Tips and Best Practices for Lysing the Toughest Plant Samples

Tough Plant Samples

Like other sample types, plant samples need to be sufficiently homogenized to extract nucleic acids, proteins, and other target analytes, however some plant samples are particularly resilient and difficult to break, such as seeds, roots, pine needles, and fibrous leaves. Homogenizing tough samples is laborious and often results in low yield of target analytes, creating frustrating bottlenecks in your sample preparation workflow.

With the right tips and practices, you can consistently break down the tough cell wall of recalcitrant plant samples, get high yields, and remove impurities to run successful downstream analyses.

You won’t find a one-size-fits-all method, but some practices are better suited for certain sample types and applications. We suggest identifying methods that have proven successful for sample types similar to yours and considering the strategies mentioned here to optimize your method.

Pretreating Your Plant Sample

A pretreatment, and sometimes multiple types of pretreatments, are required prior to homogenizing a tough plant sample. Pretreatment can involve washing, soaking, cutting, flash freezing (in liquid nitrogen), and/or lyophilizing the sample to minimize exogenous contamination and make tissue disruption easier and faster. Use liquid nitrogen to pre-treat plant seeds rich in polysaccharides and phenolic compounds to minimize contamination and degradation of key analytes.

Learn more about plant tissue sample preparation—from pretreatment through homogenization and extraction.

Effective Tools And Methods For Plant Tissue Homogenization

Direct force is the only way to effectively break down tough plant samples and this is often performed via bead beating.

Multiple lysing matrices (i.e., bead type) and lysis solutions are available to choose from and the ideal type for your sample will depend on the sample toughness and downstream application. Zirconium oxide beads (e.g., Lysing Matrix Z) and stainless steel grinding balls (e.g., Lysing Matrix SS) are commonly used for plant sample homogenization.

In certain cases, you may need to use cryogenic temperatures throughout the homogenization process. Cryogenic sample holders keep temperatures within the tube from becoming too hot and damaging to the sample. Also, for your most resilient samples, you should consider using Metal Lysing Tubes—tough enough to stand up to the most aggressive mechanical forces that could cause thermoplastic tubes to crack.

Dedicated bead beating systems, such as MP Bio’s FastPrep instruments, are designed to quickly and thoroughly homogenize samples, even the toughest plant samples. Choose systems that are proven to be effective, are adaptable to create the conditions you need, and are reliable and easy-to-use.

Pro Tips:

• Optimize DNA recovery from extremely dry samples by leaving the lysed sample at room temperature in the lysing tube for 15 minutes to 2 hours after processing with a FastPrep instrument.
• Sufficiently lyse most samples with a single 40-second run at a speed setting of 6.0 in the FastPrep instrument.
• If you need to process your sample for longer, incubate your sample on ice in the Lysing Matrix tube for at least 2 minutes between successive homogenization runs to prevent overheating the sample and tube.
• Choose the appropriate lysing solution based on secondary metabolites within your sample. For instance, in RNA extraction, the removal of genomic DNA can be performed during the cell lysis step if the appropriate solution is used, but some secondary metabolites of plants can bind DNA-binding carrier particles within lysing solutions, preventing binding to DNA. To avoid this effect, MP Bio’s FastRNA^TM Win Kit for Plants offers two different lysis solutions—one for plant material showing a high polysaccharide concentration and one for plant material with high phenol content.
• For efficient homogenization with beads, and to avoid sample loss and tube failure, you should maintain at least 5 mm air space in the tube during FastPrep processing.

Recommendations for Roots and Seeds

The difficulty with root DNA and RNA extraction is the toughness and starchiness. The high power of the FastPrep^® System used in conjunction with our lysing matrix tubes quickly and efficiently lyses tough root and seed samples.

Typical Root Sample Recommendations

Roots are very fibrous and contain high levels of polyphenolic compounds and polysaccharides, and it can be extremely difficult to extract enough usable DNA or RNA for PCR analysis and other downstream applications.

Typical Seed Sample Recommendations

Seeds are notoriously tough to lyse, due to brittle seed coats and hard cotoledyon inside. The seed coats often contain tannins that inhibit PCR. The inner seed material contains starches and lipids that make lysis difficult.