Team:UNSW Australia/Lab/Plants

Navigation

Home

Project

Description Design Experimental Protocols Lab Overview Cloning Protein Production Assembly FRET Enzyme Assays Plants Results Interlab

Modelling

Overview Enzyme Kinetics & Diffusion Molecular Dynamics

Parts

Overview Basic Part Improved Part Software

Human Practices

Overview Commercialisation Education & Engagement Law & Regulation Integration

People

Team Collaborations Attributions Experts

Documentation

Notebook Safety

Judging

Medals Judging Form

© 2018 UNSW Australia iGEM

Plants

Overview

Auxins are plant hormones which are involved in the regulation of plant growth and development. The biosynthesis of the auxin indole-3-aecetic acid (IAA) was used as a test pathway for our scaffold system, thus a protocol was developed to investigate the functionality of biosynthetically produced IAA in a plant growth assay compared to commercially available IAA. Arabidopsis thaliana seedlings were grown in media containing varying concentrations of IAA, to observe its effect on growth and development. Reduced primary root growth and high lateral root number was observed at higher concentrations of IAA, exhibiting phenotypes common with a stress induced morphology. These results, in addition to comments made by PlantBank researchers who had found that addition of exogenous IAA showed no benefit, indicated that IAA synthesis was not an appropriate pathway to purse for commercialisation with our scaffold. Despite this, the UNSW iGEM team was able to develop a protocol that could be used in the future to observe the functionality of the IAA product produced with our scaffold compared with commercially available IAA.

Introduction

Auxins are plant hormones (phytohormones) which are involved in the regulation of plant growth and development ¹. Indole acetic acid (IAA) is the most widely characterised auxin, and the primary auxin found in most plant species². It is associated with many physiological processes in plants including cell elongation, cell division, tropisms to light and gravity, and root initiation and development¹.

Plants can naturally synthesise auxins, and the biosynthesis of IAA in A. thaliana occurs predominantly through a tryptophan dependent pathway¹3. Bacteria can also synthesise IAA through a variety of pathways, and often utilise IAA to interact with and colonise plants⁴. To test our scaffold system with a proof of concept, we chose the two-step indole-3-acetamide pathway for IAA biosynthesis, the best characterised IAA biosynthetic pathway in bacteria⁴.

Although plants can synthesise IAA de novo, application of exogenous phytohormones is common in industry to stimulate specific responses⁵. Thus auxins, and phytohormones more broadly, have commercial significance. In order to understand the commercial significance of IAA we consulted with real world users of auxins (link to journal page) and conducted experiments with the model plant A. thaliana to observe the effect of IAA on plant development.

Aims

To observe the effect of the auxin indole-3-aecetic acid on Arabidopsis thaliana growth and development.

Methods

Wild type A. thaliana seeds were surface sterilised in a solution of 2.5% bleach and Tween for 10 minutes, and subsequently washed with sterile water. Seeds were then placed in a sterile solution of water and 1% sucrose and left to stratify at 4°C for two days. Seed germination occurred in the 1% sucrose solution at room temperature.

Germinated four-day old seedlings were transferred to square MS agar plates supplemented with 1% sucrose, containing IAA concentrations of 0 µM, 1 µM, 10 µM and 100 µM. Plates at each concentration of IAA were prepared in triplicate with approximately five seedlings placed in a line 2 cm from the edge of the plate. The plates were partially covered in foil, leaving only the top 2 cm exposed in order to limit IAA photodegradation and simulate the natural growth conditions of higher plants whereby only shoots are exposed to light⁷. Plates were stored upright, illuminated and incubated at room temperature for 20 days. Primary root length and lateral root numbers were measured at four, six, eleven and twenty days following the initial seedling transplantation.

Flowchart of plants methods uploaded on google drive

Figure 1: Summary of methods used for plant growth assay

Detailed protocols can be found on our experiments page.

Results

Primary root growth was inhibited in IAA concentrations from 1 to 100 µM, with the control 0 µM specimens displaying the longest mean primary root length (Figure 2).

Figure 2: Effect of IAA concentration on A. thaliana seedling primary root length.

^a Time elapsed since seedling transplanted to MS agar plate containing IAA.

Conversely, lateral root count was highest in specimens grown in the media containing the highest, 100 µM concentration of IAA (Figure 3).

Figure 3: Effect of IAA concentration on A. thaliana lateral root count.

^a Time elapsed since seedling transplanted to MS agar plate containing IAA.

Generally, plant growth (including shoots and leaf growth) was inhibited as the concentration of IAA was increased (Figure 4) ).

Figure 4: Arabidopsis thaliana seedlings growth on MS agar with varying concentrations of IAA. Seedling after initial transplant to MS agar plate with IAA concentration A: 0 µM B: 1 µM C: 10 µM D: 100 µM. Growth after 20 days incubation, illuminated at room temperature on MS agar with IAA concentration E: 0 µM F: 1 µM G: 10 µM H: 100 µM.

References