General Plant Homogenization

Like animal tissues, many plant tissues can be homogenized using a set of standard protocols.  Below are several protocols for leaf tissue and seeds.  Aside from extremely fibrous leaf tissue, homogenization is usually easily done on leaf punches with deep well plates and 5/32" stainless steel balls. Seeds are more difficult and require optimization.  Typically seeds can be homogenized in 24 well vial sets and the larger 3/8" grinding balls.  Normally stainless steel ball have sufficient energy to grind seeds, but for very tough seeds, tungsten carbide balls may be necessary.

References

Hung, H-Y.  2007.  Characterization of a gene from breeding line WX93D180 conferring resistance to leaf rust (Puccinia triticina) in wheat.  Master of Science Thesis.  Texas A&M University.

Kennedy, E.  2008.   Effects of Control of the Invasive Plant, Phragmites Australis, on Microbes and invertebrates in Detritus, Master of Science Thesis, Kent State University

Protocol for Leaf Punches

1. It is best to use round well plates.  Place the leaf punch or equal mass sample into the well.
2. Add at least 100 µl of lysis buffer but not more than 0.5 ml.
3. Using a grinding ball dispenser, add one 5/32" grinding balls to each well.  Seal the wells using a press on strip cap or some other tight fitting seal.  Do no use seal adhesive tape.
4. Homogenize the leaf punches for 2 min. on 3/4 speed in either the Geno/Grinder or HT Homogenizer. Once homogenization has finished remove the balls by using a Magnet Tip and recover the supernatant with the appropriate volume pipette.  Plates can be centrifuged first to pellet debris.

Protocol for Seed Homogenization

1. Mixer mills such as the Geno/Grinder and HT Homogenizer II are used when processing large number of samples is necessary. Seeds can be homogenized both by dry grinding and with buffer.
2. Place the seed in a 4 ml vial. Add either a 3/8" stainless steel or tungsten carbide ball to the vial. Then cap the vial and place back into a rack like a vial set. For dry grinding, an unlined cap is used and for wet grinding an lined cap is necessary.
3. For wet grinding, add no more than 500 µl of homogenization buffer.

4. Place the rack into the mixer mill (Geno/Grinder or HT Homogenizer) and process on high for 5 minutes.
5. Balls can be removed from the vial set using Magnet Tips or the 24 Pin Magnet Ball Dropper.
6. Remove the supernatant with the appropriate sized pipette and transfer to a new tube if desired.
7. Batches of seeds can be processed using a 15 ml vial sets. These larger vials make use of two 7/16" stainless steel grinding balls.
8. Take note: that mixer mill disruption does generated heat and may cause denaturing of certain enzymes and protein. Heat can be mitigated by submersing the tubes and cryogenic rack into liquid nitrogen before homogenization. Cryoblocks are available and act as cold sinks for cryogenic grinding.