Name: high-throughput, microscale protocol for the analysis of processing parameters and nutritional qualities in maize (zea mays l.)
Uploaded: 2018-06-16T15:00:00.000+00:00
Duration: 5 min 55 s
Description: Watch this Scientific Journal Video about High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize Zea mays L. at JoVE.com

The authors would like to thank Tom Patterson and the Analytical Technologies Team at Dow AgroSciences for the use of their laboratory facilities and for their mentorship. This work was funded in part through gifts from the Kellogg Company and Dow AgroSciences and through USDA Hatch Grant, award ILLU-802-354. Student support for CJBW was provided by the Illinois Distinguished Fellowship and the William B. and Nancy L. Ambrose Fellowship in Crop Sciences.

1. Produce Cooked Grits
<ol>
	<li>Place a 15 L canning pressure cooker on an electric hotplate.</li>
	<li>Add 1 L of tap water into the canning pressure cooker and heat to 100 &#176;C.</li>
	<li>While the water heats, place a 100 g sample of industrial flaking grits or flaking grit material (12% moisture content, wet basis)7 in a 1 quart canning jar. 
	NOTE: The representative results of this study are based on the flaking grit materials produced by Macke et al.9 using the laboratory scale dry milling protocol outlined by Rausch et al.7</li>
	<li>Add a sugar-salt solution consisting of 200 mL of distilled water, 2 g salt, 6 g granulated white sugar, and 2 g liquid malt extract. 
	NOTE: Multiple samples can be analyzed at once, although the exact number of samples will depend on the size of the canning pressure cooker.</li>
	<li>Mix the solution with the flaking grit material using a glass stirring rod.</li>
	<li>After the water in the canning pressure cooker begins to boil, add 1 L of tap water to cool the water in the canning pressure cooker.</li>
	<li>Place the canning jars in the canning pressure cooker such that they are equidistant from each other and from the wall of the canning pressure cooker.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57809/57809fig1.jpg" /> 
Figure 1: Placement of canning jars in canning pressure cooker. Canning jars should be placed equidistant from one another and from the sides of the canning pressure cooker to ensure even cooking and to avoid damage to canning jars. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57809/57809fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="8">
	<li>Allow the water to reach a rolling boil. Place the lid on the canning pressure cooker.</li>
	<li>Cook the large flaking grits or flaking grit material at 15 psi for one h. Allow the canning pressure cooker to cool and depressurize completely before opening.</li>
	<li>Remove the lid from the canning pressure cooker using heat resistant gloves.</li>
	<li>Remove the canning jars from the canning pressure cooker using tongs. Place the jars on a heat resistant surface. 
	NOTE: The resulting intermediate product at this point is cooked grits.</li>
	<li>If using flaking grit materials as produced using the protocol outlined by Rausch et al.7, remove non-endosperm material with a spatula after cooking. If using industrial flaking grits, skip this step.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57809/57809fig2.jpg" /> 
Figure 2: Removal of non-endosperm material. (a) Cooked grit sample prior to the removal of non-endosperm material that has risen to the top during cooking. (b) Cooked grit sample after the removal of non-endosperm material. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57809/57809fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="13">
	<li>Place 30 g of cooked grits (per processed sample) in a weigh boat and dry in an oven at 65 &#176;C for 12 h. After drying, grind the cooked grit sample to a fine powder using a coffee mill and store in a cool dry place for phenolics analysis.</li>
</ol>
2. Produce Baked Grits
<ol>
	<li>Place the remaining cooked grits on a foil-lined baking sheet.
	<ol>
		<li>To improve throughput, bake two samples concurrently. To do this, create two foil boats on a baking sheet. This eliminates the possibility of cross-contamination between the samples.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57809/57809fig3.jpg" /> 
Figure 3: Placement of cooked grits on baking sheet. Two different cooked grit samples are placed in individual foil boats on a baking tray prior to baking. The boats are labeled with green tape in the photo. This increased the throughput of the protocol while also ensuring cross-contamination did not take place. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57809/57809fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Place the baking dish containing the two samples in a pre-heated convection oven at 107.2 &#176;C (225&#176;F) for 50 min.
	<ol>
		<li>Stir the samples after 25 min of baking to ensure even baking.</li>
	</ol>
	</li>
	<li>At the end of the 50 min time period, remove the baking dish containing the first two samples and allow to cool at room temperature for 30 min.</li>
	<li>At the end of the cooling period, take a 30 g sample from the baked grits intermediate product. Place this sample in a weigh boat in an oven at 65 &#176;C for 12 h. After drying, grind the baked grit sample to a fine powder using a coffee mill and store for phytochemical analysis.</li>
</ol>
3. Produce Final Toasted Cornflake Product
<ol>
	<li>Roll the baked grits through a tortilla press.
	<ol>
		<li>Remove the remaining baked grits from the foil-lined baking dish and place these on a piece of parchment paper approximately 1 m in length. 
		NOTE: To increase throughput, it is helpful to fold the parchment paper length-wise into a pouch. This minimizes the amount of sample lost during the rolling stage that follows.</li>
		<li>Slowly feed the baked grit sample in the pouch through a tortilla press. Be careful to avoid pinching fingers in the press.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57809/57809fig4.jpg" /> 
Figure 4: Parchment paper pouch. (a) The parchment paper is folded lengthwise. (b) The long, open side of the pouch is folded over. (c) The long side is folded over again at a 10&#176; angle. (d) The short, open side of the pouch is folded over. This will be the side of the pouch that is fed through the tortilla press. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57809/57809fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Cut the rolled baked grits into 2.5 cm x 2.5 cm (1 in2) squares. Use a tool such as a pizza cutter to cut/score the rolled dough through the parchment paper.</li>
</ol>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57809/57809fig5.jpg" /> 
Figure 5. Cutting rolled dough into flakes. The rolled dough is scored through the parchment paper. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57809/57809fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Open the parchment paper and allow the rolled, cut, baked grits to dry at room temperature for 12 h.
	<ol>
		<li>To increase throughput, store the dried sample in a foil-covered weight boat at room temperature until multiple samples (typically 24 or more) are ready for toasting.</li>
	</ol>
	</li>
	<li>Pre-heat a convection oven to 204.4 &#176;C (400 &#176;F). Place the dried untoasted flake sample on a flat baking sheet. Spread the sample so that minimal overlapping of the sample occurs. This ensures even toasting.</li>
	<li>Place the sample in the oven for 60&#8211;90 s until it achieves the proper color (see Figure 6).</li>
</ol>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57809/57809fig6.jpg" /> 
Figure 6: Correct color of final toasted cornflakes. The cornflakes on the left side of the picture were toasted for the appropriate amount of time. The cornflakes on the right side of the picture were toasted for too long. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57809/57809fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="6">
	<li>Allow the sample to cool for approximately 5 min at room temperature. This yields the final toasted cornflake.</li>
	<li>Grind the toasted cornflake sample into a fine powder using a coffee mill.</li>
</ol>
4. Phytochemical and Statistical Analyses
NOTE: Depending upon the exact phytochemical of interest and the laboratory equipment available to researchers, these analytical protocols may change.
<ol>
	<li>Determine phytochemical content using a protocol such as that outlined in Butts-Wilmsmeyer et al.3 Follow all safety procedures provided in the protocols.</li>
	<li>Analyze the data using an appropriate statistical model. 
	NOTE: These example data were analyzed using a split-plot in an RCBD where the whole plot unit was the field plot from which grain was harvested, and the subplot unit was the processing stage. Analyses were conducted in PROC MIXED of SAS (version 9.3), and figures were produced in R.</li>
</ol>

