The overall goal of this procedure is to down-regulate the lignin content in maze via RNAi to facilitate the production of digestible biomass for bioethanol production and to measure the lignin content using the Clayson lignin measurement protocol. This is accomplished by first constructing the maze CCR one RNAi plasmid to downregulate the lignin biosynthesis related gene syn oil COA reductase. Next maize high two embryo calli are transformed with the ZM CCR one RNAi construct Using particle bombardment and homozygous transgenic lines are generated.
Then the phenotypic changes are observed by means of a histological assay and by scanning electron microscopy performed on the first, second, and third generations of the transgenic maze. Finally, acid insoluble or clason lignin from the transgenic maze lines is measured and compared to the lignin content in non transgenic maze plants. Ultimately results are obtained that show a 10%lignin reduction in CCR one transgenic maze plants compared to wild type plants using particle bombardment and as determined by the Clayson lignin measurement End Visual Demonstration of this method is critical because some of the steps are difficult to learn and these steps difficult to learn include preparing Embry corn, Cali under sterile condition coating of tanks on particle with plasmid DNA and particle bombardment of the Cali, Dr.Hossein Aladin, and Dr.Gli Brew Han and undergraduate.
Donald would be demonstrating this research by showing the procedures through particle bombardment. The implications of the clayson lignin measurement technique for bioethanol production are that it not only provides information regarding clayson lignin quantities in different lignano cellose materials, but also provides insight into how resistant lano cellose biomass is to cellulite enzymes during pretreatment. To prepare tungsten particles begin by placing 60 milligrams of tungsten beads into a 1.5 milliliter tube.
Wash them by adding one milliliter of 70%ethanol and vortexing for two minutes. Then incubate at 23 degrees Celsius for 10 minutes and centrifuge at 18, 894 times G for two minutes before discarding the supernatant. Use one milliliter of 100%ethanol to wash the beads three times.
Then add one milliliter of sterile, 50%glycerol to bring the microparticle concentration to 60 milligrams per milliliter. To prepare DNA for bombardment, place 50 microliters of the tungsten beads in glycerol into a 1.5 milliliter tube and add the following vortexing between each edition. One microgram of IV 1.1 ZM CCR one RNAi plasmid DNA, prepared according to the text protocol.
50 microliters of 2.5 molar calcium chloride, and 20 microliters of 0.1 molar sperm aine. After a final vortex, centrifuge the mixture at 18, 894 times G for 30 seconds. Then pour off the supernatant and use 140 microliters of 70%ethanol to wash the pellet two times after discarding the final wash, add 48 microliters of 100%ethanol to the beads and use them immediately or store on ice for up to four hours prior to bombardment at least four hours prior to bombardment.
Place a three to five centimeter high, two embryo genic maze Calli in the middle of a 100 millimeter Petri dish containing N six OSM medium. Prepare the PSD 1000 helium particle delivery device according to the manufacturer's instructions and use 70%ethanol to sterilize the chamber wall. Next, load a sterile 650 PSI rupture disc into the sterile retaining cap.
Then spread five to six microliters of the M 10 DNA solution onto the surface of a macro carrier and dry briefly. Before loading the macro carrier and stopping screen into the micro carrier launch assembly, place the micro carrier launch assembly and maze calli in the chamber at a selected distance from the stopping screen and close the door. Accelerate in a vacuum of 27 PSI against the wire mesh screen.
Press the fire button until the rupture disc bursts and the helium pressure drops to zero on the gauge. Then release the fire button. Transfer the bombarded calli into a Petri dish containing N six OSM and incubate for 16 hours in the dark at 27 degrees Celsius.
Then break the Calli into about 10 pieces and transfer them to N six E or callous induction medium in Petri dishes and incubate for five days in the dark at 27 degrees Celsius. After about eight to 10 weeks, white fast growing sectors will grow out of the non proliferating and partially necrotic. Mother C eye excise the white fast growing tissues and transfer them to fresh selection medium, continuing to incubate them, transfer the white and fast-growing embryonic Cali onto regeneration medium and incubate in the dark at 27 degrees Celsius for one week before switching to a period of 16 hours daylight and eight hours dark at 25 to 27 degrees Celsius.
