Reference no: EM132622207

BIO3318 Plant Microbe Interactions - University of Southern Queensland

Report for Plant Microbe Interactions will be focussed on arbuscular mycorrhizal colonisation in plants.

Given the timing of the residential school, you will be provided with data with which to write this report. You will conduct the actual exercise of harvesting plants and processing roots at the residential school so that you gain experience of this important technique.

EXERCISE - DEVELOPMENT OF ARBUSCULAR MYCORRHIZAS IN PLANTS

1.1 Growth of plants (to be done for you)

1. Soil was collected from near the surface and from underneath vegetation to ensure that there will be plenty of AM spores in it. Soil was sieved to produce a fine consistency for planting and to minimise heterogeneity caused by stones, pieces of debris or large clods of soil. The soils used included red soil (lateritic krasnozem; Toowoomba) and sandy soil (Flagstone Creek-Preston). Two different plant hosts will be used namely oats (Avena sativa) and mungbean (Vigna radiata).

2. Twelve 10cm pots were filled with soil and eight seeds planted in each as appropriate, that is, 2 soils x 2 hosts x 3 reps x 1 sampling time = 12 pots with at least 8 seeds in each.

1.2 Harvesting plants and processing roots
1. Samples are to be taken at 12 weeks after planting. At this time, 3 pots from each treatment are taken (12 pots) and the soil washed from the root system. Then the root systems are placed in the empty pots containing the appropriate treatment label and taken up to the laboratory. The roots are then cleared and examined for mycorrhizal colonisation. Four slides per pot are prepared with 10 root segments per slide.

2. Select pieces of root system about 5cm long and no greater than 1mm thick. Wash gently under the tap taking care not to damage the cortex. Cut into 1cm segments. Four slides will be prepared, each with 10x 1 cm segments for each pot. At least 40 segments are needed for each pot (it is a good idea to prepare some extra ones to allow for losses during clearing and staining).

3. Place the root segments into a separate labelled microfuge tube and add enough 10% KOH to cover the root segments. Place the tubes into a 90°C hotplate for 20 minutes. Remove the tubes and decant off the KOH. Rinse once with tap water.

4. Rinse with 0.1M HCl and then distilled water. Cover root segments in the microfuge tube with the stain, 0.5% trypan blue in lactoglycerol (1:1:1 lactic acid:glycerol:water) and incubate at 95°C for 5 minutes.

5. Remove tubes from the incubator, decant stain from segments and transfer them to a watch glass. Add clear 50% glycerol-water and shake segments gently to remove excess stain.

6. Mount ten segments per slide in clear 50% glycerol-water and place a long coverslip over the segments. Squash gently and examine under the microscope. Record the number of colonised root segments per slide. Take photomicrographs of clear views of AM structures within roots eg. arbuscules, intercellular hyphae.

7. Pool all the class results and compare the data obtained for the two species grown in each of the soils for twelve weeks. Present this data in an appropriate table and perform suitable statistical analyses to help define any differences in the extent of AM development in the different treatments. Discuss the class results in terms of what is known about the nature of AM associations.

Attachment:- Plant Microbe Interactions.rar