Many methods have been described for preparing leaf tissue for nucleic acid isolation and like most laboratory protocols, there are as many variations as researchers. Generally, leaf tissue is harvested and processed fresh, frozen and processed cryogenically, or frozen, freeze dried, and then homogenized. Each variation can impact the quality of the DNA, such as the size of the fragments isolated. The protocol used for isolating the DNA will also greatly affect DNA quality, especially relating to contaminating polysaccharides and polyphenols. Depending upon the need, harvesting and homogenization are matched for optimal yield.
There are three common routes by which leaf tissue is harvested prior to disruption. The first involves harvesting leaf tissue followed by freezing. Placing the tissue in a -80°C freezer provides a suitably cold environment that preserves DNA and many proteins, but is unsuitable for preserving RNA. Even at -80°C there is sufficient water activity and nuclease action to degrade RNA, albeit slowly. To harvest leaves and preserve RNA, samples must be frozen rapidly, usually by submersing in liquid nitrogen. To preserve the RNA the samples must be held below -120°C, the glass transition temperature of water. At this temperature all biological activity ceases. The second option of preparing leaf tissue prior to homogenization is to harvest, freeze and then freeze dry the samples. Freeze drying allows for long-term storage of DNA and protein (though not all proteins will remain active), but once again RNA typically doesn't survive the freeze drying process. Freeze drying of leaves removes water which if present can alter the concentration of analytes in extractions buffers. The third options for preparing leaf tissue for disruption is to simply harvest the leaves and homogenize them while they are fresh. With the advent of buffers which preserve DNA and RNA, such as Trizol, disrupting leaf tissue when harvested is often practical.
Once leaves are harvested, one of several grinding/homogenization methods is used to break open the cells. Below is a brief summary of each methods.
CryoCooler™, a tool for collecting and transporting temperature sensitive samples ( video).
Grinding frozen leaf tissue using a mortar and pestle chilled with liquid nitrogen is a common technique.
One of the most traditional and common methods for harvesting nucleic acids from plants involves grinding leaves in liquid nitrogen with a mortar and pestle. Either the mortar and pestle can be pre-chilled and the grinding performed dry on frozen leaves, or the leaves can be submersed in liquid nitrogen for the grinding. Cryogenic grinding is a very effective technique for taking hard substances, like plant and animal tissues, and turning them into dust. The tough carbohydrates of plant tissues become very fragile at -196°C and easily shatter. The two concerns with cryogenic grinding is that the sample may warm up, and secondly, throughput is very low. Preventing sample warming can be done be adding additional liquid nitrogen to the mortar while pulverizing the sample. Low throughput is a more difficult issue as a mortar can only be used once before it must be cleaned. As mortar and pestles are typically ceramic, this means that the set must be warmed gradually before it is cleaned. Materials ground into the surface of the mortar may be difficult to remove and hence may act as a contaminant.
Freezing is often used to store harvested leaves
For samples which are fresh or freeze dried, homogenization can be attained by shearing leaves with a blade. The simplest bladed homogenizer is a blender, which at times can be completely adequate for disrupting samples. Though efficient for milkshakes, blenders are best used for course shearing while rotor-stators are the preferred tool for efficiently disrupting tissues. Rotor-stators have a spinning circular blade called a rotor inside of a tube with slits, known as the stator. As the blade passes the slits it acts like a fine scissor and shears whatever straddles the slit. As the rotors can turn at 20,000 rpm, this makes the rotor-stator very efficient at tearing open cells. The problem with both blenders and rotor-stators is throughput. These homogenizers must be cleaned between use, and in both cases, this may require taking apart the blade assembly.
Where mortar and pestle and homogenizers fall short in throughput, bead beating makes it up. Bead beating is accomplished using a mixer mill, which is basically a machine that rapidly shakes samples which have been mixed with balls. The balls crash around and effectively shear and crack cells and tissues. For microorganisms, small beads of several hundred microns are mixed with the microbe in a microfuge tubes which can be vortexed. Some vortexers will hold multiple tubes making the processing of many samples relatively easy. However, with leaf tissues, it is most practical to use vials or deep well plates and large stainless steel balls. With Vial Sets that use 4 ml polycarbonate vials, the balls are large being 3/8" in diameter. Several hundred milligrams of tissue can be disrupted using a vial. Larger vials can also accommodate up to five grams of leaf tissue. But the most widely used method for homogenizing leaf tissue is to punch leaf holes with a paper punch and drop one disk into a deep well of a microplate along with a 5/32" stainless steel ball. Using a high throughput homogenizer that holds deep well plates, multiple samples can be processed in minutes.
Harvesting fresh tissue for immediate homogenization is dependent upon throughput and practicality.