Plant materials, bacterial strains and vectors, and fungal inoculum
Seeds of Pisum sativum L. cv. Sparkle and cv. Frisson, and seeds of its mutants P5 and P56 [generous gift from Gérard Duc (Dijon, France)], were surface-sterilized, imbibed, and planted in sterile vermiculite to germinate (see below for specifics). Unless otherwise mentioned, P. sativum plants were grown under incandescent (OGE 600 hour, 60 - 120 W, 120 Volts, General Electric) and cool white fluorescent lights (Watt-Miser GE, F96TIZ-CW-HO-WM) in a growth-room with a 23°C/18°C, 16 h/8 h light/dark regime. The total light intensity received by the plants was 280 μmol·m-2·s-1.
Agrobacterium rhizogenes AR12 and AR1193 [ and , respectively] containing SYM10 were cultured in 100 mL of Luria Bertani (LB) broth with 100 μg/mL rifampicin and 100 μg/mL spectinomycin (all from Bioshop, Burlington, ON). As a control, AR1193 with no SYM10 construct was grown in LB with 100 μg/mL rifampicin. Cultures were grown at 28°C for 24 hours on an orbital shaker (New Brunswick Scientific, Edison, NJ) at 200 rpm until the culture reached an absorbance between 0.6 and 0.8 at 600 nm on a spectrophotometer (Varian Inc., Mississauga, ON). The bacterial culture was then transferred to sterile centrifuge tubes and spun at 6000 × g for 15 min, 4°C. The pellet was re-suspended in ice-cold, 1/4 × Murashige and Skoog basal medium (Sigma-Aldrich M5524, Oakville, ON), pH 5.8, until an optical density of 0.3 at 600 nm was reached.
Rhizobium leguminosarum bv. viciae 128C53K (gift from Dr. S. Smith, Milwaukee, WI) was kept on slants at -20°C and cultured in 20 mL of yeast-mannitol broth, consisting of (g/L) mannitol 10.0, K2HPO4 0.5, MgSO4 0.2, NaCl 0.1, yeast extract 0.4 and of pH 6.8. The medium inoculated with a loop-full of stock culture was grown at 25°C in Erlenmeyer flasks on an orbital shaker at 100 rpm. Cells were grown to late log phase (48 hours) at which time the viable cell density was 1.2 × 106 colony forming units (CFUs)/mL.
Phytagel™media plugs containing spores of the AM fungus Glomus irregulare (DAOM 197919) were obtained from Agriculture and Agri-Food Canada's Glomeromycota in vitro collection. The fungus was propagated using root-organ cultures  of transformed carrot (Daucus carota) roots (Culture Collection, Mycothéque de l'Université Catholique de Louvain, Belgium).
Figures 1 and 2 highlight the important steps of the protocol with which we obtained the highest transformation efficiencies.
1| Surface-sterilize seeds in 8% bleach for 5 minutes to eliminate microbial and fungal contamination during germination. Wash seeds in three rinses of sterile water for 1 minute each and store them in sterile water in darkness for 12 to 15 hours to imbibe.
2| Plant in vermiculite imbibed seeds at a depth of 1-2 cm and let grow in the dark until the seedlings are ~ 8 cm tall [about 7 days after planting (DAP)].
NOTE: The dark treatment promotes etiolation and thus facilitates the shoot excision at a later step.
3| Set plants into a controlled growth-room for 3 days to provide time for the leaves to develop (Figure 2A).
4| Cut Fibrgro® cubes (Homegrown Hydroponics Inc., Breslau, ON) into smaller cubes (~ 2.5 cm3). Use a dissecting probe to make a small starter hole, a quarter to half way through the cube, into which the stem of the plant will be inserted (step 7). Sterilize the cubes (pre-vacuum cycle for 20 min) in autoclave.
NOTE: Without this hole the stems of the plants broke easily when inserted.
5| Prepare the culture of A. rhizogenes 9 DAP as described above.
