Greenhouse Injections
METHOD A:
Wheat seedlings are transplanted after vernalization in October and are maintained in single
plant pots on a greenhouse bench. Pots are watered daily. When an individual head reaches
anthesis this head is ready to be injected. To check for anthesis, open the middle floret of wheat
heads that have elongated out of the boot. It is preferable to inject a little early than too late. If
pollen has shed it is too late to inject. You don’t have to wait and shouldn’t wait until you see
the anthers outside of the floret.
When heads have reached anthesis they are pulled from their position on the bench and
assembled on one main bench to be tagged and injected. It is nice to have the pots in order at
this time but not absolutely necessary. It is also useful to print out labels with pot and rep on
them. Each pot gets assigned a rep number based on the previous number of injections of that
pot. Each head is tagged with the pot number, rep number, and date of injection. The central
spikelet to be injected is marked with a sharpie. The pot number and rep number of each pot to
be injected on that day is also recorded in the greenhouse data book so you know what to read
and when. Pots are injected with a 3 microliter spore suspension by using a pipetman. The spore
suspension is calibrated to 50,000 spores per milliliter, which is approximately 150 spores per 3
microliter injection (only 1 spore is needed for infection). Carefully open the marked spikelet
being cautious not to damage it or rip it off. Holding the spikelet open gently dispense the 3
microliter spore suspension into the spikelet, taking care not to damage the ovary.
The spore suspension should be made fresh weekly. Transport the suspension from the lab to the
greenhouse in a cooler. Heat will cause the suspension to deteriorate. Also, the fungal spores
will settle to the bottom of the vial over time. BE SURE TO SHAKE THE VIAL OF
SUSPENSION PERIODICALLY- after every 20th injection.
After the injections have been made and all information is recorded properly in the data book,
the pots are moved into a mist chamber for three consecutive nights. If you put pots in on
Monday afternoon they would come out on Thursday afternoon. Try and keep on a schedule by
using a calendar at the greenhouse. This will be very useful in the thick of things when on a
single day some pots will need to be injected while other groups will need to be taken out of the
chamber and when still other groups are ready to be read. Once pots are removed from the mist
chamber they are returned to the greenhouse bench. Pots are kept in order by injection dates to
make it easier when reading.
Pots are read 21 days after injection. Total spikelet counts can be made at the time of injection
or at 21 days post injection. When reading, simply count the total spikelets and infected
spikelets per head and record this in the book. Be sure you are reading the correct pot and rep.
Everything should match up with the data book (date of injection, pot, rep).
METHOD B:
Once plants come out of vernalizer transplant into greenhouse. Knowing how the inoculation and
subsequent irrigation will take place will allow for decisions to be made transplanting the
seedlings. If placing the plants in the mist, use pots or cones. The one drawback on using cones
is the movement of individual cones into the mist tent instead of the cone tray holder. That could
be a large task when many entries flower on the same day. A better option would be using the
pots since they are easier to manipulate than cones. Cones are useful when using overhead
irrigation. The cones are able to be water when growing then removed after several days of
irrigation. The cones would still be moved, but the cones would be organized by the day
inoculated. Cones are best used when placed in a float pond but movement can be difficult as
many roots grow together and become entangled. One way to lessen this problem is to allow
space between cones by leaving every other row empty.
Remember when planting always plant resistant and susceptible checks. Variability in genotype
performance in the greenhouse is high compared to the field environment.
When transplanting make sure no more than ¾ of the cones are filled with soil before beginning.
Put stake into cones before transplanting to limit mistakes. Place seedling in soil and press down
the roots, then lightly cover the seed with soil. If using pots make sure there is about 1” of soil
below the pot lip. Again, place stake into soil before beginning. Place seed into soil and press
down roots. Cover lightly with soil. For both containers soak soil. This is especially important
for the cones. Water cones for several days until the roots can develop. It is also important to use
weather data recorders to monitor conditions. In our greenhouse, the back house can have
problems with temperature regulation, which affects scab inoculation and disease development.
Instead of inoculating and putting the pots in the mist tent for 3 days, an alternative method is to
inoculate the plants then put on a glycine bag for 2 days. After 2 days remove the bag and read
symptom development after 21 days. The number of spores used in injection is the same (50,000
spores/ml). Many universities use this method for greenhouse screenings. We have tried the
method and found it work for us as well. This method allows for cones to be used as well as pots.