The overall goal of this procedure is to quantify the symbiotic phenotypes of medic cargo Tula plants inoculated with different mutant variants of the nitrogen fixing bacterium sino oum me latte, using single plant sterile microcosms made from standard laboratory plates. This is accomplished by first scarifying Mula, a 17 seeds with acid and germinating the seeds on agar plates. The second step is to prepare single plant sterile microcosms using a bent hot spatula notch each solidified plate and its lid closing the plate.
Later, we'll leave an oval hole in the side. Next, lay out individual Mula seedlings on the microcosms with the root tip, contacting the agar and the co leadin and 0.5 centimeters of the chute protruding out of the U notch in the plate. The final step is inoculation with SME latte and incubation of the plates as foil wrapped stacks in a lighted growth chamber.
Ultimately quantification of plant shoot length and weight and nodule type is used to show the relative symbiotic success of each S milliot high strain or ULA mutant. The main advantage of this technique over other methods that allow continuous visual examination of root growth like growth pouches and cason, is that there's very little risk of cross-contamination of plants inoculated with different Cy Melo strains. This method can help answer key questions in the Zoia legum symbiosis field, such as the timeframe of root nodule development in mego trica plants inoculated with a collection of s rosa Melodi strains that invade roots at different efficiencies.
Visual demonstration of this method is critical as the construction of the microcosms and the transfer of seedlings is difficult to describe in text. Assisting me in demonstrating this procedure will be Haji Mendez, a grad student and Clotilde Caru a postdoc in my laboratory. This procedure begins with sterilization of the M trunk aula.
A 17 seeds by acid scarification first weigh sufficient seeds for the desired number of seedlings. Mula A 17 seeds are about four milligrams each transfer seeds to sterile 125 milliliter flasks with sterile foil covers and pipette. 20 milliliters of concentrated sulfuric acid along the side of the flask pipetting the acid along the side of the flask will raise, sterilize the inside of the flask that is come into contact with unsterilized seeds.
Break up clumps of seed with a sterile glass pipette. Swirl each flask frequently for 10 to 12 minutes. Small brown pits will become visible on the seeds.
These are tiny holes in the seed coat when at least 10%of the seeds have small brown pits or 12 minutes have passed, whichever comes first, remove all acid from the flask with a sterile glass pipette. Tilt the flask to collect the remaining acid in the curve of the flask and use a small glass pipette to remove all the remaining acid rinse seeds by quickly pouring 100 milliliters of sterile ultrapure water into each flask. The excess volume of water will dilute the acid and prevent overheating and seed damage during the reaction of the acid with the water, pour off the water and flame sterilize the lip of the flask.
Add 50 milliliters of sterile ultrapure water into each flask and swirl. Pour off the water, flame the lip of the flask. Add another 50 milliliters of sterile ultrapure water and swirl in this manner.
Rinse seeds eight to 10 times after the last rinse. Pour 50 milliliters of sterile ultrapure water into each flask and replace the sterile foil cover place at four degrees Celsius in the dark overnight. To let the seeds imbibe on the following day take the two flasks to a sterile laminar flow hood.
All subsequent steps must be performed in a sterile laminar flow hood because airborne mold spores are the most problematic contaminants. Pour off the water from each flask and rinse twice with approximately 25 milliliters of ultrapure water. After the second rinse, add 20 milliliters of sterile ultrapure water to each flask swirl to resuspend seeds and pour seeds into a 1.1 to 1.2%Agar germination plate swirl the plate to distribute the seeds.
The seeds should be distributed evenly in a four to five centimeter band. At one end of the plate, this will be the top of the plate. The bottom five to six centimeters should be kept free of seeds.
Draw off the water with a sterile pipette. Tilt the plate at a 45 degree angle for 10 to 20 minutes to let the remaining water drain to the bottom of the plate. Next, remove all water that has collected at the bottom of the tilted plate with a sterile pipette.
