M. truncatula Seedling Transformation
Germinate seeds on sterile
plates.
Prepare sterile 1% agar petri dishes in advance (10 g agar per 1L water; use ~ 10 ml per plate).
Transfer imbibed,
cold-treated seeds to the surface of a 1% agar plate using a sterile spatula
or scalpel, up to 50 seeds per plate.
Invert plates and incubate
at 20-22oC in the dark for 20-24 hours. Keep plates horizontal at all times so that roots will
grow straight downwards.
These plates can be used up
to a month after preparation.
Use the tapered blade on
the end of the spatula; dip in alcohol and flame to sterilize. Scoop seeds out of tube and onto agar
surface. Separate the seeds and
move them so that they are evenly spaced. Plates should be wrapped in foil
and placed in a drawer or a growth chamber at 21oC. Uneven germination may result if the
temperature drops overnight, as can happen in a room. The newly emerged roots should almost
touch the petri dish lid, around 1 cm is the best length for ease of
manipulation. Seedlings can be refrigerated (up to 24 hours) to inhibit
growth until transformation you are ready to proceed with
transformation.
Grow A. rhizogenes lines for plant transformation.
Heavily streak fresh A.
rhizogenes containing the pHellsgate
8 (pHG8) construct on YEP plates containing spectinomycin (100 mg/ml) and streptomycin (50 mg/ml).
Incubate the plates overnight at 25oC (room temperature us
OK).
Use empty pHG8 or pHG8
containing human myosin or GUS sequences as negative controls. Plates can be
sectioned into fourths or sixths to allow streaking of Agrobacterium strains.
Plates are heavily streaked to obtain confluent or dense growth;
individual colonies are not appropriate for the transformation protocol. This can be accomplished using the
wide end of a sterile flat toothpick.
Overnight incubation is usually sufficient when started from recently
grown plate. Longer incubation
and/or incubation at 28-30oC may be necessary if started from
older plate. Although healthy,
actively growing cells are needed for transformation, the plate can be stored
for up to 2 days at 4oC after overnight growth.
Spectinomycin selects for
presence of pHG8 and streptomycin maintains A rhizogenes ARqua1.
Remove tips of M.
truncatula roots
Use sterile scalpels and
forceps in a laminar flow hood for this and subsequent steps.
Place 8-20 drops of sterile
deionized H2O in separate spots on internal surface of sterile
Petri dish. Using sterile
forceps, pick germinated seedlings from plate (see above) by the seed coat or
cotyledons and place a root tip (about 1 cm) in each drop, keeping the cotyledons
outside of the water drop.
Cut the root about 3 mm
from the tip. Be sure that the cut site and root remain wet.
Hood should be wiped down
with alcohol and/or UV-light treated prior to work. Dip spatula and forceps into ethanol and burn off alcohol
to surface sterilize. Use new,
sterile scalpel blades; Repeat resterilization of the blade dulls its edge.
Allow implements to cool before using them to protect plants from exposure to
excessive heat.
Alternatively, place one
large water puddle in center of dish and place seedlings in a circle with
roots pointed towards center of puddle.
Roots of seedlings must not
be allowed to dry out – keep plates containing seedlings covered and
wrapped when not in use. Have at
least two forceps sterilized so that one has a chance to cool while other is
being used.
Cut needs to be in the
elongation zone to ensure that apical meristem has been removed.
Inoculate wound site with A.
rhizogenes
Hold seedling by the
cotyledon using sterile forceps.
Rub the end of the cut root through the overnight A. rhizogenes culture.
Bacteria should be visible
on the end of the root. We have
tried a variety of methods to introduce bacteria to the root (cell
suspension, etc.) and found this to be the most effective. Avoid contaminating forceps with
bacteria. Resterilize the forceps before going back to get additional
seedlings if there is any concern that this has occured. Always resterilize forceps before
inoculating roots with a new Agrobacterium line.
Grow inoculated plants on
modified Fahraeus media.
Modified Fahraeus media
containing kanamycin (27.5 mg/ml) is poured in square petri plates (100 mm x 100
mm x 15mm; Fisher #0876711A or equivalent) at an angle such that about
two-thirds of the plate is covered with agar.
Using sterile scalpel or
spatula, draw or score 8-10 evenly spaced ÒnotchesÓ several cmÕs long in the agar to hold 8-10
seedlings inoculated with same A. rhizogenes line.
Place the seedlingÕs root along the notch. Once all seedlings are placed on agar, use forceps to push
root into notch, pulling seedling up to position cotyledons at top
space. Label plates with a
big Sharpie (extra fine Sharpie ink fades under lights).
Wrap plate edges in
Parafilm and poke holes at corners to allow gas exchange. Alternatively, wrap several plates
together in plastic wrap and use a rubber band to hold the plates together.
Incubate plates vertically
in 21oC growth chamber with a 16:8 hour light:dark cycle. Transgenic roots should start to
appear at wounding site between 7 and 10 days. Check for Òswollen bumpÓ at cut tip which indicates
callus-like tissue developing.
See separate file ÒModified
Fahraeus Plates for Medicago Transformation.Ó
Alternatively, 1/2x GamborgÕs B-5 (see below) plates can be used,
although transformation efficiency may be reduced. These plates should be made up no more than 1-2 days
before use since the effective kanamycin concentration is critical. Older
plates will allow more growth of nontransgenic roots. Kanamycin selects for
transformed roots as the nptII
gene is located in the T-DNA region of pHG8 and is transferred to plant
genome.
Note: 0.8% agar used here
allows easier transfer of plants at subsequent steps, but may allow roots to
grow into the agar. Higher concentrations
of agar (e.g. 2.5% ) will force root growth on agar surface, but will make it
impossible to remove any agar sticking to the root without damaging the
seedling.
Can have second row of
plants staggered with first, allowing up to 18 seedlings per plate.
Want 16-20 seedlings
transformed with each clone. Can
use pHG8::GUSi as a negative control for root phenotype and pHG8::CDPK1i as a
positive control for root phenotype.
If plastic wrap is used
small slits through the wrap of about 1 inch in length can be made at the
upper corners of each plate.
Fluorescent bulbs in growth
chamber are Philips F96T12/CW/VHO, 215 watt, 1500. Alternative incubation is at 14oC for 24 hours and then at
24oC for 12-14 days under light.