Procedure Outline

Notes and References

M truncatula Seed Sterilization and Imbibition

Add counted seeds to sterile Falcon tube

Use 15 ml Falcon tube for 200 seeds or less; 50 ml tube for 200-300 seeds.

Protocol outlined here works for both sterile seedling production (used for transformation experiments) and pot growth (used for seed collection).  For root transformation, start with 24-36 seeds per construct being tested. We generally want to analyze 16 transgenic roots.

Scarify with concentrated H₂SO₄

Completely cover seeds with concentrated H₂SO₄ (~2 ml) and mix well to ensure all seeds exposed to the acid.

Incubate 6-7 min.

In laminar hood or equivalent sterile space, remove H₂SO₄ with sterile pipet and dispose of appropriately.  Wash seeds 5x using 10-12 ml sterile deionized H₂O for each wash. Mix the tube several times during each wash. During first wash, disperse seeds quickly after adding the water to prevent a rapid temperature increase.  Water can be decanted from seeds.

Wearing a lab coat while doing this is a good idea.  Use a sterile 1 ml platic pipet tip to mix acid and seeds.  Seeds will stick to plastic (sides of tube, etc.) and need to tapped down to bottom of tube.  Six minutes is minimum time necessary to break through waxy cuticle.

This and all subsequent steps need to be performed under sterile conditions for seedling use in transformation.  Remove as much H₂SO₄ before adding water since the acid/water mixture will generate large amounts of heat. 

Sterilize in commercially available bleach (sodium hypochlorite)

Add 2-3 ml commercial bleach with sterile pipet so that seeds are covered and gently mix for 2 minutes. 

Remove bleach and wash in sterile deionized H₂O 5x as described previously. 

Keep working amount of bleach in a covered bottle instead of using large jug.  Seeds will float so decant H₂O carefully or remove with sterile pipet.

Imbibe seeds

Add 10 ml of sterile deionized H₂O and cover tube in foil (dark treatment).  Place on orbital or rocking platform (~ 50 rpm or equivalent) for 2 hrs, changing H₂O every 30 min.

If growing plants in pots for seed production, go to M. truncatula Growth for Seeds following this step.  If growing plants for transformation, continue to next step.

Cold treat seeds

Store covered tube with seeds and H₂O at ~4^oC for 48-96 hours. Alternatively, plate seeds on sterile 1% agar (see below) and store inverted plates wrapped in aluminum foil in refrigerator for 24-48 hours.

Temperature of typical refrigerator is fine.  Seeds can be treated at 4^oC for 1-7 days, but germination may be poorer at the two extremes. Cold treatment will result in greater and more synchronized germination.  Proceed next to M. truncatula Seedling  Transformation.

Total time:

2.5 hours (30 minutes for scarification and sterilization; 2 hours for imbibition)

M. truncatula Seedling Transformation

Procedure Outline

Notes and References

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 20-22^oC 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 21^oC.  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 25^oC (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 28-30^oC 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 4^oC 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 21^oC 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 14^oC for 24 hours and then at 24^oC for 12-14 days under light. 

Single temperature works well with L416 plants (about 90% efficiency of transformation); temperature change works better for A17 wild type plants.  A17 often gives lower (~50%) transformation efficiency.

Phenotypic Analysis of Transformants

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.

• Root length: Use ruler to determine average root length per transformed plant.
• Root branching: Are some roots ÒbushierÓ relative to controls?
• Root hair – length and morphology
• Root hair –growth density?
• Root color.

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 21^oC 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 28-30^oC.

Grow an overnight culture of S. meliloti strain Rm 1021 in 5 ml LB, 500 mg/ml streptomycin at 28-30^oC 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.