Replace the lid and seal the plate with param. Stack the germination plates and then turn them all up. At a 90 degree angle, the seeds will be lying on the vertical surface of the agar.
Fully imbibed seeds should easily adhere to the agar. The roots will grow downward. Wrap the stack of germination plates in foil to block light and keep at 25 degrees Celsius for three days.
To begin this procedure, prepare jenssen's medium following the recipe and directions in the accompanying manuscript. After the medium has cooled, supplements have been added and pH has been checked, begin pouring the medium into round 100 millimeter diameter. 15 millimeter deep plates.
Plates should be poured approximately 0.9 to one centimeter deep. Using about 70 milliliters of medium per plate. Stack plates during pouring to prevent the formation of condensation on the plate lids.
Components of jenssen's medium can form a cloudy precipitate, so it's important to return the pitcher to the magnetic stir plate periodically to prevent components from settling out. After the jenssen's plates have solidified both plates and lids must be notched with a weighing spatula that has been bent into a U shape. Heat the bend of the spatula with a burner until red hot notch, five to six plates and then heat and notch again, also notch the plate lids.
When the notched lid is placed on the notched plate, an oval portal will be created through which the chute of the seedling will grow. Three days after Mula seeds have been plated for germination as shown earlier, open a germination plate and immediately flood it with sterile ultra pure water. Dip forceps in ethanol and flame briefly to sterilize.
Use the sterile forceps to gently push the root tips of all seedlings under the water to prevent drying, select seedlings with straight roots and no obvious defects. Check the lids of jenssen's medium microcosms for the buildup of condensation. Flick the lid to remove the condensation using forceps.
Gently pick a seedling from the germination plate by the edins and lay the root on a jenssen's. Medium microcosm. Make sure the root tip is in contact with the agar.
Gently remove the seed coat if it is still present. Gently tease the seed coat with the forceps until it gives way and discard the co leadin and approximately 0.5 centimeters of chute. Should protrude out of the U notch in the plate carefully replace the lid of the plate with the U notch of the lid over the seedling, creating a portal for the seedling prior to inoculation of microcosms esil latte inoculum suspensions are prepared from S mil latte cultures as described in the accompanying text.
Each suspension is diluted in sterile ultrapure water to an OD 600 of 0.05 and the cloudiness of the suspension should be just barely perceptible by eye. Open each microcosm and inoculate 100 microliters of the appropriate salvati suspension onto the seedling root. Replace the lid and set aside in horizontal stacks of six to 10 microcosms for each SEL strain to be compared.
Inoculate at least 15 plants for strains that have subtle differences in symbiotic productivity. Inoculate 30 to 35 plants Leave at least 10 plants un inoculated as a negative control inoculate 30 to 35 plants with Esme Lata 1 0 2 1 or another appropriate wild type strain to serve as the positive control after at least 20 minutes to allow the Esme Lata suspension to absorb onto the root surface and the suspension liquid to soak into the agar wrap each plate individually with paraffin film plates should be wrapped up to the very edge of the notch, but the paraffin film should not block the growth of the seedling. Line up the seedling portals of each stack of six to 10 plates.
Lay the stack at a 90 degree angle with the seedlings pointing upward. The stickiness of the paraffin film will hold the plates in place long enough to wrap the entire stack in foil, leaving a one to two centimeter strip unwrapped where the seedlings emerge. Place the foil wrapped stacks in a lighted growth chamber or growth room set to appropriate conditions.
Plants can be maintained in microcosms for up to nine weeks. Nodule number can be quantified at a series of time points by unwrapping the foil from each stack and counting nodules. Developing nodules on plants inoculated with SME 1 0 2 1 wild type are apparent by 10 to 14 days after inoculation.
Symbiotic productivity can also be measured at each time point by measuring the length of the emerging chute. Final quantitation of symbiotic productivity is usually performed at seven weeks after inoculation by detaching the chute and measuring the fresh weight of the ch young mula seedlings. Growing in microcosms in an incubator are shown in this photo.
