Protocol for nodulation assay in pouches

1. Sterilize pouches by placing them between wet paper towels then wrapping them in aluminum foil wrap. If you do not autoclave them moist, then they become quite brittle.
2. Sterilize soybean seeds, and germinate 2-3 days on wet paper or water agar. Place 3-5 seedlings per pouch; place seeds in the trough formed in the paper wick (there is a germination paper inside of the pouch and there are holes within the trough). For soybean, given the size of their roots, it helps to use a sterile needle or spatula to make holes in the paper trough for the roots. Note it is useful to select the seedlings so that they have straight roots…just makes it easier to insert them and they tend to grow better in the pouches. We plant seedlings since it allows us to better select the plants and also to eliminate any that may have fungus, etc. Fungus is one of the primary problems you have in pouch grown plants. If you like, you can just plant the seeds in the paper trough and this can work fine but this gives you less of an opportunity to select the best seedlings.

If you do use seeds, then we usually put 5 seeds per pouch as at least 1 of them will not germinate properly

3. Add 8-10 ml sterile H₂O or ½ strength plant nutrient solution(c/o nitrogen) to the pouch, wrap it in alufoil (to keep in dark) and place them into growth chamber (soybean conditions) for 3 days/dark (if you use seeds in step 2) or straight into the light, if you use seedlings. When we do a lot of these, then we have some old 45 rpm record racks (probably impossible to find now) and place one pouch in each slot with cardboard on each side. This eliminates the need for the alufoil and keeps the roots dark. Again, for larger scale use, we also use sterile soda straws (the ones you buy individually wrapped are supposedly sterile) and place one down one side of each pouch. This allows you to water the plants from the bottom up without removing them from the record racks. Important to use this bottom up approach to keep from washing contamination into the pouches by watering from the top.
4. Inoculate 3-days old seedlings, with rhizobia of OD₆₀₀~ 0.05-0.1 by spraying or pipetting. We make every attempt to make sure that the inoculant is applied to the roots. Note if you use seedlings then inoculation can be done at the time of sowing, otherwise, when the seeds have germinated.

Note: if you used seeds, not seedlings, then remove seedlings that are not properly germinated or seem to have fungi. Also, if the root is not going towards inside of the pouch, make them fit into hole (within the trough of the paper wick) and ensure that the roots are facing the bottom of the pouch.

5. Cover the pouch (downwards from the troughto ensure that the roots are covered and are in dark). Place the pouches into pouch-rack (paper racks, come with the pouches) or, in our case, the record racks.
6. Never let the paper inside the pouch get dry but it is also important not to over water. You can water with either dH₂O or with ½ plant nutrient solution, sometimes we alternate just to be sure we do not get salt buildup. Normally, plants will grow nicely in the pouches from 14-21 days without any problem but usually tend to yellow if you extend beyond this period (likely associated with drop in Ph). However, we normally grow plants to 28 days in pouches. Depending on overall conditions, you can see nodule formation within 12 days or so, sometimes sooner, sometimes a bit later.

The biggest issues with the pouches is that the pH will normally drop to quite low unless you make efforts to prevent this. For example, using a well buffered plant nutrient solution for watering. In the olden days, we even added solid calcium carbonate to the pouches, which would dissolve overtime and maintain pH but of course changes other parameters, such as ionic strength. Hence, keep an eye on how damp the plants are and water as needed. Check on the medium every day, medium needs to be replenished approx. every other day but more often as plants get bigger.

7. Important to check the pouches every day, sometimes twice as day, since water usage will vary significantly based on the size of the plants and other growth conditions (chamber humidity, light intensity, temperature, etc).

Pouches – cyg seed germination pouches (Mega-International of Minneapolis)

https://mega-international.com/

Straw – plastic drinking straws sanitary wrapped; 7¾” jumbo

Fahraeus medium (nothing special about this…you can use other nutrient solutions…indeed, something that is better buffered than Fahraeus is probably better)

• Prepare Fahraeus medium(Fahraeus, 1957) from concentrated stock solutions (autoclaved and stored at 4°C as follows):
• 5 mM MgSO4·H₂O
• 7 mM KH₂PO₄
• 8 mM Na₂HPO₄·2H₂O
• 50 μM Fe-EDTA [prepared using FeSO₄and disodium EDTA; alternatively use 20 mM ferric citrate (stored in the dark) to 50 μM final concentration]
• 1 μg/liter MnSO₄
• 1 μg/liter CuSO₄
• 1 μg/liter ZnSO₄
• 1 μg/liter H₃BO₃
• 1 μg/liter Na₂MoO₄
• Milli-Q-purified H₂O up to 1 liter
• Adjust pH to ∼5
• Autoclave