<ol>
	<li>United States Department of Agriculture. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> National Agricultural Statistics Service. , (2017).</li><li>USDA. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ERS. , (2017).</li><li>Butts-Wilmsmeyer, C. J., Mumm, R. H., Bohn, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+of+Beneficial+Phytochemicals+in+Harvested+Grain+of+U.S.+Yellow+Dent+Maize+(Zea+mays+L.)+Germplasm.">Concentration of Beneficial Phytochemicals in Harvested Grain of U.S. Yellow Dent Maize (Zea mays L.) Germplasm.</a> Journal of Agricultural and Food Chemistry. 65 (38), 8311-8318 (2017).</li><li>Butts-Wilmsmeyer, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+phenolic+acid+content+in+maize+during+food+product+processing.">Changes in phenolic acid content in maize during food product processing.</a> Journal of Agricultural and Food. , (2018).</li><li>Dewanto, V., Wu, X. Z., Liu, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processed+sweet+corn+has+higher+antioxidant+activity.">Processed sweet corn has higher antioxidant activity.</a> Journal of Agricultural and Food Chemistry. 50 (17), 4959-4964 (2002).</li><li>Fast, R. B., Caldwell, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breakfast+cereals+and+how+they+are+made.">Breakfast cereals and how they are made.</a> Breakfast cereals and how they are made. , (2000).</li><li>Rausch, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+measurement+of+yield+and+composition+of+dry-milled+corn+fractions+using+a+shortened,+single-stage+tempering+procedure.">Laboratory measurement of yield and composition of dry-milled corn fractions using a shortened, single-stage tempering procedure.</a> Cereal Chemistry. 86 (4), 434-438 (2009).</li><li>Kandhola, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Processing and Genetic Effects on Resistant Starch in Corn Flakes. , (2015).</li><li>Macke, J. A., Bohn, M. O., Rausch, K. D., Mumm, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+factors+underlying+dry-milling+efficiency+and+flaking-grit+yield+examined+in+us+maize+germplasm.">Genetic factors underlying dry-milling efficiency and flaking-grit yield examined in us maize germplasm.</a> Crop Science. 56 (5), 2516-2526 (2016).</li><li>Somavat, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+lab+scale+corn+dry+milling+protocol+generating+commercial+sized+flaking+grits+for+quick+estimation+of+coproduct+yield+and+composition.">A new lab scale corn dry milling protocol generating commercial sized flaking grits for quick estimation of coproduct yield and composition.</a> Industrial Crops and Products. 109, 92-100 (2017).</li><li>Adam, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bioavailability+of+ferulic+acid+is+governed+primarily+by+the+food+matrix+rather+than+its+metabolism+in+intestine+and+liver+in+rats.">The bioavailability of ferulic acid is governed primarily by the food matrix rather than its metabolism in intestine and liver in rats.</a> Journal of Nutrition. 132 (7), 1962-1968 (2002).</li><li>Rumpagapom, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Structural Features of Cereal Bran Arabinoxylans Related to Colon Fermentation Rate. , (2011).</li><li>Wong, J. M. W., de Souza, R., Kendall, C. W. C., Emam, A., Jenkins, D. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colonic+health:+Fermentation+and+short+chain+fatty+acids.">Colonic health: Fermentation and short chain fatty acids.</a> Journal of Clinical Gastroenterology. 40 (3), 235-243 (2006).</li></ol>

The authors have nothing to disclose.

