The overall goal of this procedure is to measure herbicide metabolism in a dichot weed using an excised leaf assay. This is accomplished by first planting and cloning water hemp populations in the greenhouse and growth chamber. The second step is to incubate excised waterhemp leaves with radio labeled herbicide and conduct a time course study.
Next, the herbicide and its metabolites are extracted from the incubated leaves. The final step is the analysis of herbicide and its metabolites by reversed phase high performance liquid chromatography. Ultimately, this excised leaf assay is used to determine accurate rates of herbicide metabolism in different weed populations or species.
The main advantage of this technique or existing method like spotting radio, labeled herbicide on leaves or whole plant is that genetically identical or harm seedlings are analyzed in independent or whole plant translocation. This method can help answer key questions in the plant biochemistry field, such as determining herbicide resistance mechanisms in weeds or herbicide tolerance mechanisms in crops. Demonstrating the HPRC procedure will be judged.
Skeleton, a grad student from our laboratory In preparation, collect and suspend waterhemp seeds in an aqueous auger medium. Then store the seeds in the medium at four degrees Celsius for at least 30 days to break their dormancy and thus improve their germination. When the seeds are ready, germinate them in the greenhouse with a 16 eight photo period, a 28 degree Celsius day temperature, and a 22 degree Celsius night temperature plant.
The seeds in 12 by 12 centimeter trays provide supplementary light from a mercury allied lamp for a photon flux of 500 micromoles per square meter per second. At the canopy level, prepare to clone out six seedlings from each population to neutralize experimental variability. When the seedlings are two centimeters tall, transplant them into 80 CC pots.
When the seedlings are four centimeters tall, prepare a three to one to one to one mixture of potting. Mix soil, peat and sand. Load the mixture into 950 cc pots and add five grams of slow release fertilizer granules to each pot.
Then transplant the seedlings one seedling per pot. When the plants are seven centimeters tall, cut and remove the chute. Apical Maris stem containing the three youngest leaves to eliminate apical dominance and guarantee enough axillary shoots for cloning purposes.
Later, when the plants reach 14 centimeters, excise five three centimeter long axillary shoots from each water hemp population in the study. To minimize water loss, remove most of the leaves from the harvested chutes, retaining the two youngest leaves to allow further growth upon transplanting Next transplant each chute into an A D CCC pot with potting medium, the same medium used for germination ensure that potting medium is fully saturated until roots form in the growth chamber. Third, plants are very sensitive at this point and should not be stressed, so the potting medium should be fully saturated with water until roots form.
Gently transfer the plants to a growth chamber with environmental settings like the greenhouse there. Keep the soil moist for seven days, so the roots get established. Use a mix of lights for 550 micro MO photons per square meter per second.
After the roots have formed, and once the plants are four centimeters tall, transfer them to the greenhouse. Then transplant the seedlings into 950 CC pots with the three to one to one to one mixture and slow release fertilizer granules prepared as before. Then repeat the cloning cycle once to expand the populations.
For this experiment, use cloned water hemp plants that are 10 to 12 centimeters tall to begin excise. The third youngest leaves, they are two to three centimeters long. Include a half centimeter of the attached peole.
Next underwater, cut the leaf petioles from each water hemp plant a second time leaving about 0.3 centimeters of the peole. Now immerse the cut leaves into the pre incubation buffer for an hour in the growth chamber. If desired, treat the leaves with a metabolic inhibitor at this point.
After an hour transfer each leaf into a 1.5 milliliter plastic tube containing 200 microliters of incubation buffer with radio labeled herbicide. Incubate the leaves at 28 degrees Celsius for an hour, so they absorb the herbicide to complete the treatment. Wash the leaves with deionized water and transfer them to 1.5 milliliter tubes containing quarter strength MS.Salt solution.
Keep the tubes in the growth chamber. Incubate the leaves for several different time durations sufficient to determine rates of metabolism.Later. Grind each leaf tissue in liquid nitrogen using 15 milliliter polypropylene tubes and glass pestles.
Then extract the radio labeled herbicide and its metabolites from each leaf. Add seven milliliters of 90%acetone to the tube and make a suspension using a tissue. Homogenizer homogenize the tissue until it is thoroughly pulverized.
Usually this takes a few minutes. Then rinse off the tissue. Homogenizer with another seven milliliters of acetone.
Store the homogenate for 16 hours at minus four degrees Celsius for added extraction. The next day, spin down the tubes at 5, 000 GS for 10 minutes and collect the supernatants. Then concentrate the supernatants using a rotovap at 40 degrees Celsius until a half milliliter of solution remains.
Transfer this volume to a 2.0 milliliter plastic tube. Increase the volume to 1.25 milliliters with one-to-one a cedar nitrile water solution, and spin this down at 10, 000 GS for 10 minutes to remove the particulates. Next, resolve the total extractable radioactivity using RP HPLC.
For most non-polar herbicides, use a C 18 column at a flow rate of one milliliter per minute. For LUNA use 0.1%formic acid for LUNB, use HPLC grade acetonitrile. For further details on this and a statistical analysis method, consult the text protocol using the described protocol.
Large differences in rates of meso trione metabolism were detected between three populations of waterhemp. For example, meso trione metabolism and MCR was significantly faster than a CR or WCS as indicated by its short DT 50 value. As a specific example of the data, the time course for mistri metabolism in clonal line five is seen here.
A separate study investigated the resistance mechanism to a LS inhibitors, primi sulur on methyl and a LS inhibiting herbicide of the sulfonylurea Family was used to treat the leaves. Typically, the amount of primi sulur on methyl in MCR plants was lower than the other populations in the study. Additionally, two polar metabolites with shorter retention times than primi sulur on methyl were detected in the excised leaf extracts from all three populations.
Their forms are consistent with ring hydroxylation of primi sulur on methyl followed by glucose conjugation. Next, the amount of parent herbicide remaining at each time point was quantified and used to determine the DT 50 values for primi sulfur. Methyl shorter values were found in the MCR plants as expected.
Curiously, the DT 50 values in the other plants were similar. Despite each displaying a different response to primi sulur methyl Following this procedure. Other methods like RNA sequencing can be performed in order to answer additional questions about the identification and expression of genes involved in herbicide detoxification in plants After its development.
This technique will pave the way for researchers in the field of plant physiology to determine accurate breath or herbal metabolism.
