Hi, my name is Oliver Yu at the Dan Plant Science Center. Today we're going to show you how to generate Ute Compote Plants by Agrobacterium Ogn. This technique was brought to Dan Center by Dr.Chris Taylor, currently at Ohio State University.
Several of my former group members improved these techniques over the years and today my current group member, Dr.Stone and Mao, put together this video to show how Harry Root Composite Plans is produced. Agrobacterium ZOS are very similar to agrobacterium tumor fis in that they introduce foreign DNA into plants, but the ZOS can induce higher root formation of wild type tissues forming composite plants as this one. So on the, on these campy plants, some of the roots are transgenic while the shoots are still wild tap.
Therefore, this technology will not produce transgenic seeds. However, there are three major advantages of high root campy plants that make this technology very useful. First, many plant species, especially dicho species, can be transformed by agro bacterial ous.
Second, we do not need to establish tissue culture prior to transformation. We can take many parts of plants such as seedlings, shoots and co to generate campy plants directly. And finally, we can get transgenic root tissue in a matter of weeks.
For soybean and medic cargo trans, we can get transgenic roots in as short as three weeks faster than a OPIS transformation. Overall, the higher root cyte plant technology is very useful tool to study gene functions and the root related phenotypes. No.Hello, my name is Ong.
Hello, my name is, There is my cameraman bill. Today we'll introduce Harry root Composite Plant protocol. Yeah, we use this protocol in our several projects.
I already, we Started, yeah.Yeah. For our demonstration, we'll use meta cargo Tula as an example. These are meta cargo mature pods.
Put the pods on a grinding box and then grind the pods with a long firm Motion. Separate the seeds from the seed pods by gently shaking place Seeds in a 50 milliliter centrifuge tube. Then add five milliliters of concentrated sulfuric acid.
Very Carefully scarring. Takes about 10 minutes. An acid Vortex the tube two to three times.
During the acid treatment, the seed coat will change to a yellow color. Remove sulfuric acid from the tube and rinse the seeds at least five times with sterile water. Soak The seeds in deionized water at room temperature for six hours, and then keep them at four degrees centigrade for two days.
To synchronize germination. Spread 20 to 30 seeds on moist filter paper in the Petri dish. After five to six days, transfer the seed links to soil and then grow them in the greenhouse for four weeks.
For composite plant transformation, we normally use the agrobacterium Zony K 5 99 strain. The shuttle vector or binary vector can be transformed into the K 5 99 following the standard agrobacterium tumor facit transformation protocol Inoculate the agrobacterium, Grow them at 30 Degrees centigrade for 16 to 24 hours after the growth period. Collect the cells by placing the culture solution in a 50 milliliter centrifuge tube.
Run it in the centrifuge at 5, 000 G for 30 minutes. Resuspend the pellet with nitrogen free nutrient solution. Make sure to dilute the agrobacterium cells to 0.3 at OD 600.
This concentration is important for maximum transformation. We use rock wall as a supporting matrix. Using this material is important for high frequency transformation.
Cut the rock wall into the right size.Plug. Poke a hole at the top about one and a half to two centimeters in diameter. Using a pipette tip, add five milliliters of Resuspended Agrobacterium Zony solution into each plug.
Select a stem section with two to three auxiliary buds and make a slanting cut. Insert the plant sections into the hole in the rock wall. Place the Cut plants into a growth chamber or lab bench for three weeks.
Add five milliliters of nutrient solution into each plug every 10 days. After three weeks, the roots are regenerated from the cut side. Soak the plant in water.
Remove the softened rock wall carefully and do not damage the roots. Be sure to remove all of the rock Wall. You can transfer the hairy root composite plants to sand or pearlite For symbiosis experiments, grow the rhizobium in a yeast extract manito medium for seven days at 30 degrees Celsius.
Collect the zoia cells Resus. Suspend the pellet with the nitrogen free nutrient solution. Dilute the solution to 0.08 at OD 600.
Add 10 milliliters of ZOA solution to each composite plant culture, the plant for an additional two to three weeks to allow nodules to form. Remove sand from the composite plants by uprooting and washing them. Use green fluorescent protein expression to check the transgenic root.
You'll use a fluorescent microscope to do this. All our hairy root constructs have a constitutive GFP cassette for screening transgenic lines. This picture shows the GFP transgenic hairy root composite plants observed in the fluorescent microscope.
The transgenic roots with the brighter green fluorescence are easy to distinguish from the non transgenic roots. Nodules are formed in the hairy root of the composite plants. Here is the complete protocol.
Once again, generating hairy root composite plants is a quick and easy method to obtain large quantities of transgenic tissue, especially for diop species. Although this method cannot give you transgenic seeds, it can produce transgenic material in a few weeks. It's especially suitable for plants that have difficulty in establishing tissue culture and transformation.
Over the years, we have used this technology to study gene functions, promoter functions, microRNAs, root and lateral root developments, defense and antibiotic stress response, nodulation and other symbiotic process. We studied hormone response, metabolic profiling, gene profiling, and the proteomic analysis using the higher root composite plants. We hope this video will help you adapt this system to your favorite species.
You are welcome to contact us for questions related to this technology and Good luck.