Changes in the nutritional content of maize-based food products throughout processing are likely due to the removal of gain components and thermal stress5,10. However, exactly how processing affects various nutrients had been studied in relatively little detail prior to the development of this protocol4,8. Furthermore, due to the large scale of most laboratory processing protocols, it has often been impossible to study the genetic basis of sensory and nutritional characteristics in maize grain8. Here, we present a microscale laboratory method for studying nutritional and sensory traits in maize throughout food product processing.
This protocol allowed sampling to take place at the flaking grit stage, after cooking, after baking, and after the shearing forces encountered during rolling. Thus, with the additional analysis of harvested corn grain, the protocol facilitates the analysis of the initial stage substrate and well as the final food product and intermediary stages of processing to elucidate changes in composition related to nutrition. This key feature of the protocol enables nutritional and sensory traits to be analyzed throughout processing while also enabling the researcher to choose which analytical chemistry protocols to use for those specific analyses. Another key feature of this protocol is the efficiency of this microscale protocol. First, this protocol uses a small sample, which is appropriate in a plant breeding setting (Table 3). One kg of grain tended to produce approximately 0.3 kg of large flaking grit constituents, and roughly one third of the large flaking grit constituents produced were needed for processing. Secondly, this protocol allowed for the laboratory processing of approximately 16 samples per day, which is much more efficient than the previous protocol that required large sample sizes6.
This protocol could easily be modified to mimic the production of other processed maize food products. For instance, large flaking grits are used in the production of various snack foods in addition to ready-to-eat breakfast cereals9. The laboratory protocol for the production of these snack foods could foreseeably include adjustments to cooking times and cooking solutions or adjustments to baking times. It is also possible that an adapted version of this protocol could be used for the study of other grains and their respective processed products. Processed grain products often include cooking, baking, or toasting processing stages that could be mimicked using an adapted version of the protocol presented here.
An important limitation of this protocol is that it has very few stopping points, i.e. once a processing step begins, it and subsequent steps must be completed (Table 3). There is a single stopping point after the production of the cooked grits from the flaking grits. Only if necessary, the cooked grits could be placed in a sealed container (e.g. a sealed canning jar) and refrigerated for at most two days. However, storing the cooked grits for longer time periods appeared to alter the sample. Furthermore, once baking begins, there are no stopping points until after the baked grit dough has been rolled, cut, and dried.
Conclusion
Through these example results (see Butts-Wilmsmeyer et al.4 for more information), we demonstrated that nutritional content could be monitored throughout processing. Furthermore, key processing stages where nutritional changes occurred were identified. Additionally, the small sample size required for this processing protocol enabled the study of multiple hybrids within the context of a plant breeding program. Using these hybrids, we identified which set of hybrids maintained the highest concentrations of insoluble-bound ferulic acid and p-coumaric acid throughout processing. These traits are important indications of the final toasted cornflakes&#39; prebiotic potential.11,12,13 These results could be used directly to help plant breeders establish breeding populations for improved prebiotic potential of processed maize products.
One of the major advantages of this processing protocol is that it does not limit the nutritional analyses that can be conducted. If a phytochemical protocol exists for analysis of the grain, then it can be used to study the processed products. Furthermore, because this processing protocol enables laboratory-scale food processing and nutritional analyses to be conducted independently, multiple phytochemicals can be studied. The analytical protocols for the study of phytochemical content should use small sample sizes, however, due to the small amount of intermediate and final processing products generated using the laboratory-scale processing protocol.

Maize (Zea mays L.) is the most widely cultivated grain crop in the United States1. In 2016, 71.12 billion kg (2.8 billion bushels) of maize were devoted to human consumption2, indicating the importance of maize in the American diet. One of the great benefits of maize grain is that it is a relatively inexpensive commodity, but it also contains beneficial phytochemicals such as phenolics, unsaturated fatty acids, and protein3. As such, maize-based food products may be relatively inexpensive sources of beneficial phytochemicals for humans.
However, maize must be processed prior to human consumption. As a result, processing activities often impact the nutritional value of the final processed food product4. For instance, during the production of snack foods and ready-to-eat breakfast cereals (i.e., cold cereals), maize kernels are dry milled to produce large flaking grits. During dry milling, the bran and germ are physically removed, leaving only endosperm material. Since many phytochemicals are predominantly located in either the bran or the germ (e.g., phenolics and unsaturated fatty acids, respectively), this may result in a significant decrease in the nutritional value of the processed food product4. Conversely, downstream processing steps may improve the nutritional value. For instance, many food product processing techniques include cooking, baking, or toasting. The thermal stresses encountered during these stages may improve the bioavailability of beneficial phytochemicals5.
From a food science and human nutrition perspective, it would be interesting to know how processing affects not only the nutritional value of processed food products but, foreseeably, also how adjustments to processing parameters may impact other sensory qualities, including color, texture, and taste. A protocol that allows such qualities to be monitored throughout processing could be used to select maize varieties for the improvement of the final processed maize food product. Two of the major obstacles to analyzing such characteristics in the past were the scale and throughput of available protocols. For instance, during the production of breakfast cereals for laboratory analysis, Fast and Caldwell6 suggested the use of 45.4 kg of large flaking grits. This mass of large flaking grits far exceeds the amount of large flaking grits or large flaking grit materials7 that can be produced from small plot field trials that are typical in plant breeding programs. Thus, the development of a microscale laboratory protocol for the production of processed food products could enable (1) plant breeders to improve maize varieties for nutritional and sensory traits that are of importance to food processors and (2) processors to efficiently design and test alternative processing strategies.
In this manuscript, we describe a high-throughput modification of the microscale processing protocol described in Kandohla8 that was used to produce toasted cornflakes from large flaking grit materials. We present the results of an example experiment that used this processing protocol to study the change in insoluble-bound ferulic acid and p-coumaric acid in maize. Our objectives in that specific study were to determine (1) how the phenolic acid content of maize changed during the production of ready-to-eat breakfast cereals, (2) at which processing stages those changes occurred, and (3) whether any of our experimental hybrids responded differently to processing stresses This protocol can be coupled with high-throughput analytical chemistry protocols for the efficient analysis of nutritional traits. This protocol can also be adjusted to mimic the production of other processed maize food products or processed food products that are produced from other grains.

