple integration and unstable transgenes within a transformant (Filipecki and Malepszy, 2006). They work by attaching to a fragment of DNA of interest,The results of GUS-PCR was expected to be present in lanes 1 and 3, therefore a band appeared at 401bp. These expected bands were shown in the PCR output. In lane 1 there was also a band of smaller bp which indicates DNA fragments of a smaller size were also present in this sample. Identically GFP-PCR in the second set of wells, the GFP gene was expected to be seen in lanes 6 and 7, therefore a band was produced at 714bp. The expected band was shown in lane 7 of the PCR output but very faint in lane 6. Light microscope examination of blue-coloured transgenic hairy roots suggested that they expressing the blue stain constitutively confirming their transformation to express 35S GUS geneMicroscopic examination of GFP stained tomato hairy roots were visualised under a UV where by GFP expressing cells will fluoresce. These transgenic hairy roots showed constitutive green fluorescence proved their successful transformation (Fig. 2D). These results demonstrated the incidence of transformation with 35S GFP

Intro: In response to the threats caused by phytopathogens and plant disease, this investigation looks at the potential of genetic modification technology as a strategy to protect plant crops globally. Specifically, it will explore the usefulness of GUS (β-Glucuronidase) and GFP (Green fluorescent protein) as reporter genes in Agrobacterium-mediated transformation of tomato (Lycopersicum esculentum L.) and potato (Solanum tuberosum L.) hairy roots. Transformation of Agrobacterium with GUS and GFP will be analysed using PCR to evaluate the efficacy of this vector. Compiling knowledge of genetic markers and transformation techniques will allow for high yield and efficient crop plant transformations.

GFP transgenic potato roots did not show as much green fluorescence as tomato roots, and some regions of roots showed up red colour indicating the absence of transformation in those cells. However, microscopic observations indicated that most of potato roots had successfully transformedGFP shows low toxicity, no interference with normal cellular activities, and is easy to assayGFP has been used extensively in plant systems, in localization studies and as a screenable marker for gene transfer