Switchgrass Lab Protocols

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Diploid Panicum crossing technique

Crossing the diploid Panicum species P. hallii, P. filipes, and P. capillare is difficult because the species are strongly inclined to self-fertilize. In order to make successful crosses, hand emasculation must be used. Because flowers are small I have always used a dissecting microscope. I confirm all crosses with microsat genotyping. Even when I am very careful, about 5-15% of my putative hybrids end up being the product of self fertilization instead.

The method I have found most successful is the following:

1) Place plants in the dark over night. This prevents flowers from opening too quickly the next morning.

2) Remove plants from dark the next morning around 8-10am.

3) Search for flowers that look as if they are about to open (i.e. lemma and palea starting to separate).

4) Maneuver plant so that flower is under dissecting microscope.

5) Using a fine forceps, carefully and gently separate lemma and palea. This will help the flower to fully open. The anthers and stigma should pop out of the flower and be accessible for emasculation and fertilization. If you damage the lemma or palea in the process you are unlikely to be successful, so only flowers that are almost open will work.

6) Use the fine forceps to remove all three anthers. Make sure that the anthers have not opened yet as this will lead to self-fertilization. Examine the stimgas carefully to make sure that no pollen is on them.

7) Clean forceps with ethanol to prevent any contamination

8) Once anthers have been removed, find nearly open flowers on the plant that you want to be the pollen donor (sire) of your cross. Remove the anthers from that fresh flower and wait until the anthers open. Usually, the anthers open only a minute or two after the flower opens. Note: Fresh pollen is absolutely necessary for a successful cross. I have tried crosses with older pollen and all of these failed. A researcher working on other Panicum species told me that pollen may only be viable for 15-30 minutes after the flower has opened, so move quickly.

9) Use the forceps to transfer pollen to the stigma. The stigmas look like little purple Christmas trees. I generally cover them with 50-100 grains of pollen such that the stigmas look like they are decorated with many ball ornaments.

10) Mark crosses with a small piece of lab tape

11) Clean forceps with ethanol and move onto the next cross.

CTAB DNA extraction technique

This method works for extraction of Panicum virgatum as well as Diploid Panicum species

Things to do first:

1) Turn on water bath (60 degrees C).

2) Get liquid nitrogen.

3) Get sample tube/plates from -80 degree freezer. Keep samples chilled on ice.

4) Add one bead to each tube.

DO IN HOOD (except for freezing, grinding, and spinning steps)

1) Make Extraction Master Mix: Measure out 500 uL of CTAB extraction buffer per sample into trough. Add 1 uL B-mercaptoethanol per 500 uL of CTAB extraction buffer to same trough. This “master mix” should include: [(# of samples + 10%) x 500 uL of CTAB] + [(# of samples + 1) x 1 uL B-mercaptoethanol].

• For one 96 well plate: 52mL buffer + 104 uL B-mercaptoethanol
• For two 96 well plates: 104mL buffer + 208 uL B-mercaptoethanol

2) Remove bottom of tube rack. Dip tubes/plates in liquid nitrogen (for up to 10 seconds). Clamp plates into shaker rack in Genogrinder. ALWAYS CLAMP PLATES INTO BOTH SHAKER RACKS AND MAKE SURE TUBES ARE EVENLY DISTRIBUTED ACROSS PLATES. Shake at 500-700, 1x setting (1550-1700 rpm) for 20 seconds to 2 minutes (check periodically and don’t grind for longer than necessary).

3) When tissue is ground to a fine powder, centrifuge plate fast and briefly @ 4000 to get powder off lids.

4) Add 500 uL of Extraction Master Mix to each tube and dip in water bath to thaw. For tubes, shake until buffer is thawed and beads are mobile. For plates, return to Genogrinder for 10 seconds at 500-700, 1x setting (1550-1700 rpm).

5) Incubate tubes for 20 minutes in 60 degree C water bath. VERY IMPORTANT: Invert tubes/plates every ~5 minutes during incubation. Turn off water bath when you are done!

6) Centrifuge for ~10 seconds at 4000 to separate solids.

7) Transfer 400 uL liquid from each tube to a new 96-well Costar Plate. Reserve tubes with beads. [*Clean beads for reuse with soap and water, then rinse with bleach or ethanol and autoclave.] *this can be done later.

8) Cool plate to room temperature. Add 400 uL of chloroform to each tube and place in the genogrinder on 0.5x rate at 00 (lowest setting) for 5 minutes.

9) Centrifuge for 10 minutes at 4000. A band of tissue debris will separate the aqueous (upper) and chloroform (lower) layers.

10) Carefully pipet off 280 uL of the aqueous layer and transfer to a new 96-well plate. Avoid drawing up debris or chloroform (if so, recentrifuge briefly and transfer again).

11) Add 280 uL isopropanol and mix well by inverting 10 times. Place in -20 degree freezer for 20 minutes or as long as overnight.

12) Centrifuge for 10 minutes at 4000. A grayish gelatinous pellet should form.

13) Pour off supernatant, using a Kimwipe or paper towel to wick out as much as possible from tube.

14) Add 200 uL 70% ethanol. Flick tubes multiple times to mix (Do not Vortex).

15) Centrifuge at 4000 for 10 minutes.

16) Pour off ethanol, using a Kimwipe or paper towel to wick out as much as possible from tube.

17) Place open tubes in a 37 degree C incubator overnight to dry. If no incubator is available, tubes can be left out on bench to dry. Make sure all ethanol is gone before proceeding to next step.

18) Add 50-100 uL TE or distilled water to each tube. Flick tube to mix well.

CTAB Leaf Buffer (Recipe to make 100 mL Buffer)

• 1 M Tris Buffer, pH 8.0 10 mL
• NaCl 8.3 g
• EDTA 0.744 g
• CTAB 2 g
• Polyvinyl-Pyrrolidone (PVP) 2 g
• Asorbic acid 0.088 g

Needed for Extraction:

• Genogrinder
• Centrifuge (Plate Centrifuge if you are using plates)
• B-mercaptoethanol
• Isopropanol
• 70% Ethanol
• Chloroform
• Liquid Nitrogen
• Fume Hood
• CTAB Leaf Buffer
• Water Bath
• Incubator (optional)

Microsat Primers

Coming soon

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