<table><tbody><tr><td>Canning pressure cooker</td><td>Wisconsin Aluminum Foundry Co.</td><td>Model 921</td><td>Any can be used, but it should be large enough to accommodate multiple canning jars</td></tr><tr><td>Single burner or large hot plate</td><td>Waring Professional</td><td>Model SB30</td><td>Any can be used, but it should be large enough so that canning pressure cooker can securely be placed on burner or hot plate</td></tr><tr><td>1 quart wide mouth canning jars</td><td>Ball</td><td>1440096258</td><td>Any can be used, but they should be wide mouthed quart jars</td></tr><tr><td>1 L Beaker</td><td>Fisher Scientific</td><td>09-841-104</td><td></td></tr><tr><td>Stir plate</td><td>Corning</td><td>6796420D</td><td></td></tr><tr><td>Magnetic stir bar</td><td>Fisher Scientific</td><td>14-513-67</td><td></td></tr><tr><td>1 L Graduated cylinder</td><td>Kimble</td><td>20027500</td><td></td></tr><tr><td>Spatula</td><td>Wal-Mart</td><td>552145280</td><td></td></tr><tr><td>Hot pads</td><td>Wal-Mart</td><td>556501140</td><td></td></tr><tr><td>Scale</td><td>Any</td><td>NA</td><td>Mettler Toledo Model MS105DU or Similar</td></tr><tr><td>Weigh boats</td><td>Fisher Scientific</td><td>08-732-113</td><td></td></tr><tr><td>Sugar</td><td>Wal-Mart</td><td>9259244</td><td></td></tr><tr><td>Salt</td><td>Morton (Purchased at Wal-Mart)</td><td>9244849</td><td></td></tr><tr><td>Liquid malt extract</td><td>By the Cup (Purchased on Amazon)</td><td>NA</td><td>https://www.amazon.com/Barley-Malt-Extract-Syrup-Bottle/dp/B01N4SK72C</td></tr><tr><td>Labeling tape&amp;nbsp;</td><td>Fisher Scientific</td><td>15966</td><td></td></tr><tr><td>Permanent marker</td><td>Wal-Mart</td><td>55529894</td><td></td></tr><tr><td>Convection oven</td><td>Wal-Mart</td><td>1598495</td><td></td></tr><tr><td>Baking pan (usually included with oven)</td><td>Wal-Mart</td><td>1598495</td><td></td></tr><tr><td>Cooking foil</td><td>Wal-Mart</td><td>564264789</td><td></td></tr><tr><td>Tortilla press</td><td>E&amp;amp;A Hotel &amp;amp; Restaurant Equipment and Supplies</td><td>CTM-2000</td><td></td></tr><tr><td>Parchment paper</td><td>Reynolds (Purchased at Wal-Mart)</td><td>551219672</td><td></td></tr><tr><td>Pizza cutter</td><td>Farberware (Purchased at Wal-Mart)</td><td>&amp;nbsp;553012200</td><td></td></tr><tr><td>Cooling racks</td><td>Flytt (Purchased on Amazon)</td><td>NA</td><td>https://www.amazon.com/dp/B075HQY627/ref=sspa_dk_detail_7?psc=1&amp;amp;pd_rd_i=B075HQY627&amp;amp;pd_rd_wg=WaJol&amp;amp;pd_rd_r=SF07KCHMP753WAPG6ED4&amp;amp;pd_rd_w=2BOwf</td></tr><tr><td>SAS Version 9.4</td><td>SAS Institute</td><td>Version 9.4</td><td></td></tr><tr><td>R</td><td>R Foundation for Statistical Computing</td><td>Version 3.4.0</td><td></td></tr></tbody></table>

high-throughput, microscale protocol for the analysis of processing parameters and nutritional qualities in maize (zea mays l.)

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole grain composition and processing characteristics vary among maize hybrids and can impact the quality of the final processed product. Therefore, in order to produce healthier processed food products from maize, it is necessary to know how to optimize processing parameters for particular sets of germplasm to account for these differences in grain composition and processing characteristics. This includes a better understanding of how current processing techniques impact the nutritional quality of the final processed food product. Here, we describe a microscale protocol that both simulates the processing pipeline to produce cornflakes from large flaking grits and allows for the processing of multiple grain samples simultaneously. The flaking grits, the intermediate processed products, or final processed product, as well as the corn grain itself, can be analyzed for nutritional content as part of a high-throughput analytical pipeline. This procedure was developed specifically for incorporation into a maize breeding research program, and it can be modified for other grain crops. We provide an example of the analysis of insoluble-bound ferulic acid and p-coumaric acid content in maize. Samples were taken at five different processing stages. We demonstrate that sampling can take place at multiple stages during microscale processing, that the processing technique can be utilized in the context of a specialized maize breeding program, and that, in our example, most of the nutritional content was lost during food product processing.

