Homogenization Options of Leaf Tissue for Nucleic Acid Isolation

Updated Webpage: This page was updated February, 2024. The original version was archived February 2024.

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 1) harvested and processed fresh, 2) frozen & processed cryogenically, or 3) frozen, freeze dried, and then homogenized. Prior to grinding, freezing leaf samples for easier handling and most importantly for inactivation of cellular nucleases is common. Some protocols homogenize leaves dried on silica, or herbarium samples. Each variation can impact the quality of the nucleic acids, such as the size of the fragments isolated. Additionally, the protocols used for isolating the nucleic acids will greatly affect quality, especially relating to contaminating polysaccharides and polyphenols. Depending upon the need, harvesting and homogenization methods are coordinated for optimal yield.

Once leaves are harvested, there are several grinding/homogenization methods can be used to breakdown cell walls and release its cellular contents. Below is a summary of common methods.

Smashing

Hard leaf tissues placed inside a plastic sleeve or bag are bashed with a rubber mallet or small metal masher depending on the size of the sample, along with the lysis buffer. It is important though to avoid creating holes in the plastic sleeve or bag.

Bead Beating

Bead beating is the easiest and fastest method for homogenizing leaf tissue. Bead beating is accomplished using a mixer mill, basically a machine that rapidly shakes samples placed in tubes with grinding balls or beads. The balls crash around and effectively shear and crack cells and tissues. The method can be done in individual tubes or high throughput 96 well plates. Though most bead beating is done under ambient conditions, cryogenic bead beating is possible. A variety of disruption tubes and vials can be used for bead beating.

Bead beating also has the advantage of lysing the associated microbiome of the sample (e.g., leaves or roots). Mixing grinding balls with small beads (100-800 microns) will disrupt both sample and microbes. Bead beating also allows for the rapid isolation of nucleic acids using SYNERGY™ isolation chemistry, a novel CTAB based method that uses the bead beating itself in the purification process. This has been effective for the isolation of nucleic acids from plants, and microbes and viruses from leaves.

Using a high throughput homogenizer that holds deep well plates, multiple samples can be processed in minutes. This widely used method for homogenizing leaf tissue punched with a paper punch with one disk placed into a well with a 5/32" stainless steel ball. This same approach can be used with SYNERGY™ chemistry.

Grinding in Liquid Nitrogen with Mortar & Pestle

Grinding frozen leaf tissue using a mortar and pestle chilled with liquid nitrogen is a traditionally common technique. 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 powder. The tough carbohydrates of plant tissues become very fragile at -196°C and easily shatter. The two concerns with cryogenic grinding are that the sample may warm up, and throughput is very low. Sample warming can be prevented by 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 act as a contaminant. It is possible to efficiently transfer small amounts of tissue (10 mg or less) to a microfuge tube after grinding the tissue in a cold mortar with powdered dry ice as a carrier for the tissue powder. When grinding large bits of hard tissue (like Vicia faba cotyledons), a tiny coffee grinder with dry ice worked better than a mortar and pestle (Rogers and Bendich, 1985).

To increase throughput of cryogenic grinding, bead beating with polycarbonate vials is an alternative. For example, a specialized device, the AC Block™, holds twelve polycarbonate vials which are cooled with liquid nitrogen during bead beating. It provides a balance between cryogenic processing of samples and higher throughput.

Disruption via Homogenizer: Rotor-Stators and Blenders

For samples which are fresh or freeze dried, homogenization can be achieved by shearing leaves with a blade. The simplest blade homogenizer is a blender, which at times can be completely adequate for disrupting samples. Though efficient for milkshakes, blenders generate a relatively course homogenate. For a more thorough homogenate, 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 or shaft assembly.

Microneedle (MN) Pathch

A disposable polymeric MN patch is a recent method used for rapid extraction of DNA from a fresh plant leaf by applying gentle pressure of MN patch on the leaf surface. To isolate intracellular DNA during DNA extraction, the polymeric MN patch pierces through tough plant cell walls and enters plant leaf tissues. MN patch which is made up of polyvinyl alcohol can be fabricated, and the recovered DNA could be used directly for amplification processes without the need for additional purification (Paul et al., 2019).

Focused Ultrasound Extraction (FUSE)

FUSE is a novel technique for extracting DNA from plant tissues using focused ultrasound. A dense cloud of sonic cavitation bubbles created by FUSE breaks apart the targeted tissue into acellular debris. Leaf samples from American chestnut oak, red maple, tulip poplar, and American chestnut have been processed successfully using this method. The use of FUSE improves the efficiency and speed of DNA extraction of leaves compared to conventional methods and shows that the quality and quantity of the extracted DNA are sufficient for amplification and sequencing (Stettinius et al. 2023).

Impact of Processing on DNA Quality

Leaf tissues are typically mechanically disrupted by methods such as homogenization, grinding, or bead beating. While it works well, too much mechanical force can shear DNA, lowering its yield and quality. The mechanical force applied to the plant samples can cause the DNA molecules to break into smaller pieces. In addition, bruised leaves quickly release polyphenols, which lead to DNA deterioration (Couch and Fritz 1990). Plant samples can also contain impurities such as dirt, metal ions, nucleases and organic solvents from the surrounding area, equipment, or other samples that might hamper the extraction of DNA. DNA loss may also occur via incomplete recovery or adsorption to the surfaces of the equipment or the disruption beads while being processed.

The condition of the starting plant material also affects DNA quality. Comparing both senescing and mature leaves, and fresh and stored leaves of a tough leathery plant Encephalartos, Jones et al. (2023) showed differences in DNA extraction efficiency. Senescing leaves and/or tissue that have been stored in silica for long periods can still produce significant amounts of DNA.

Summary

There are several options for homogenizing leaf tissue for nucleic acid extraction. Most methods use some form of grinding or shearing. The homogenization method, and the previous handling of the sample, can dramatically impact the quality of the purified DNA. Rapid freezing of leaves, followed by cryogenic grinding will generate the largest DNA fragments. This is useful for long read sequencing. Other methods, such as bead beating in buffer, will yield shorter DNA fragments, however completely adequate for most PCR applications.

References

Couch J, Fritz P. Isolation of DNA from plants high in polyphenolics. 1990. Plant Molecular Biology Reporter. 8:8-12.

Jones, M. P., Nagalingum, N. S., and Handley, V. 2023. Testing protocols to optimize DNA extraction from tough leaf tissue: A case study in Encephalartos. Applications in Plant Sciences. 11

Paul, R., Saville, A., Hansel, J. C., Ye, Y., Ball, C., Williams, A., Chang, X., Chen, G., Gu, Z., Ristaino, J. B., and Wei, Q. 2019. Extraction of plant DNA by microneedle patch for rapid detection of plant diseases. ACS Nano. 13:6540-6549

Rogers, S. O., and Bendich, A. J. 1985. Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Molecular Biology. 5:69-76

Stettinius, A., Holmes, H. R., Zhang, Q., Mehochko, I., Winters, M., Hutchison, R., Maxwell, A. D., Holliday, J. A., and Vlaisavljevich, E. 2023. DNA release from plant tissue using focused ultrasound extraction (FUSE). Applications in Plant Sciences. 11

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