Rhizobial Spot-Inoculation of Pea
Figure 4 highlights the important steps in the protocol for successful spot-inoculation.
1| As 1| above.
2| Plant seeds in 7.5 cm pots filled with sterile vermiculite and let grow for 3 days (Figure 4A).
NOTE: Growing seeds in vermiculite is crucial to obtain plants with straight roots for easy transfer to pouches. It is important to allow the pea seedlings to develop for 3 days as the length of the radicle is optimal at this time.
3| Three days later, prepare to transfer one seedling per germination pouch (cyg™ seed germination pouch, Mega International, St-Paul, MN). Make a hole with a clean razor blade in the top centre of the pouch filter paper, so that the root enters the pouch but the cotyledons do not fall through the hole.
4| Wet the pouch with sterile low-nitrogen nutrient solution [as in [44]] until the entire surface of the filter paper has absorbed liquid (~ 7 mL).
NOTE: Micro-nutrients such as boron are required for nodules to develop [46]. It is essential to supply these nutrients instead of water alone as the seedling will not have access to them in a soil-free environment.
5| Remove gently the seedlings from the pots, and the seed-coat from the seedling, and place the primary root through the hole previously made (Figure 4B). The root must be in contact with the moist filter paper of the growth pouch.
6| Tape the top of the pouch closed, leaving a hole for the shoot to emerge (Figure 4B). Taping the pouch helps to maintain a moist inner environment preventing the roots from drying out. It also prevents vermiculite from entering the pouch (see below).
7| Cut a piece of the growth pouch 2.5 cm from its bottom with a pair of scissors (Figure 4B). The filter paper within the pouch will thus be in contact with the moist vermiculite (see below); this will ensure a constant supply of water to the developing roots while at the same time prevent any water-logging. The sides of the pouch can be trimmed for a better fitting when the pouches are inserted into the pots.
8| Place the growth pouches with the seedlings in 15 cm diameter pots (Figure 4C) and fill those with sterile vermiculite to cover completely the pouches (Figure 4D).
NOTE: Covering the pouch with vermiculite prevents light from reaching the roots; this is important as exposure to light inhibits pea nodulation [47].
9| Moisten the vermiculite with water and place the pots in trays in a controlled growth-room. Water the plants by adding water to the tray. The vermiculite maintains a humid environment for the pouch and filter paper.
10| Two days before inoculation, grow a rhizobial culture as above.
NOTE: Do not water the plants during this time to reduce the amount of residual water in the pouch. If the pouches are too wet, the inoculum drop will not remain on the root. It is of prime importance to time the earlier watering so that at this stage the pouches are only damp.
11| After 5 days of growth in the pouches, lateral roots will be of an optimal length for spot-inoculation (Figure 4E). Remove the pouches from the pots and find under the dissecting microscope the zone that is the most susceptible to infection, i.e., the region where roots hairs are starting to emerge from the root. This location is usually 0.5-1 cm away from the root tip.
NOTE: Condensation on the inside of the plastic pouch can impede the viewing of roots. Gently lift the top layer of the pouch from the bottom and use a sterile filter paper to remove the water droplets.
12| Mark the zone of the most susceptibility on the outside of the pouch with a water-proof marker (Figure 4E). Do not damage the root when applying the marker; if needed, hold the top surface of the pouch away from the root and apply the mark to the desired location.
13| Lift the top plastic sheet at the bottom end of the pouch and apply with a micro-pipette onto the root a 0.5 μL drop of rhizobial suspension adjusted to the viable cell density of 6.4 × 104 CFUs/mL.
NOTE: Do not place the top of the pouch back onto the root right away; let the drop sink in the root and paper before closing the pouch.
14| Return the pouches to the pots and cover with vermiculite (Figure 4F). Water the plants when needed; a good indication to do so is given when the colour of the pouch changes as it starts to dry. Harvest at the desired time (Figure 4G).
15| In our experiments carried out under the described conditions, we observed nodulation events, such as divisions of pericycle cells, as early as 24 hours after spot-inoculation; pink nodules are seen at 10 DAI.