Objectives
The overall purpose of this experiment is to isolate a specific endophyte that has a desired plant growth promoting trait. The endophyte colonies were observed and cultured, screened on selective media for PGP, screened for antibiotic resistance and tagged with fluorescent isolates, and screened for nitrogen fixing abilities. The reason for this experiment is to ideally attain plant promoting traits that would help communities of plants thrive in toxic or otherwise unhealthy environmental conditions.
Methods
Each strain of endophytes were studied under these conditions. This report is to discuss strain HE10.
Start by streaking a couple of isolated colonies on MG/L plates to grow that colony. Wait a couple of days for growth. After a couple of days, check the plate for growth. Ensure there is no contamination in the plate visible with the eye. Then, using sterile technique, use the three-stick rule to streak a couple of colonies onto new plates for growth. Streak colonies in four different quadrants of plates onto plates with Nitrogen-free media, Phosphorous, and Siderophore media. Then, streak isolates onto separate plates containing antibiotics Gentamicin and Kanamycin. After a couple of days, examine the plates to see isolate growth and check for growth selective media plates.
To examine the growth of the Siderophore solubilization plates, photograph the plate with a ruler in the background. Upload the photograph onto ImageJ, and measure the diameter of the colony and the diameter of the halo. If a halo is present, your isolate can solubilize phosphate and iron.
To examine and measure the growth on the Nitrogen Free media, photograph your plate with a ruler in the background. Select three healthy colonies and measure the diameter with ImageJ. Calculate the mean of the colony diameters with x-bar = (colony) / 12. Compare this to your Azobacter+ and Ecoli- controls.
To characterize the antibiotic plates, record cell growth on plates and document whether cells grew, grew weakly, or didn’t grow at all. Determine which fluorescent protein plasmid worked with your strain.
To prepare inoculum, wash the cells from the growth medium. Transfer a few isolated colonies to 1.5mL of culture medium (NL-CCM) in a glass culture tube using sterile technique. After inoculating a cultures in media for a couple of days, spin down cells at 1200 rcf for ten minutes in a centrifuge. Remove the supernatant, and make a one to ten dilution from the pellet of the cells and media. Measure in the spectrophotometer at OD600 to get an idea of the amount of chlorophyll and cell density in your new culture. Now, inoculate plants with one mL of your culture onto the shoot base of each seedling. Include a set of control plants and pipette one mL of Nitrogen Free Media.
Results
HE10 was successfully grown in media and on plates. HE10 plates had healthy growing colonies with no contamination. HE10 colonies grew to 3.375mm for the mean diameter of the colonies.The Group 13 HE10 strain grew on the Iron plates with a mean of 6.831mm for the colony diameter and 1.17mm mean diameter for the halos. Halos present demonstrated that HE10 is phosphate soluble. For HE10 antibiotic resistance plates, HE10 was susceptible to both Gentamicin and Kanamycin.
For HE10 antibiotic resistance, HE10 was not antibiotic resistant to Km or Gm, therefore, both are being currently tested for both GFP and RFP because both are susceptible to gm and km, but resistant to cb, tc, and and sp.
Conclusion
In conclusion, the experiment to isolate a specific endophyte that has a desired plant growth promoting trait is off to a good start with strain HE10. Categorizing the endophyte has left us with valuable knowledge about phosphorus solubility and antibiotic resistance, which will be helpful in our next experiment of tagging plasmids. We still need to finish our experiment on whether they fixate nitrogen by completing the acetylene reduction analysis, and also run a PCR on NIF genes.
