Hello Rieseberglers.

Almost all of us collect tissue for DNA but there are always new people in the lab, some of whom may have never worked with plants, or even DNA I suppose, and who might benefit from the experience of seasoned plant geneticists. This post contains my advice on collecting sunflower, or other plant, tissue for DNA work. If you have any tips, or alternatives please contribute in the comments.

Objective

You want to collect tissue such that decent yields of high quality DNA can be extracted from it at a later date.

The problem

DNA, while not particularly delicate, is easily damaged while its in a degrading “cellular” environment. In a healthy plant cell, nuclear DNA is safely quarantined from endogenous cytoplasmic enzymes, enzymes produced by pathogens and a host of problematic chemicals – sunflower (and a lot of other plants) have leaf, and other, cells full of all sorts of weird and wonderful chemicals, many of which are bad for DNA. The thing is that once you collect leaf (or other) tissue, you remove that tissue from the “healthy” system that was preserving the DNA in it – you have just started the cellular degradation process that will expose the DNA in the tissue to all of the enzymes and chemicals that will destroy it. In a nutshell, you now have to protect the DNA.

What you need to do

You need to preserve the DNA in the tissue you collect. In the briefest possible terms:

You need to either dry out, freeze, or chemically preserve the tissue you collect as soon as you can.

As mentioned above, there are three options for preserving the tissue/DNA you collect. Drying and freezing are the preferred methods (for simplicity) and, for our purposes, these can be considered the go-to options but bear in mind that there are tried and true chemical preservation methods that you can opt for – Google “saturated CTAB” to find one that I know works. Also note that chemical preservation jumps right up in relevance if you are collecting in humid places where accessible refrigeration is unlikely – i.e. a lot of the tropics.

Drying

Why: Drying works because the reactions, enzymatic and otherwise, that destroy DNA occur in aqueous solution. If you get rid of the water that is in your sunflower tissue quickly enough you can effectively preserve the DNA in it for a very long time.

How: The standard method is to collect a small amount of leaf tissue into paper envelopes then bury those envelopes in dry silica gel. Any procedure that dries the tissue without “cooking” it is fine. If you are collecting somewhere dry enough, or hot enough, you can simply air-dry the tissue. I have extracted DNA that was perfectly fine for PCR from fig leaves that were collected in the tropical north of Australia, air dried – i.e. left out in the sun, and then posted to me in the south in the normal mail.

How not to do it: The obvious way to blow this is to overwhelm your drying capacity with tissue. You might do this by collecting too much tissue, for example. Another way to do this badly would be to start with “wet” silica gel – make sure its dry before you start.

Freezing

Why: Freezing works because reactions, enzymatic and other, are, to state the obvious, temperature dependent – they don’t kick off at really cold temperatures.

How: If you are not drying your tissue collections, anything you can do to cool them is probably worth doing. The gold standard is a liquid N bath. The silver medal would go to submergence in dry ice. Your ultimate objective should be to get your tissue into a reliable minus 80 freezer in the lab ASAP. If you are opting for freezing you really should do whatever is the most practical-extreme thing you can do to cool your samples as quickly as possible. In the field, you should get your samples into a freezer, liquid N dewar, cooler full of dry ice, or whatever you’ve got as soon as possible – within hours if within minutes is not an option. At UBC you should take a cooler with liquid N or dry ice in it to the glass-house or growth chamber or field location that you are collecting at so you can freeze tissue immediately.

How not to do it: This is obvious – if you freeze plant tissue, you destroy the cellular infrastructure of it, and then, if you let it warm back up to reaction-friendly temperatures, all hell will break loose – everything in that tissue is now mixing in a big DNA-hostile soup. This is bad. Once you freeze plant tissue it has to stay frozen. DO NOT let frozen tissue thaw!