This experimental system uses agro infiltration to effectively deliver gene constructs to leaves for the production of high levels of recombinant protein. First, grow end hamana plants in a controlled environment for six weeks, three days before infiltration. Prepare the agrobacterium cultures which contain the target gene to be expressed in the plants on the day of infiltration.
Introduce the agrobacterium cultures into the plant tissue by either syringe or vacuum infiltration. Results like the intensity of green fluorescence of GFP in tobacco leaves under UV light demonstrate a robust expression of recombinant proteins in plants that rivals that of current mammalian bacterial and insect based expression systems. Agro infiltration is a effective way to introduce trans genes into plant cells, which lead to high level expression of recombinant proteins.
It can be used for a broad range of plant species. Most importantly, it can be scaled up for commercial production of pharmaceutical proteins. So this method can provide insight into the kinetics and expression of GFP and can be applied to other proteins like subunit vaccines, recombinant monoclonal antibodies, and can be used in treatments for cancer and infectious disease.
A successful experiment requires optimal conditions for both the bacteria culture and the plants material. This infiltration process is easier to replicate after visual demonstration. Begin by placing up to 60 peat pellets into a propagation tray.
Add four liters of tap water and let the pea pellets absorb the water for two hours. Now, add two and hamana seeds into each peat pellet and cover the tray with a transparent plastic dome for germination. Place the seeds in a 25 degree Celsius, 84%humidity environment.
Two weeks after seeding, remove the dome and transfer the plants to a new tray. Then add two liters of 1.48 grams per liter. Jack's fertilizer Continue to grow the plants in a 25 degrees Celsius.
50%humidity environment every two days. Supply two liters of jack's fertilizer per tray. At week four, transfer the plants with peat pellets to a new tray that hosts six plants for further growth to six weeks of age.
Choose four, six week old and hamama plants with five to six leaves each three leaves for complete infiltration with the agro bacterial strains harboring the genes of interest. One leaf for the negative control and one leaf for spot infiltration. Create a small nick with a needle in the epidermis on the backside of the leaf, making sure not to pierce the leaf through both sides.
Take a firm hold of the front side of the first leaf while applying gentle counter pressure to the neck. With the thumb of one hand. Inject the agrobacterium mixtures in infiltration buffer into the neck with a syringe, a needle.
Continue to inject the agrobacterium mixtures into the nick until the darker green circle stops to expand. Then create another nick and repeat the injections until the entire leaf is infiltrated and the whole leaf turns darker green. Complete infiltrations of the first three leaves and the last leaf for the fourth leaf of each plant infiltrate with vector combinations of either all four Gemini viral vectors or three magcon vectors.
Also, make one nick for each combination of agrobacterium strains and infiltrate each nick with one combination. After infiltration, move plants back to the growth room and monitor protein expression between two and 15 days. Post infiltration, place a tub into a vacuum desiccate and transfer in three liters of infiltration buffer containing the agrobacterium strains.
Then connect the desiccate to a vacuum brand diaphragm vacuum pump. Now place a plant upside down on the desiccate plate and lower the plate with the plant until the entire leaf and stem system is submerged into the infiltration buffer with the plate resting on top of the tub. Next position the desiccate O ring along the rim.
After putting the desiccate lid on the chamber, turn on the vacuum pump and start timing when the vacuum reaches 100 millibar. After turning off the vacuum pump, slowly open the release valve on the desiccate after one minute at 100 millibar to allow entrance of agro bacteria into the interstitial spaces of submerged plant tissue. Repeat this infiltration.
Step ones to ensure good infiltration. Then remove the plant from the desiccate and put it back in its upright position. Move plants back to the growth room and monitor protein expression between two and 15 days post infiltration.
To demonstrate the effectiveness of syringe, infiltration of agrobacterium into plant tissue, we tested the expression of two fluorescent proteins, GFP and Ds red by two different deconstructed plant viral vectors, Gemini, viral and magcon for end. Hamana leaves that were entirely infiltrated with agro bacteria containing Gemini viral vectors. GFP expression was observed over the entire leaf area under UV light, starting from as early as 2D PI and reached peak accumulation at four DPI.
In contrast, leaves infiltrated with magcon vector containing agrobacterium combinations showed GFP fluorescence only after five DPI and reached its maximum accumulation at seven DPI. No green fluorescence was observed from leaves infiltrated with negative control agro bacteria mixtures of EP one 10 plus P 19, indicating that the fluorescence was specific to the GFP gene and was not the result of background fluorescence from the leaves. At its peak accumulation, the fluorescence of magcon vector expressed to GFP is more intense than that of Gemini viral vector.
No major necrosis was observed on leaves infiltrated with GFP constructs when both fluorescent proteins were expressed on the same leaf via Gemini viral vectors with syringe spot infiltration, they were detected with their expected fluorescent color in the spot where they were infiltrated. Interestingly, co infiltration of GFP and DS RED resulted in yellowish fluorescence. Vacuum infiltration was also examined to develop a scalable agro infiltration method that can be used for large scale production of recombinant proteins by plant transient expressions systems.
As expected GFP fluorescence was observed for all leaves of the infiltrated plant compared with syringe infiltration. It is more robust and can achieve infiltration of each plant with a much shorter timeframe. Once mastered, this technique can be performed in 48 hours.
Most of the time it's for agro bacterial culture as the actual infiltration time is only a few minutes long For optimal agro infiltration efficiency and targeted protein expression. Follow the protocol. Remember to control the critical parameters, including plant growth conditions, agro bacterial concentration, vacuum pressure, and duration In the field of plant biotechnology.
This experimental approach can be used to explore the development and bioproduction of novel pharmaceutical proteins in a broad range of plant species. After watching this video, you should have a good idea of how to use agro infiltration to deliver target gene constructs into the plant and to allow plant transient expression systems to produce recombinant protein, rivaling that of mammalian bacterial and insect cell cultures.
