Floral dipping is used to transform arabidopsis plants with a reporter construct. The resulting transgenic plants are then examined for the expression of the reporter gene. First young flowering arabidopsis plants are dipped in a solution of agrobacterium to Ephesians containing the reporter construct.
Next, the plants are returned to their normal growth conditions until they produce seeds a small percentage of which will be transformed with the foreign gene. The third step of the procedure is to collect the seeds from plants and sow them on selection medium containing antibiotics. Finally, the seedlings growing on the selection medium with antibiotics are examined.
Transgenic seedlings containing the foreign gene are resistant to the antibiotics and remain alive while the non transgenic seedlings eventually turn white and die. Reporter gene expression in the transgenic seedlings is observed by staining the transgenic seedlings with the substrate of the reporter gene and observing the cells and tissues under a dissecting microscope. Hi, I'm Chloe Mara from the laboratory of Dr.Zou in the Department of Cell Biology and Molecular Genetics at the University of Maryland.
I'm Wna gva also from Dr.Z Chile's lab. Today we'll show you the floral depth method for transforming arabidopsis plants. We'll then show you how to examine reported gene expression in transgenic plants.
Our laboratory routinely uses these procedures to study gene expression and functioning plants. So let's get started. Begin this procedure approximately one month prior to transformation.
Sow a rabbit ops of seeds into four to eight five inch square pots with approximately 10 to 15 plants per pot, and then place the sode seeds at four degrees seed in a cold room for three days before placing them in. Plant growth chambers for transforming mutant arabidopsis plants with reduced fertility. Increase the number of pots adding six to 10 more pots.
Grow the plants under standard conditions. Plants grown under a shorter day or lower temperature regimen, take longer to flower and longer to be ready for transformation. Good plant care ensures healthy plants which are essential for successful transformation.
Spraying growing plants with pesticide is recommended when the plants have just bolted and begun to flower. They're ready for transformation to increase transformation efficiency. Three to four days before the transformation trim off the main in fluorescence shoots as soon as they have bolted.
To encourage more secondary shoot formation two days prior to transformation, inoculate 40 milliliters of LB containing 50 micrograms per mil. Canin with agrobacterium Tema Fasion strain GV 3 1 0 1 containing the PTs oh two Gus construct grow overnight in a shaking incubator at 20 to 30 degrees Celsius. Gus is cloned into the PIN 20 vector, which also carries can mycin resistance.
The TSO two promoter drives cell cycle regulated expression of Gus the following day, which is the day before transformation. Transfer eight milliliters of the overnight culture into 400 milliliters of LB containing 50 micrograms per milliliter. CIN grow overnight in a shaky incubator at 28 to 30 degrees Celsius on the same day.
Also water the plants. This ensures that the soil does not soak up too much. Infiltration media during floral dipping on the day of transformation followed the absorbance of the bacteria at 600 nanometers.
When the OD 600 of agrobacterium culture reaches approximately 0.8, spin down the agrobacterium overnight culture for eight minutes. At 5, 000 RPM at four degrees centigrade Resuspend, the agrobacterium pellet in one liter of freshly made infiltration media. The medium does not need to be autoclave.
Pour the one liter agrobacterium containing infiltration medium into an eight inch by 15 inch by two inch pyx glass dish. So we're about to dip the plants into the infiltration solution. While doing this.
Pay particular attention to avoid dipping the soil of the pot into the infiltration solution. Now that the bacteria are ready, invert the pot and dip all aerial parts of the plants into the infiltration filtration solution. Hold for five minutes.
Dipping one pot at a time while all the aerial parts of the plant are immersed in the infiltration medium. Avoid letting the soil soak up any of the infiltration media to reduce chances of later fungal growth on the soil. After dipping place all dipped pots on their side on several layers of paper towel in a tray.
The paper towels soak up excess amount of infiltration media. Cover the tray with plastic wrap to ensure a high humidity. Place the tray into the plant growth chamber on the following day.
Remove the plastic wrap in paper towels and place the pots upright. Do not water these plants for four to five more days afterwards. Maintain the plants normally until they set seeds about three weeks later and collect the seeds in bulk.
Proceed to select the transgenic seeds. First set out plates with Ms.Medium containing 50 micrograms per milliliter. Can mycin then weigh and estimate the amount of seeds.
Knowing that 0.1 gram of seeds equals about 5, 000 seeds, one needs to screen a minimum of 20, 000 seeds to select for antibiotic resistant transgenic plants. Next, sterilize the seeds by washing them with 70%ethanol in a 15 milliliter Falcon tube for 30 seconds. Then washing the seeds with sterile water.
Once soak the seeds for 30 seconds in a solution containing a one to 10 dilution of store-bought 5%bleach. And finally rinse the seeds with sterile water three to six times. After sterilizing the seeds work in the sterile hood and pipette about 5, 000 seeds into each MS plate.
Containing 50 milligrams per milliliter. Can mycin spread the seeds evenly on the plate and seal the plates with micropore 3M tape to avoid contamination. Incubate the plates at four degrees centigrade in the dark for three to four days and transfer the plates to the plant growth chamber.
After about 14 days, transgenic seedlings stay green. They're bigger and develop true leaves in addition to the cos, compared with the non transgenic seedlings, which turn pale green arrest growth, and eventually die. Transformation efficiency should be approximately 0.1 to 0.2%on average.
About 100 transgenic lines can be obtained by transforming four pots of wild type plants. Identify the transgenic plants and slowly pull their roots out of the medium. Transfer the individual seedlings to soil cover with plastic for one to two days.
Then remove the cover. Allow the plants to grow after harvesting the transgenic seedlings. Place them in glass scintillation vials or einor tubes containing cold.90%acetone.
Keeping the samples on ice polystyrene but not polypropylene. Microtiter plates can also be used for analyzing a large number of samples. After all the samples are harvested, place the vials at room temperature for 20 minutes.
During this time, make up fresh staining buffer without xgl and place on ice. Also, prepare staining buffer containing xgl by diluting a 100 millimolar stock solution of xgl to a final concentration of two millimolar xcl. At the end of the 20 minute incubation, remove the acetone from the samples and add the staining buffer without the xgl.
Then remove the staining buffer without xgl from the samples and add the staining buffer containing xgl to the samples. Proceed to infiltrate the samples under vacuum for 15 to 20 minutes. Release the vacuum slowly and verify that all the samples sink beneath the surface of the staining solution.
If necessary, repeat the infiltration until all the samples sink beneath the surface. After the vacuum is released, after the vacuum infiltration, incubate the samples at 37 degrees centigrade overnight in the staining buffer containing xgl. The next day, take the samples out of the incubator and remove the staining buffer.
Wash the samples in a successive ethanol series at room temperature for 30 minutes for each change. Finally, fix the tissues in FAA fixative for 30 minutes at room temperature. Then remove the FAA fixative and add 70%ethanol.
Examine the tissues under a dissecting microscope equipped with a digital camera or store at four degrees centigrade for later viewing in the plant shown here. Dark blue color reflects GUS activity driven by the TS O2 promoter. The chute apex has young leaf primordial with actively dividing cells.
This is reflected by highly expressed TS O2 gene for DNDP biosynthesis indicated by the dark blue color of Gus activity. Lateral root primordial that stained blue can also be observed and staining can be seen in the root tip as well. The non-uniform sporadic pattern is characteristic of cell cycle phase specific expression.
We've just shown you how to use the floral dip method to transform arabidopsis plants and examine reporter gus expression in transgenic seedlings. When doing this procedure, it is important to have healthy plants. So that's it.
Thanks for watching and good luck with your experiments.
