Isolating (peppermint) glandular trichomes by the bead-beater method

Sean Johnson

April 26, 2017

Simplified from: Gershenzon et al., ANALYTICAL BIOCHEMISTRY 200,130-138 (1992)

 

Wash buffer (1 Liter):

50 mM NaH2PO4

pH 7.4

Autoclave it.

 

Isolation buffer (1 Liter):

Sorbitol 200 mM

50 mM NaH2PO4

pH 7.4

Autoclave, then add

Methyl Cellulose 0.6 % (4,000 cP, sigma M0512-100G)

Stir over night (preferably in the cold room). Methyl cellulose takes a while to dissolve.

 

Trichome isolation in a cold room seems to result in less RNA degredation than at room temperature.

 

Add 50-100 g of 0.5 mm glass beads to the bead beater chamber (beads are easier to pour if they are wet, as dry beads jump all over the place), set up nylon meshes by attaching them with clothes pins or document clamps to large (500 mL or larger) plastic beakers. The first two meshes should be 350 microns and 110 microns (I’ve never seen a glandular trichome be retained on a 110 micron mesh, but there is probably some species with trichomes that big). The third mesh depends on the size of the trichome you are trying to purify, 20 microns is a safe size to choose, as most glandular trichomes are at least 20 microns across, bigger meshes run a little faster, so can be more convenient.

 

Once everything is set up in the cold room, go harvest the tissue.

 

Harvest young (less than half-fully expanded) leaves into a bag on ice. The method also works with stems, flowers, and calyxes. For species with very long leaves (2.5 cm or longer) cut the leaves into 1.5-2 cm sections during harvesting.

 

Weigh the leaf material by weighing the empty bag, and the bag with leaves in it. The bead beater can accommodate anywhere from 3-25 grams of leaves. 5 grams of leaves will usually be sufficient to get at least 1 microgram of total RNA.

 

Put the harvested leaves into the bead beater chamber, and fill the rest of the chamber with isolation buffer (isolation buffer can be diluted at least up to 50% with wash buffer without much effect on the trichome isolation).

 

Close the bead beater chamber with the rotor, and beat as three one minute pulses, with 1 minute of cooling on ice between pulses. A variac should be used to keep the bead beater at between 50% and 70% maximum voltage (I don’t know if/how this has to be adapted to account for differences between American and European power standards), the point is to mix the leaves and glass beads vigorously without destroying the trichomes.

 

Pour the contents of the chamber onto the 350 micron filter, then the flow-through onto the 110 micron mesh, then the flow-through onto the 20 micron mesh. The trichomes will be retained on the 20 micron mesh. Flow through the 20 micron mesh is usually very slow. It can be sped up by stirring the trichomes, tilting the beaker, undoing some of the clips bunching up the mesh and applying gentle pressure, or by wicking the flow-through away with a paper towel. At any stage, wash buffer can be used to wash the bead beater chamber or any of the beakers to slightly increase yields, or dilute the isolation buffer so it passes through the meshes more quickly.

 

Once most of the liquid has passed through the 20 micron mesh, unclip it completely and place it onto some paper towels. Scrape the trichomes off the mesh using a metal spatula, and stir them into 4 mL of wash buffer in a 15 mL plastic conical tube. Centrifuge the tube at low speeds (about 250 G) for 5 minutes to pellet the trichomes (higher speeds and lower times can also be used without risk of damaging the trichomes). Pull off the supernatant, put a small sample of the pellet onto a microscope slide for visual inspection. Either immediately freeze the rest in liquid nitrogen and/or a -80 freezer, or resuspend it in 1 mL of wash buffer, transfer it to a smaller tube, re-pellet it, and then freeze it.