If packed tightly greater than 220 microcosms will fit on a 73.8 centimeter by 67.5 centimeter incubator shelf. These next four images show several different views of microcosms containing Mula. A 17 plants inoculated with wild type S, Malta 1 0 2 1 panels B and C show microcosms in a foil wrapped stack with plants emerging from the portals in the microcosms.
The shoot length can be measured from the point of emergence from the microcosm without disturbing the plant. Panel D shows a single microcosm with the foil removed and the root and nodules showing through the front of the plate. Panel E shows the same microcosm lying flat with the plant emerging from the portal in the plate.
Growth in the microcosms is ideal for collecting a series of time points of nodule development and shoot length. This graph shows shoot length data for emran atula. A 17 plants inoculated with wild type Es Malta 1 0 2 1 compared with un inoculated plants at seven weeks and nine weeks post inoculation.
At nine weeks post inoculation shoots are clipped off for measurement of shoot fresh weight. Un inoculated plants will cease to grow and turn yellow and their shoots will attain a weight of only approximately 0.01 grams. Plants inoculated with ssta strains that invade Mula more efficiently will grow faster and will attain a weight of 0.10 to 0.12 grams by nine weeks post inoculation.
This figure shows examples of three symbiotic phenotypes at seven weeks post inoculation. The relative symbiotic productivity of MTR aula. A 17 plants inoculated with different smeta streams is quantified by shoot length in centimeters, meters, and shoot fresh weight in grams.
Asterisks over graft bars denote a statistically significant difference from the 1 0 2 1 wild type reference strain in an unpaired T-test. The number of root nodules on plants inoculated with these strains is shown in Panel C.A pink color indicates that nodules are functional and capable of nitrogen fixation while small white nodules are usually non-functional and brown nodules are senescent and necrotic. The comparisons in this figure showed that the symbiotic productivity of M trunk aula A 17 plants inoculated with SML Lavati 1 0 2 1 carrying the P-S-T-B-L-A FFR five XOY plasmid, which over expresses the XOY ecop parental phosphate galactose phospho transferase is greater than that of wild type S me 1 0 2 1 and strains carrying the negative control plasmid P-S-T-B-L-A-F-R five.
These ex oy over expressing strains produce more of the symbiotic exo polysaccharide SAG glycan than the control strains. The ability to quantify differences in symbiotic phenotypes that take several weeks to manifest is a major advantage of this method for all measures of symbiotic productivity. On MTR Aula A 17, the Sano Zoia Medicaid strains WSM 4 1 9 and ABS seven perform better than Smeta 1 0 2 1 plants inoculated with an XO YN five null mutant that does not produce any suc and O glycan had similar shoot lengths and fresh weight to un inoculated plants and produced only ineffective nodules at the time that the microcosm is disassembled and shoot fresh weights are measured shoots can also be removed.
For imaging representative views of M trunk atula. A 17 roots after removal from microcosms are shown in this.Figure. Panels A and B are images at 10 days post inoculation and panel C is an image at 14 days post inoculation, the bar in each image corresponds to 100 microns.
The roots shown here were inoculated with smeta 1 0 2 1 wild type, an smeta strain carrying an egg, C Gus Fusion and an smeta strain carrying an SMC 0 0 9 1 1. Gus Fusion in Panel C gusts staining of STI cells carrying the SMC 0 0 9 1 1 Gus fusion is visible on the root hairs and the root surface. While attempting this procedure, it's important to remember to keep the seedling root we or in contact with the agar at all times Following this procedure.
Other methods like csection and staining of nodules are preparation of total RNA from roots and nodules can be performed in order to analyze the differences in anatomy, physiology, and gene expression that are associated with each symbiotic phenotype. After watching this video, you should have a good understanding of how to set up single plant sterile microcosms for observation of the Mego Tula Sino Rosa Melo symbiosis, including seed germination, microcosm preparation, and seedling inoculation.