After three to four weeks transfer the regenerating shoots onto the rooting medium in a glass tent tube and continue to incubate in a light dark cycle After substantial root development appears, use tap water to wash the roots carefully. Then transplant the plantlets to four inch pots with soil. Cover the pots with plastic bags to keep the plants moist.
After two days, make small holes in the plastic bags. Then after five to six days, remove the plastic bags. Continue to incubate for another five to six days.
Transfer the seedlings into 18 inch pots with soil and maintain in full summer sunlight or greenhouse light. The initial regenerated plants are called T zero while the first seeds belong to the T one generation. To take clayson lignin measurements, mill the samples through a two millimeter screen.
Then use a moisture analyzer to determine the moisture content of each sample and record the value. Weigh out about 1.5 grams of each sample and record the weight with an automated solvent extractor. Use water to extract the samples.
Then use ethanol to extract them a second time. Dry the extracted samples at 45 degrees celsius overnight. Then allow them to cool in the desiccate before weighing them again.
Next, measure 0.3 grams of each dry extracted sample into three screw top high pressure tubes, and record the weights to the nearest 10th of a milligram. Then add three milliliters of 72%sulfuric acid to each pressure tube. Use glass or Teflon stir rods to mix the samples and leave the stir rods in the tubes.
Incubate the vials at 30 degrees Celsius for 60 minutes at 150 RPM. Then add 84 milliliters of deionized water to dilute the acid concentration to 4%mixing with the stir rod. Take care not to leave large amounts of sample on the sides of the vial above the waterline.
After tightly sealing the stoppers on all vials, place them into a metal rack or large beakers and autoclave them at 121 degrees Celsius. Using a liquid sterilization cycle for one hour, allow them to cool to room temperature before opening. Pre ash the filtering crucibles in a furnace at 575 degrees Celsius for at least four hours.
Then allow the crucibles to cool in a desiccate for at least one hour using a rubber adapter to secure each crucible vacuum, filter the solution from each tube through a separate crucible. Use deionized water to rinse the particles from each tube. Dry the lignin residue at 105 degrees Celsius for a minimum of four hours before recording the weight of the dry crucible and the residue.
Using a 575 degrees Celsius furnace. Pre-fire the samples over a bunsen burner until there is no visible smoke or ash before placing them in the furnace for 24 hours. Remove the crucibles from the furnace and cool in the desiccate.
Finally, calculate the acid insoluble residue using the following equation where M pre equals the mass of the pre-ex extracted biomass M post equals the mass of the postex extracted biomass. M vial equals the mass of the extracted biomass added to the vial M residue equals the mass of the crucible and lignin residue. M ash equals the mass of the crucible and ash and mc equals the moisture content of the pre-ex extracted biomass.
Total weight basis reduction in lignin content in maze via DS RNAi particle bombardment transformation yielded approximately 30%gene silencing of zm CCR one was consistently observed in T zero through T two generations compared to control plants. The transgenics grew similarly except for brown coloration in the leaf, mid rib husk and stem as shown here. Histological analysis revealed that the mutant lines exhibit a significant reduction in cell wall thickness of the scle enma fibers.
However, the xylem vessel flow and sheath cells appeared similar to wild type plants, suggesting that there are no detrimental effects to water transport, nutrient transfer, or mechanical strength to the stems in the mutant lines. This figure shows the amount of acid insoluble clason lignin in wild type maze and zm CCR one RNAi transgenic lines. Three transgenic lines showed a statistically significant reduction in lignin compared to wild type plants to determine whether carbon flow was shifted from the lignin biosynthetic pathway to cell wall carbohydrate biosynthesis pathways.
Cellulose was analyzed via the graph method as seen in this plot. Two zm CCR one RNAi mutant lines contained significantly increased levels of crystalline cellulose. Analysis of hemi cellulose content, however, revealed no changes in the mutant lines After its development.
This technique paved the way to researchers in the field of plants transgenic visual visualizing. This technique will provide guidance to researchers who need to introduce a gene of interest in high transformation frequencies. In addition, the particle bombardment technology we use could be also applied to transfer of genes to other monocots such as wheat, rice, sorghum, and other even dichot plants.
After watching this video, you should have a good understanding of how to measure clayson lignin from li, no cellulose biomaterials. It is important to mention that working with 72%sulfuric acid can be extremely hazardous, so any personnel handling the chemical should be very cautious and keep the chemicals and safety cabinets and work with it only under a fume hood.
