Jun 26, 2022
Version 2
In vitro germination of Austropuccinia psidii urediniospores V.2
- 1School of Life and Environmental Sciences, The University of Sydney, Camperdown NSW 2006, Australia;
- 2School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane City QLD 4072, Australia
Protocol Citation: Alyssa M Martino, Rebecca M Degnan 2022. In vitro germination of Austropuccinia psidii urediniospores . protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4b73jvo5/v2Version created by Alyssa M Martino
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: June 26, 2022
Last Modified: June 26, 2022
Protocol Integer ID: 65406
Abstract
Optimisation of Austropuccinia psidii urediniospore germination for use in RNA extraction and cytogenetics.
Spore collection, desiccation, and storage
Spore collection, desiccation, and storage
2d
2d
Harvest fresh spores from heavily infected leaves by shaking into a paper bag.
Move fresh spores to a glass petri dish with no lid. Transfer petri dish to a desiccator with silica gel beads for 24 - 48 hours to dry the spores.
Sieve spores to remove any plant matter or dirt.
Aliquot spores into Nunc CryoTubes or similar for short or long term storage.
Note
Spores can be frozen at -80ºC or in liquid nitrogen for long-term storage but may reduce germination rates. Use of spores immediately after desiccation is recommended.
Inocula preparation and plating
Inocula preparation and plating
2h
2h
Create 2% water agar plates.
In a 15 mL centrifuge tube, make up inocula to a concentration of approximately 1 mg/mL in sterile distilled water (SDW) with 5ul/mL Tween 20 (0.05%). Mix thoroughly by gently inverting the tube several times.
Note
A haemocytometer can be used to count and confirm spore concentration.
Rehydrate spores in inocula for 30 minutes prior to use.
Pipette 2 uL aliquots of inocula onto agar plate, covering the entire plate with distinct 2 uL droplets and seal the plates with parafilm.
Incubation
Incubation
8h
8h
Germinate in incubator for a minimum of 8 hours in the dark at 18°C and 75% relative humidity.
Determine Germination Percentage
Determine Germination Percentage
1h
1h
For each aliquot, count the number of germinated spores under a dissecting microscope. A germinated spore is considered to be a spore with a germination tube at least double the length of the urediniospore.
Note
Germination rates of approximately 70 - 80% are expected when using fresh spores.