Maize Kernel Disruption Methods

Maize can be pulverized by employing several methods depending on the part of the plant and the desired end result.  Adult leaves, dry kernels, and stems are some of the toughest parts to homogenize into fine powders, because of their durability and toughness.  Yet young seedlings may be less challenging and can be done in a 96 well format. They may require a combination of techniques including; treatment with liquid nitrogen, pulverizing by bead beating, or freeze drying and grinding with a mortar and pestle to render the best overall outcome.

Protocols

• Bead beat a single kernel
• Bead beat multiple kernels pooled
• Traditional grinding using liquid nitrogen, with a mortar and pestle
• High Throughput homogenization using a 96 well plate

Individual corn kernels are too large to homogenize in 96 well plates.  Even if a kernel can fit in a well, the 5/32" grinding balls normally used with grinding in deep well plates have insufficient mass to disrupt intact kernels. Consequently, for dry grinding kernels are best homogenized in 4 ml polycarbonate vials. Though vials can be made in different materials, such as polyethylene, best results are attained with polycarbonate vials due to their rigid design. For dry grinding, kernels should be low in water content otherwise a starchy paste may result from the grinding. Moisture can be removed from kernels by air drying (normally a prolonged process) or by freeze drying.

1. Place one dry kernel into a 4 ml polycarbonate vial. Vials will need to be placed in a rack or used in a vial set so it can be homogenized in a mixer mill.
2. For nearly all kernels, drop a 3/8" stainless steel grinding ball on top of the kernel. Sometimes kernels can be very tough and require a more dense 3/8" tungsten carbide ball. The tungsten carbide balls have a much greater density and may possibly split the vial, so use only when necessary. Furthermore, tungsten carbide balls are very expensive.
3. Cap the vial with either a lined or unlined cap. With vial sets, a 24 well mat can be used to seal the vials much like a 96 well sealing mat. Place the vial set into a high throughput homogenizer, such as a Geno/Grinder or HT Homogenizer, and homogenize for 2 minutes on high.
4. Examine the resulting meal generated from the grinding. If small pieces of kernel remain, homogenize for an additional 2 minutes. Occasionally a piece of kernel will wedge between the vial and grinding balls and impede the movement of the ball. If this occurs, free the ball with a thin spatula and repeat the processing.
5. The homogenized kernel can be extracted for analysis or stored in a well sealed vial until needed.

Field trials often make use of pooled kernels for analytical procedures. Using a high throughput homogenizer, up to 60 kernels can be effectively homogenized by dry grinding. This is accomplished by using larger polycarbonate vials and small jars with larger grinding balls. The protocol is very similar to single kernel dry grinding but with vial size and processing times increased.

Generally kernels can be dry ground at a ratio of 1 kernel/ 1 ml of vial. Thus in a 15 ml polycarbonate vial, up to 15 kernels can be processed. The most common format for dry grinding pooled samples is the 15 ml vial set.  Like the single kernel method, polycarbonate containers provide the best results.  In addition to 15 ml vials, small polycarbonate jars can be used for dry grinding.  There are a limited number of jars available commercially (i.e., 30, 60, and 125 ml), and all must have the bottoms reinforced to prevent cracking.  In our lab, we have reinforced the bottoms using a two-part silicone mixture that fills-in and supports the bottom from the impact of the grinding balls.

1. Once the proper vial/jar is selected (and modified if necessary), add the kernels and 7/16" balls.  For 15 ml vial sets, two 7/16" stainless steel grinding balls are provide with each vial. For larger jars, use two balls for 30 ml jars, four balls for 60 ml jars, and six balls for 125 ml jars.
2. Cap the vial, place it into the Geno/Grinder or HT Homogenizer and homogenize for 5 minutes on high.
3. Examine the resulting meal. If large pieces remain, process for another 5 minutes. Undoubtedly the larger jars will take longer to process than the 15 ml vial.
4. Once homogenized, the meal can be analyzed or stored. For storage, seal in a tight container to prevent the meal from picking up moisture.

A traditional method of homogenizing any plant material is by grinding cryogenically using a mortar and pestle. This method is typically very low in throughput, but it can generate very fine, controllable powders. An advantage of cryogenic grinding is that kernels do not require drying prior to processing (like the dry grinding methods above).

Though this protocol is for kernels, it can be used on virtually any part of the plant, i.e., leaf punches, kernels, root, and stem. The materials needed will include a porcelain mortar and pestle, Styrofoam bucket or box, and liquid nitrogen.

1. Put the mortar into the Styrofoam container and set the sample and pestle into the mortar.  NOTE:  Liquid nitrogen is extremely cold (-196°C) and can cause burns.  Handle all objective while wearing protective gloves (latex gloves do not protect from the cold).  Additionally, as the liquid nitrogen vaporizes, it can displace oxygen from enclosed areas.  Make sure you work in a well ventilated area while working with liquid nitrogen.
2. Fill the mortar well with liquid nitrogen. It will boil (bubble and splatter) when the liquid nitrogen is first added, but as the temperature of the mortar, pestle, and sample decrease, the boiling will stop.  Several applications of the liquid nitrogen might be necessary to get the items down to cryogenic temperatures.
3. Hold the pestle with a gloved hand (use a cryogenic glove or at minimum a cotton gardening glove) and press down on the sample while turning the pestle.  As the kernels shatter, small pieces may fly out of the mortar, so proceed with the grinding slowly.  Continue the grinding until the sample is finally pulverized.

4. Add more liquid nitrogen throughout the process as needed. 
5. Once grinding is completed the sample can be extracted or stored.  Note that fresh kernels contain a significant amount of water and upon thawing, the powder will take on a paste-like characteristic.  If it is desirable to keep the sample powdered, then keep it frozen at all times.

The protocol below describes homogenizing kernels in a 96 well format, however most small samples from maize (e.g., 100 mg or so) can be processed using 96 well plates and small grinding balls. Kernels are the most laborious to process in 96 well plates simply because of the initial preparatory work that is required. As kernels tend to be very hard and the grinding balls used in 96 well plates are small, kernels must be soaked overnight and then sectioned (i.e., dissected) before homogenizing. The homogenization is typically done in an extraction buffer as the endosperm can create a thick paste if not diluted during processing.

1. The day before processing samples, soak kernels in water, TE buffer or other suitable buffer to soften. Just before processing, section off a piece of the kernel, ideally no more than 100-200 mg of tissue, and place it in a 96 well plate. The type of plate is relatively important, as it should have sufficiently this bottoms which can take the beating from grinding balls and they should also seal well with cap mats. OPS Diagnostics has 1.2 ml deep well plates and 2.5 ml deep well plates that work well with the high throughput homogenizers used to process samples.
2. An extraction buffer should be added to the sample and should be 3-4X the sample mass. For example, a 100 mg sample can be supplemented with 400 µl of buffer. However, the total volume of the sample and buffer should not exceed half the volume of the well. Grinding balls need room to move and impact the sample, and over-filled well will impede the effectiveness of the grinding balls.

3. Using a ball dropper, dispense a 5/32" stainless steel grinding ball into each well. Seal the plates with press on cap mats (round well or square well).  Sealing mats must fit tightly to avoid leakage during processing.  Additionally, heat or tape sealed plates will not hold up to the impacting grinding balls.

4. Place the prepared deep well plate into the GenoGrinder or HT Homogenizer. Set at 2/3 maximum speed and process for 2 minutes.  To avoid cross contaminating wells when the cap mat is removed, it is advisable to centrifuge the plate first to push liquid and homogenate to the bottom of the wells.  If subsequent process is to be done in the deep well plate, the grinding balls can be removed using magnet tips.