This protocol allowed for the sampling and nutritional analysis of a processed maize food product, cornflakes, beginning with large flaking grits and continuing through intermediate stages of processing to the final product. This protocol was coupled with the protocol outlined by Rausch et al.7 to produce flaking grit components from hybrid grain samples. Thus, information regarding the nutritional content of hybrid samples analyzed at the whole grain, large flaking grit, cooked grit, baked grit, and toasted cornflake processing stages are presented. Regardless of the hybrid cultivar under evaluation, most of the insoluble-bound ferulic acid and p-coumaric acid was removed during dry milling (Figure 7). Another decrease in the insoluble-bound ferulic acid and p-coumaric acid occurred during cooking. The decrease in the insoluble-bound ferulic acid and p-coumaric acid content observed during cooking may be due to the removal of the small amount of non-endosperm material that remained in the large flaking grit material. Multi-degree of freedom contrasts indicated that both the ferulic acid and p-coumaric acid content remained stable throughout the remainder of processing, regardless of the hybrid (Table 1).
Furthermore, the initial ranking of the hybrid cultivars in terms of their insoluble-bound ferulic acid content and p-coumaric acid content were not indicative of the ranking of the hybrids at the final processing stage (Table 2 and Figure 8). In other words, the initial content in the whole kernel was not indicative of which hybrid would possess the most insoluble-bound ferulic acid or p-coumaric acid at the end of processing. Thus, in order to study the genetic traits underlying the nutritional characteristics of processed food products, microscale processes must be used to study maize grain.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57809/57809fig7.jpg" /> 
Figure 7: Change in insoluble-bound phenolic acid content throughout processing. (a) Change in insoluble-bound ferulic acid content throughout processing. (b) Change in insoluble-bound p-coumaric acid content throughout processing. WK: Whole Kernel, FG: Flaking Grit, CG: Cooked Grit, BG: Baked Grit, TO: Toasted Cornflake. Different color points represent different hybrids. Figure originally published in the supplemental information of Butts-Wilmsmeyer et al.4 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57809/57809fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57809/57809fig8.jpg" /> 
Figure 8: Interaction plot of the hybrid-by-processing stage interaction. (a) Interaction plot for insoluble-bound ferulic acid content. (b) Interaction plot for insoluble-bound p-coumaric acid content. The intersecting lines indicate a change-of-rank interaction, meaning that neither the insoluble-bound ferulic acid nor the insoluble-bound p-coumaric acid content of the final toasted cornflake can be predicted based upon the initial content of either of these phytochemicals in the whole kernel. WK: Whole Kernel, FG: Flaking Grit, TO: Toasted Cornflake. Figure originally published in the supplemental information of Butts-Wilmsmeyer et al.4 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/57809/57809fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2" style="text-align: center;">Ferulic Acid</td>
			<td colspan="2" style="text-align: center;">p-Coumaric Acid</td>
		</tr>
		<tr>
			<td>Hybrid</td>
			<td style="text-align: center;">F-value</td>
			<td style="text-align: center;">p-value</td>
			<td style="text-align: center;">F-value</td>
			<td style="text-align: center;">p-value</td>
		</tr>
		<tr>
			<td>B73xMO17</td>
			<td style="text-align: center;">0.07</td>
			<td style="text-align: center;">0.93</td>
			<td style="text-align: center;">0.34</td>
			<td style="text-align: center;">0.72</td>
		</tr>
		<tr>
			<td>B73xPHG47</td>
			<td style="text-align: center;">0.02</td>
			<td style="text-align: center;">0.98</td>
			<td style="text-align: center;">0.61</td>
			<td style="text-align: center;">0.55</td>
		</tr>
		<tr>
			<td>LH1xMO17</td>
			<td style="text-align: center;">0.08</td>
			<td style="text-align: center;">0.93</td>
			<td style="text-align: center;">0.14</td>
			<td style="text-align: center;">0.87</td>
		</tr>
		<tr>
			<td>PHJ40xLH123HT</td>
			<td style="text-align: center;">0.32</td>
			<td style="text-align: center;">0.73</td>
			<td style="text-align: center;">0.74</td>
			<td style="text-align: center;">0.48</td>
		</tr>
		<tr>
			<td>PH207xPHG47</td>
			<td style="text-align: center;">0.15</td>
			<td style="text-align: center;">0.86</td>
			<td style="text-align: center;">0.24</td>
			<td style="text-align: center;">0.79</td>
		</tr>
		<tr>
			<td>PHJ40xMO17</td>
			<td style="text-align: center;">0.01</td>
			<td style="text-align: center;">0.99</td>
			<td style="text-align: center;">0.31</td>
			<td style="text-align: center;">0.74</td>
		</tr>
		<tr>
			<td>PHG39xPHZ51</td>
			<td style="text-align: center;">0.06</td>
			<td style="text-align: center;">0.94</td>
			<td style="text-align: center;">0.07</td>
			<td style="text-align: center;">0.93</td>
		</tr>
	</tbody>
</table>
Table 1: Multi-degree of freedom contrasts testing the difference in insoluble-bound phenolic acid content in the cooked grits, baked grits, and toasted cornflakes.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2" style="text-align: center;">Ferulic Acid</td>
			<td colspan="2" style="text-align: center;">p-Coumaric Acid</td>
		</tr>
		<tr>
			<td></td>
			<td style="text-align: center;">F-value</td>
			<td style="text-align: center;">p-value</td>
			<td style="text-align: center;">F-value</td>
			<td style="text-align: center;">p-value</td>
		</tr>
		<tr>
			<td>Hybrids</td>
			<td style="text-align: center;">7.15</td>
			<td style="text-align: center;">0.001</td>
			<td style="text-align: center;">8.7</td>
			<td style="text-align: center;">&#60;0.001</td>
		</tr>
		<tr>
			<td>Inbreds</td>
			<td style="text-align: center;">4.07</td>
			<td style="text-align: center;">0.007</td>
			<td style="text-align: center;">6.57</td>
			<td style="text-align: center;">&#60;0.001</td>
		</tr>
		<tr>
			<td colspan="5">NOTE: All year-by-genotype-by-processing stage intereactions were non-significant at &#945; = 0.05.</td>
		</tr>
	</tbody>
</table>
Table 2: Significance of genotype-by-processing stage interaction.
<table align="center" border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td style="text-align: center;">Protocol Step</td>
			<td style="text-align: center;">Critical Information</td>
			<td style="text-align: center;">Troubleshooting</td>
			<td style="text-align: center;">High-Throughput Recommendations</td>
		</tr>
		<tr>
			<td style="text-align: center;">1.2 and 1.6</td>
			<td style="text-align: center;">The coupling of these two steps enables water to be heated without breaking the canning jars.</td>
			<td style="text-align: center;">NA</td>
			<td style="text-align: center;">The heating of half the water prior to the addition of the canning jars increases throughput.</td>
		</tr>
		<tr>
			<td style="text-align: center;">1.4</td>
			<td style="text-align: center;">NA</td>
			<td style="text-align: center;">NA</td>
			<td style="text-align: center;">Pre-measure ingredients.&#160; The given amount is for one jar, so multiply the volume or mass of ingredients needed by the number of jars used in step 1.4.2. Divide the resulting mixture equally among the canning jars.</td>
		</tr>
		<tr>
			<td style="text-align: center;">1.4 note</td>
			<td style="text-align: center;">Do not allow jars to touch edge of canning pressure cooker or each other. They will break, and sample will be lost.</td>
			<td style="text-align: center;">NA</td>
			<td style="text-align: center;">NA</td>
		</tr>
		<tr>
			<td style="text-align: center;">1.9 and 1.10</td>
			<td style="text-align: center;">Water should reach a rolling boil prior to cooking.</td>
			<td style="text-align: center;">If grits are not thoroughly cooked after one h, then check to ensure the pressure weight was set to 15 psi and that water is reaching a full boil before the lid is placed on the canning pressure cooker and the timer is set.</td>
			<td style="text-align: center;">NA</td>
		</tr>
		<tr>
			<td style="text-align: center;">1.13</td>
			<td style="text-align: center;">Remove the non-endosperm material that floats to the top during cooking.&#160; This will skew any phytochemical results if left in the sample.</td>
			<td style="text-align: center;">If non-endosperm material does not rise to the top during cooking, then the grits did not cook thorougly.&#160; See information regarding steps 1.9 and 1.10.</td>
			<td style="text-align: center;">NA</td>
		</tr>
		<tr>
			<td style="text-align: center;">1.15, 2.4, and 3.7</td>
			<td style="text-align: center;">Grind sample to a fine powder.</td>
			<td style="text-align: center;">If phytochemical analyses do not appear to work, ensure that the sample has been ground to a fine powder such that there is a greater surface area exposed to solvents.</td>
			<td style="text-align: center;">NA</td>
		</tr>
		<tr>
			<td style="text-align: center;">2.1</td>
			<td style="text-align: center;">Do not allow samples to touch one another.&#160; They will become cross-contaminated.</td>
			<td style="text-align: center;">NA</td>
			<td style="text-align: center;">Bake two samples concurrently by making individual foil boats for them on a cooking sheet.</td>
		</tr>
		<tr>
			<td style="text-align: center;">2.2.1</td>
			<td style="text-align: center;">Stir the sample after 25 min to ensure even baking.</td>
			<td style="text-align: center;">If the sample does not appear to have baked evenly, stir at more frequent intervals (e.g. every 15 min).</td>
			<td style="text-align: center;">NA</td>
		</tr>
		<tr>
			<td style="text-align: center;">3.1</td>
			<td style="text-align: center;">Place the baked grit dough in a parchment paper pouch.&#160; This ensures sample will not be lost during pressing.</td>
			<td style="text-align: center;">If sample starts to come out of the end of the parchment paper pouch, make the pouch longer. We found that 1 m appeared to be sufficient.</td>
			<td style="text-align: center;">NA</td>
		</tr>
		<tr>
			<td style="text-align: center;">3.2</td>
			<td style="text-align: center;">Leave the parchment paper pouch closed.</td>
			<td style="text-align: center;">If the cutting tool cuts through the parchment paper, use a duller tool.</td>
			<td style="text-align: center;">We found that a pizza cutter was the best tool for cutting the baked grits into squares.&#160; We did not cut through the parchment paper using this tool, but the baked grits still were able to be cut very quickly into squares.</td>
		</tr>
		<tr>
			<td style="text-align: center;">3.5</td>
			<td style="text-align: center;">Become very comfortable with the color and do not toast for too long.</td>
			<td style="text-align: center;">If the sample becomes too dark, reduce the amount of time used to toast.</td>
			<td style="text-align: center;">Store multiple dried baking grit samples in individual foil-covered weigh boats until multiple samples are ready for toasting.</td>
		</tr>
	</tbody>
</table>
Table 3: Table of critical steps, troubleshooting steps, and recommendations.