6| Place 2 to 4 sterile Fibrgro® cubes, with the holes facing up, in 12 cm sterile Petri plates. Inoculate the cubes with the diluted A. rhizogenes culture until complete saturation; 4 to 7 mL of inoculum is usually sufficient per cube (Figure 2B).
7| Cut off the shoot of 10-day old plants above node 2 with a clean razor blade and place their stem gently in the hole of the Fibrgro® cube (Figure 2C).
NOTE: When the distance between node 2 and 3 is too short, the plant is cut above node 1 instead so that the leaves are never in contact with the inoculated cube.
8| Place the Petri plates containing the plant/cubes in a plastic tray with drainage holes and cover with a transparent dome with ventilation holes closed (Homegrown Hydroponics Inc., Breslau, ON). Leave tray on lab bench for 24 hours (temperature of 21°C and light intensity of about 10 μmol·m-2·s-1). Remove the lid on the following day and leave the plants on the bench for an additional 24 hours to promote wilting of the shoots (Figure 2D).
NOTE: The wilting period is required for a callus to develop as it likely draws the inoculum into the plant ).
9| Add sterile water to the Petri plates when the stems are wilted, and replace the dome for an additional 24 hours (Figure 2E). Place the plastic tray into a metal watering tray and set the plants in the growth room (Figure 2F). Add water to the metal tray to cover the bottom of the plastic tray.
NOTE: Keep the plastic dome snug to the plastic tray and the ventilation holes closed to enhance humidity, which increases transgenic yield .
10| Dissect carefully with a pair of tweezers the Fibrgro® cube away from the roots when the roots are visible (~10DAI) (Figure 2G). Excise all roots with a sterile blade.
NOTE: At this stage most of the roots are adventitious and their removal promotes root growth from the callus.
11| Transfer the callus-forming shoot with its epicotyl to a pot filled with sterile vermiculite; tightly pack the vermiculite around the rootless callus so that the shoot is kept upright (Figure 2H).
12| Two days before inoculation, grow a culture of R. leguminosarum as explained above. Inoculate the plants with 5 mL of a 5% bacterial solution 3 days after moving them to vermiculite.
13| Inspect plants 21 DAI and check for nodules.
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 ] 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 . 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 .
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.
Fungal Spot-Inoculation of Pea
The protocol used in the fungal spot inoculation of pea begins with steps 1-9 of the rhizobial spot-inoculation protocol as stated above, except for step 4 where a low-phosphate nutrient solution [as in ] is used instead of a low-nitrogen one.
10| After 5 days of growth in pouches, prepare enough freshly autoclaved Phytagel™ (Sigma P8961) medium [0.3% in minimal medium [23, 49]] so that there is approximately 1 mL of liquid per plant to be inoculated.
NOTE: The medium should be kept warm (60°C) to maintain its liquid form until the time of inoculation.
11| Remove each pouch from the pots; under axenic conditions, choose a location along one or more lateral roots for inoculation to occur. Mark this location on the top of the plastic sheet with a waterproof marker.
12| Lift the top plastic sheet at the bottom end and gently lift the lateral root chosen using tweezers. Apply 1 mL of liquid Phytagel™ to the filter paper of the growth pouch in the desired location below the selected lateral root. Using another set of tweezers, place a media plug containing the fungal inoculum onto the Phytagel™ and lower the root into place over the top of the plug, gently pushing the root into the medium of the plug.
13| Allow a minute or so for the plug to set in place and for the Phytagel-™ to solidify before lowering the plastic sheet. Return the pouches to the pots and cover them with fresh vermiculite. Water the plants when needed; however, be mindful of overwatering which can encourage fungal contamination of the pouches. Harvest plants at the desired time.
14| When harvesting the plants, remove the pouch from pots with special care so that none of the roots within the pouch are disturbed. Note the location of the plug and make cuts in the plastic to expose the root system. Remove the fragment of lateral root that was placed on top of the plug using a scalpel blade. Clear the root fragment and stain it with ink-vinegar  in order to observe fungal infection.