Allow root development for
14-21 days on modified Fahraeus plates with appropriate antibiotic selection
until transgenic roots are 1.5–2 cm in length.
Do
initial phenotype observations during 14-21 days post-transformation or on
GamborgÕs B-5 plates (see below).
Make sure that new root growth is occurring from severed root Òstump.Ó
Remove all but a single root.
Some variations in root development are obvious and are visible using low magnification microscopy.
Note: Some of changes noted
may be due to growth on kanamycin, so observations are also needed on GB-5
(see below).
Transfer one-half of plants
(8-10) to 1/2x GamborgÕs B-5 media for 5-10 days.
At 2-3 weeks
post-transformation (root length 1-2 cm), transfer plants to plates of
GamborgÕs B-5 Basal Salt Mixture (GB-5) without kanamycin. Again, be certain that each plant has
a single root. Add sterile water to plants on plate to loosen roots and keep
moist. Be careful not to let
plants dry out at any point. Use a sterile spatula and forceps to ÒcombÓ away
excess agar – this will eliminate source of kanamycin and allow better
visualization and photography of roots.
Make sure that roots are in direct contact with agar. Recheck for
phenotypes 5-10 days post-transfer.
These plants can now be moved to Turface and assayed for nodulation
and shoot morphology (see below).
GB-5 is from Sigma
(#G5768). Use half of recommended amount listed on label. Bring to pH 7-7.4 and add agar as for
Fahraeus plates.
At this point RNA can be
isolated from the roots for determination of suppression level. For RT-PCR, extract RNA from 1 or 2
roots using TRIzol reagent (Invitrogen). Northern blots require RNA from many
more roots (12-20).
Place a Sharpie ÒdotÓ on
the outside of the plate at the root tip in order to assay the extent of root
growth relative to the controls.
Transfer one-half (8-10)
plants to BNM media for inoculation with Sinorhizobium meliloti and assay for nodulation.
Transfer plants (root
length 1-2 cm) to BNM media containing 1 mM a-aminoisobutyric acid (AIB) in 120 mm x 120 mm x 15
mm square plates and grow for 2-3 days in 21oC growth chamber with
a 16:8 hour light:dark cycle.
S. meliloti strain Rm 1021 is streaked on LB agar containing 500
mg/ml streptomycin and
grown at 28-30oC.
Grow an overnight culture
of S. meliloti strain Rm 1021 in
5 ml LB, 500 mg/ml streptomycin at 28-30oC with shaking (12-16 hours if
colony from an actively growing plate).
Pellet cells 4000 rpm in JA20 for about 7 min.
Remove LB and resuspend
cells in 50 ml sterile water (10x dilution of original cell culture). Repeat
centrifugation to wash cells.
Cover roots with cell suspension for 1-10 min.
Tip plates to collect
excess suspension at bottom of plate and remove.
Immediately stand plates
upright to ensure root growth on surface of agar and not into agar. Grow plants as before in growth
chamber.
Evaluate phenotype twice
from day 5 (nodule primordi) to day 21 (e.g. days 10 and 21). Mature nodules will appear around day
14.
See separate file BNM
Plates for Nodulation. Nodulation protocols are based on
those available at Long lab web site: (http://cmgm.stanford.edu/biology/long/). Important to transfer seedlings with
1-2 cm roots as faster growing roots nodulate better.
Up to 15 plants can be
transferred to these larger sized plates. Plants transformed with different
constructs can be put on the same plate in order to utilize the plates
maximally, but origin of each plant must be clear. Want to inoculate roots before growth to bottom of plate.
Growth will take two days
from a refrigerated plate; 3 days from stored frozen stock. Overnight cultures can be started
directly from frozen S. meliloti
stock.
Can use a sterile 50 ml
Falcon tube.
Want the resuspended cell
OD600 @ 0.05 (bacteria barely visible). If culture grown for 24 hours this may mean diluting 50x.
Directly pipet several mls
of bacteria on to growing roots.
Transfer plants previously
placed on GamborgÕs B-5 plates to Turface MVP (Profile) to assay nodulation
and shoot morphology.
Use fresh (or previously
used and autoclaved) Turface.
Use 2 ¼Ó square
pots (3² tall) and trays. Place Turface in pot to within about ½Ó of
being full. Add about 40 ml of water.
Cover roots on plate with
deionized water and tease plant from plate, being careful not to damage root.
Do not allow the roots to dry.
Poke a hole into the
Turface (a large Sharpie works well). Place the root into the hole and gently
fill-in it in. Place pots in long flat and cover with clear plastic dome.
Incubate plant in a growth chamber for 2-3 days.
Inoculate plants by adding
several ml of diluted rhizobia to pots.
Gradually acclimatize
plants to room (growth chamber) humidity by off-setting the dome for several
hours once, twice and three times on consequtive days. On the next day, the
dome can be removed. Water plants sparingly once the surface of the Turface
appears dry.
Evaluate nodulation
phenotype between days 10 and 21.
Plants grown on Turface
need to be watered about every other day (approx. 20 ml each plant). Fertilization is generally not
necessary, but 1/2x BNM can be used.
Do not keep plants too wet
(i.e. no standing water in flat) as they tend to develop root rot.
Can do additional
inoculation 1-2 days after first inoculation with several mls of diluted
bacterial suspension per plant.