Watch this Scientific Journal Video about High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize Zea mays L. at JoVE.com

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize Zea mays L.

Here, we present a microscale protocol for processing grain samples and for incorporating this microscale approach into a high-throughput analytical pipeline. This is a higher throughput adaptation of currently available protocols.

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize (Zea mays L.)

Maize is an important grain crop in the United States and worldwide. However, maize grain must be processed prior to human consumption. Furthermore, whole ...

high-throughput-microscale-protocol-for-analysis-processing

Universidad San Sebastian

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6.8K Views.  University of Illinois at Urbana-Champaign. Here, we present a microscale protocol for processing grain samples and for incorporating this microscale approach into a high-throughput analytical pipeline. This is a higher throughput adaptation of currently available protocols.

Video: High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize Zea mays L.

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks to human and animal health. Among cereal production, maize is arguably the most affected crop, due to pathogen-induced losses in grain integrity and mycotoxin seed contamination. The two most prevalent and problematic mycotoxins for maize growers and food and feed processors are aflatoxin and fumonisin, produced by Aspergillus flavus and Fusarium verticillioides, respectively.
Recent studies in molecular plant-pathogen interactions have demonstrated promise in understanding specific mechanisms associated with plant responses to fungal infection and mycotoxin contamination1,2,3,4,5,6. Because many labs are using kernel assays to study plant-pathogen interactions, there is a need for a standardized method for quantifying different biological parameters, so results from different laboratories can be cross-interpreted. For a robust and reproducible means for quantitative analyses on seeds, we have developed in-lab kernel assays and subsequent methods to quantify fungal growth, biomass, and mycotoxin contamination. Four sterilized maize kernels are inoculated in glass vials with a fungal suspension (106) and incubated for a predetermined period. Sample vials are then selected for enumeration of conidia by hemocytometer, ergosterol-based biomass analysis by high performance liquid chromatography (HPLC), aflatoxin quantification using an AflaTest fluorometer method, and fumonisin quantification by HPLC.

The devastation of cereal crops by seed-infecting fungi has prompted numerous research efforts to better understand plant-pathogen interactions. To study seed-fungal interactions in a laboratory setting, we developed a robust method for the quantification of fungal reproduction, biomass, and mycotoxin contamination using kernel bioassays.

The rotting of grains by seed-infecting fungi poses one of the greatest economic challenges to cereal production worldwide, not to mention serious risks ...

