Arbuscular Mycorrhizal fungi are ancient and essential plant fruit symbiotes, and they enhance the nutrient intake of the host and are also key players in cell health. While cell microorganisms are difficult to study, our protocol allows for real time monitoring, digital observation, and efficient establishment of single spoke cultures. Currently, AM fungi are propagated in pot culture, in greenhouse condition, or in-vitro in using RITDNE transform route.
Both technology have significantly contributed to the knowledge of those AM fungi in the last decade, but they have specific limitations. Assessment of successful establishment of the fungal partner relies on the presence of arbuscular in the roots or spore in the soil. In-vitro propagation allows leaves monitoring, but requires transformed roots and sterile condition, limiting its applicability to many AM fungal species.
The pot culture or in-vitro culture techniques used to study AMF have limitations in terms of live observation and successful propagation. To overcome these limitations, we have developed this super absorbent, polymer based autotrophic system, SAP AS, which is a simple and inexpensive technique that combines the advantages of in-vitro culture and pot culture. Our laboratory will focus on improving the establishment of new AM fungal species in single spoke cultures, and also studying the interaction with over cell microorganism.
This technique will allow us to study the competitive dynamics between different AM fungal species, high fault propagation mechanism, and nutrient uptake processes. To begin, use the rotary tool and the appropriate carving bit to drill two irrigation holes in the top of the lid. On the side, drill a one centimeter opening.
Use the electric rotary tool to drill a notch in the side of the Petri dish bottom for the plant stem. Next, using the electric rotary tool equipped with the appropriate drill bits, drill a 30 millimeter rectangular notch down to the bottom of the Petri dish. For high resolution observation, drill circular holes approximately 25 millimeters in diameter in the bottom of the Petri dish.
Ensure the holes are not drilled directly under the lid's irrigation holes. Using the metal guide and paperclip, wedge the piece of the nylon mesh filter membrane in place and press it firmly onto the plastic barrier on the root or notch side. Then align the top of the membrane with the top of the barrier.
Using the pyrography kit, cut along the edge of the metal guide to melt and seal the membrane to the bottom of the Petri dish and the plastic barrier, separating the two compartments. With the metal guide and paperclips still in place, use tweezers to carefully remove excess, nylon mesh filter membrane. Under the dissecting microscope, verify that the nylon mesh filter membrane is properly sealed to the plastic to prevent root penetration into the hyphal compartment.
Then apply the silicone sealant along the edge of the circular holes on the outside of the Petri dish bottom. Place the cover slip on the outside of the Petri dish bottom and gently press for perfect adhesion. To begin, weigh five grams of dry super absorbent polymer, or SAP, and mix it with 500 milliliters of MMS one nutrient solution.
Approximately one week before assembly, germinate plantago lanceolata seeds on blotting paper, moistened with MMS one nutrient solution in a Petri dish. Then incubate the Petri dish in the dark at room temperature. To start assembling the SAP AS system, place the seedling stem in the notch on the side of the Petri dish with the root inward in the cotyledons or leaves and stems outward.
Cover the roots with approximately 1.5 to two grams of vermiculite. Afterward, hydrate the root compartment with eight milliliters of MMS one nutrient solution. Clear a vermiculite free space along the nylon mesh filter on the side of the root compartment and add five grams of hydrated SAP.
Then add 15 grams of hydrated SAP to the hyphal compartment. Now align the side notches of the lid and the Petri dish bottom to avoid damaging the stem and close the lid. Then seal the Petri dish with a strip of parafilm joining the base and lid.
Weigh the Petri dish and record its average weight to determine when the SAP AS needs rehydration. Then stack the Petri dishes to optimize space. Use an opaque cover with an opening on the side to keep the seedlings in the dark.
Using a flame candle or Bunsen burner, heat the glass pipette and stretch the glass tube as it melts to separate it. Under a stereo microscope, break the tip of the glass pipette to reduce its internal diameter. Now open the Petri dish and locate where the spore is to be placed.
Clear a space among the five grams of hydrated sap along the nylon mesh filter membrane to expose a section of a root. Under the dissecting microscope, pick up a spore in a liquid suspension with the extruded pipette while pipetting some liquid before collecting the spore. After that, carefully deposit the spore on the root.
Then replace the sap over the root fragment to maintain a moist microenvironment for the root and spore. After 24 hours, if needed, irrigate the root compartment, taking care not to wash off the spore. Align the side notches of the lid with the bottom of the Petri dish to avoid damaging the seedling stem.
Enclose the Petri dish with the lid.