Immunology and Infection

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of nitrogen-containing plant defensive compounds, and variation in species-specific correlations between nitrogen and plant protein content all limit the accuracy of these inferences. Additionally, research focused on plant and/or herbivore physiology require a level of accuracy that is not achieved using generalized correlations. The methods presented here offer researchers a clear and rapid protocol for directly measuring plant soluble proteins and digestible carbohydrates, the two plant macronutrients most closely tied to animal physiological performance. The protocols combine well characterized colorimetric assays with optimized plant-specific digestion steps to provide precise and reproducible results. Our analyses of different sweet corn tissues show that these assays have the sensitivity to detect variation in plant soluble protein and digestible carbohydrate content across multiple spatial scales. These include between-plant differences across growing regions and plant species or varieties, as well as within-plant differences in tissue type and even positional differences within the same tissue. Combining soluble protein and digestible carbohydrate content with elemental data also has the potential to provide new opportunities in plant biology to connect plant mineral nutrition with plant physiological processes. These analyses also help generate the soluble protein and digestible carbohydrate data needed to study nutritional ecology, plant-herbivore interactions and food-web dynamics, which will in turn enhance physiology and ecological research.

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn Zea mays As an Exemplar

The protocols described herein provide a clear and approachable methodology for measuring soluble protein and digestible (non-structural) carbohydrate content in plant tissues. The ability to quantify these two plant macronutrients has significant implications for advancing the fields of plant physiology, nutritional ecology, plant-herbivore interactions and food-web ecology.

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of ...

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the others contain 5&#39; monophosphate generated post-transcriptionally (processed transcripts). To discriminate between the two types of transcripts, several strategies have been developed, and most of them depend on presence/absence of 5&#39; triphosphate. However, the triphosphate at primary 5&#39; termini is unstable, and it hinders a clear discrimination of the two types of transcripts. To systematically differentiate and map the primary and processed transcripts stably accumulated in maize mitochondrion, we have developed a circular RT-PCR (cRT-PCR)-based strategy by combining cRT-PCR, RNA 5&#39; polyphoshpatase treatment, quantitative RT-PCR (RT-qPCR), and Northern blot. As an improvement, this strategy includes an RNA normalization step to minimize the influence of unstable 5&#39; triphosphate.
In this protocol, the enriched mitochondrial RNA is pre-treated by RNA 5&#39; polyphosphatase, which converts 5&#39; triphsophate to monophosphate. After circularization and reverse transcription, the two cDNAs derived from 5&#39; polyphosphatase-treated and non-treated RNAs are normalized by maize 26S mature rRNA, which has a processed 5&#39; end and is insensitive to 5&#39; polyphosphatase. After normalization, the primary and processed transcripts are discriminated by comparing cRT-PCR and RT-qPCR products obtained from the treated and non-treated RNAs. The transcript termini are determined by cloning and sequencing of the cRT-PCR products, and then verified by Northern blot.
By using this strategy, most steady-state transcripts in maize mitochondrion have been determined. Due to the complicated transcript pattern of some mitochondrial genes, a few steady-state transcripts were not differentiated and/or mapped, though they were detected in a Northern blot. We are not sure whether this strategy is suitable to discriminate and map the steady-state transcripts in other plant mitochondria or in plastids.

We present a circular RT-PCR-based strategy by combining circular RT-PCR, quantitative RT-PCR, RNA 5&#39; polyphosphatase-treatment, and Northern blot. This protocol includes a normalization step to minimize the influence of unstable 5&#39; triphosphate, and it is suitable for discriminating and mapping the primary and processed transcripts stably accumulated in maize mitochondrion.

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the ...

Genetics

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &#945;-amylase Inhibitor from Lablab purpureus L.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are potent carcinogens that substantially reduce crop value in maize and other oil rich crops like peanut besides posing serious threat to human and animal health. Different approaches, including traditional breeding, transgenic expression of resistance associated proteins, and RNA interference (RNAi)-based host-induced gene silencing of critical A. flavus gene targets, are being evaluated to increase aflatoxin resistance in susceptible crops. Past studies have shown an important role of &#945;-amylase in A. flavus pathogenesis and aflatoxin production, suggesting this gene/enzyme is a potential target to reduce both A. flavus growth and aflatoxin production. In this regard, the current study was undertaken to evaluate heterologous expression (under control of the constitutive CaMV 35S promoter) of a Lablab purpureus L. &#945;-amylase inhibitor-like protein (AILP) in maize against A. flavus. AILP is a 36-kDa protein, which is a competitive inhibitor of A. flavus &#945;-amylase enzyme and belongs to the lectin&#8211;arcelin&#8211;&#945;-amylase inhibitor protein family in common bean. In vitro studies prior to the current work had demonstrated the role of AILP in inhibition of A. flavus &#945;-amylase activity and fungal growth. Fungal growth and aflatoxin production in mature kernels were monitored in real time using a GFP-expressing A. flavus strain. This kernel screening assay (KSA) is very simple to set up and provides reliable and reproducible data on infection and the extent of spread that could be quantified for evaluation of germplasm and transgenic lines. The fluorescence from the GFP strain is closely correlated to fungal growth and, by extension, it is well-correlated to aflatoxin values.&#160; The goal of the current work was to implement this previous knowledge in a commercially important crop like maize to increase aflatoxin resistance. Our results show a 35%&#8211;72% reduction in A. flavus growth in AILP-expressing transgenic maize kernels which, in turn, translated into a 62%&#8211;88% reduction in aflatoxin levels.

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the &#945;-amylase Inhibitor from Lablab purpureus L.

Here we present a protocol to analyze Aspergillus flavus growth and aflatoxin production in maize kernels expressing an antifungal protein.&#160; Using a GFP-expressing A. flavus strain we monitored the infection and spread of the fungus in mature kernels in real time. The assay is rapid, reliable, and reproducible.

Aflatoxin contamination in food and feed crops is a major challenge worldwide. Aflatoxins, produced by the fungus Aspergillus flavus (A. flavus) are ...

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

Bioengineering

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of these analyses is of crucial importance, because growth is driven by cell division and cell elongation. Kinematic analysis represents a methodology to quantify these two processes. Moreover, this technique is easy to use in non-specialized laboratories. Here, we present a protocol for performing a kinematic analysis in monocotyledonous maize (Zea mays) leaves. Two aspects are presented: (1) the quantification of cell division and expansion parameters, and (2) the determination of the location of the developmental zones. This could serve as a basis for sampling design and/or could be useful for data interpretation of biochemical and molecular measurements with high spatial resolution in the leaf growth zone. The growth zone of maize leaves is harvested during steady-state growth. Individual leaves are used for meristem length determination using a DAPI stain and cell-length profiles using DIC microscopy. The protocol is suited for emerged monocotyledonous leaves harvested during steady-state growth, with growth zones spanning at least several centimeters. To improve the understanding of plant growth regulation, data on growth and molecular studies must be combined. Therefore, an important advantage of kinematic analysis is the possibility to correlate changes at the molecular level to well-defined stages of cellular development. Furthermore, it allows for a more focused sampling of specified developmental stages, which is useful in case of limited budget or time.

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Quantifying cell division and expansion is of crucial importance to the understanding of whole-plant growth. Here, we present a protocol to calculate cellular parameters determining maize leaf growth rates and highlight the use of these data for investigating molecular growth regulatory mechanisms by directing developmental stage-specific sampling strategies.

Growth analyses are often used in plant science to investigate contrasting genotypes and the effect of environmental conditions. The cellular aspect of ...

Biology

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

Scalable Transfection of Maize Mesophyll Protoplasts

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation or polyethylene glycol (PEG). Previous methods were developed to produce tens of thousands of transfected protoplasts at once. Here,&#160;we describe a straightforward method to isolate and transfect millions of leaf mesophyll protoplasts in maize (Zea mays L.). This streamlined process removes certain common protoplasting steps, such as washing in W5. Additionally, steps such as centrifugation, PEG-mediated transfection, and incubation have been modified to work with a greater number of protoplasts. The ability to express large libraries of plasmid constructs enables genome-scale experiments, such as massively parallel reporter assays in maize.

Here, we present a high-throughput protocol to transiently transfect millions of maize protoplasts for testing large libraries of plasmids in maize mesophyll cells.

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation ...

Improved Methods for Imaging Maize Leaf Primordia

Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging

In maize (Zea mays) and other grasses (Poaceae), the leaf primordia are deeply ensheathed and rolled within the leaf whorl, making it difficult to study early leaf development. Here, we describe methods for preparing transverse sections and unrolled whole mounts of maize leaf primordia for fluorescence and confocal imaging. The first method uses a wire stripper to remove the upper portions of older leaves, exposing the tip of the leaf primordium and allowing its measurement for more accurate transverse section sampling. The second method uses clear, double-sided nano tape to unroll and mount whole-leaf primordia for imaging. We show the utility of the two methods in visualizing and analyzing fluorescent protein reporters in maize. These methods provide a solution to the challenges presented by the distinctive morphology of maize leaf primordia and will be useful for visualizing and quantifying leaf anatomical and developmental traits in maize and other grass species.

Author Spotlight: Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging  

Maize leaf primordia are deeply ensheathed and rolled, making them difficult to study. Here, we present methods for preparing transverse sections and unrolled whole mounts of maize leaf primordia for fluorescence and confocal imaging.

In maize (Zea mays) and other grasses (Poaceae), the leaf primordia are deeply ensheathed and rolled within the leaf whorl, making it difficult to study ...

Enabling High-throughput Transgene Expression Studies Using Automated Liquid Handling for Etiolated Maize Leaf Protoplasts

The field of plant biotechnology has witnessed remarkable advancements in recent years, revolutionizing the ability to manipulate and engineer plants for various purposes. However, as research in this field increases in diversity and becomes increasingly sophisticated, the need for early, efficient, dependable, and high-throughput transient screening solutions to narrow down strategies proceeding to stable transformation is more apparent. One method that has re-emerged in recent years is the utilization of plant protoplast, for which methods of isolation and transfection are available in numerous species, tissues, and developmental stages. This work describes a simple automated protocol for the randomized preparation of plasmid within a 96-well plate, a method for the isolation of etiolated maize leaf protoplast, and an automated transfection procedure. The adoption of automated solutions in plant biotechnology, exemplified by these novel liquid handling protocols for plant protoplast transfection, represents a significant advancement over manual methods. By leveraging automation, researchers can easily overcome the limitations of traditional methods, enhance efficiency, and accelerate scientific progress.

This protocol describes the creation of a randomized transfection layout using an automated liquid handler, a protoplast isolation protocol for etiolated maize leaf, and a 96-well transfection procedure using a liquid handler.

The field of plant biotechnology has witnessed remarkable advancements in recent years, revolutionizing the ability to manipulate and engineer plants for ...

High-throughput, Microscale Protocol for the Analysis of Processing Parameters and Nutritional Qualities in Maize (Zea mays L.)

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Chapters in this video

Quantification of Fungal Colonization, Sporogenesis, and Production of Mycotoxins Using Kernel Bioassays

Quantifying Plant Soluble Protein and Digestible Carbohydrate Content, Using Corn (Zea mays) As an Exemplar

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

Inhibition of Aspergillus flavus Growth and Aflatoxin Production in Transgenic Maize Expressing the α-amylase Inhibitor from Lablab purpureus L.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

Kinematic Analysis of Cell Division and Expansion: Quantifying the Cellular Basis of Growth and Sampling Developmental Zones in Zea mays Leaves

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

Scalable Transfection of Maize Mesophyll Protoplasts

Improved Methods for Preparing Transverse Sections and Unrolled Whole Mounts of Maize Leaf Primordia for Fluorescence and Confocal Imaging

Enabling High-throughput Transgene Expression Studies Using Automated Liquid Handling for Etiolated Maize Leaf Protoplasts