Optimized Method for Cultivation and Microbial Bioaugmentation of Typha latifolia (Cattail)

Our work addresses the lack of protocols for growing cattail from seed. We developed reproducible methods for seed germination, early growth, and microbial bio augmentation to support future plant microbe research. Few studies grow cattail from seed or track them to maturity, yet cattails are commonly used in wetland remediation research.

Most cattail research involves purchasing rhizomes for propagation of cattails in laboratory studies. Few studies grow cattail from seed, leaving no standardized methods. Achieving consistent germination and persistent colonization of introduced microbes has been a major challenge.

We've developed reproducible methods for seed germination and microbial bio augmentation, demonstrating how early inoculation helps support long-term colonization. To begin, use garden shears to cut the stem of a Typha latifolia plant approximately two centimeters from the base of the inflorescence. Pull the seeds off the inflorescence, transfer the seed into a laboratory blender until the blender is approximately 1/4 full, corresponding to about 250 milliliters of uncompacted seeds.

Now fill the blender with 500 milliliters of tap water, ensuring a 10 centimeter head space is maintained. Blend the mixture at medium low speed for 20 seconds, and immediately transfer the viscous contents into a one liter beaker. Transfer approximately 100 milliliters of the seed water mixture back into the blender.

Then add 400 to 600 milliliters of fresh tap water and blend on medium high speed for 20 seconds. Pour the contents into a fresh one liter beaker. Now fill the beaker with tap water up to 800 milliliters.

After letting it sit for 60 seconds, scoop off the floating sludge from the top without disturbing the seeds settled at the bottom Slowly pour out the remaining water and transfer the seeds into a 100 milliliter beaker. Repeat the blending process for the remaining seed water sludge. Next place the beaker containing the separated seeds onto a stir plate.

After stirring, scoop off any floating plant material from the surface of the beaker. Pour the seeds into a Buchner funnel with filter paper attached to a vacuum and allow them to dry overnight. Store the dried seeds at 20 degrees Celsius in 50 milliliter conical polypropylene tubes.

Prepare half strength Murashige and Skoog media plates with 1%phyto agar. Transfer approximately one milligram of the dried seeds into a 15 milliliter conical polypropylene tube. Then add 10 milliliters of sterile double distilled water to the tube.

Place the tube on an orbital shaker at medium high speed for 10 minutes. Now remove the water from the tube and add five milliliters of 0.1%polysorbate 20 solution prepared in sterile double distilled water. Shake the tube at medium high speed for 10 minutes.

Remove the polysorbate 20 solution. Perform all further steps in front of a flame or laminar flow hood. Replace the solution with five milliliters of 30%commercial bleach and 0.025%polysorbate 20 solution prepared in sterile double distilled water.

Replace the supernatant with sterile double distilled water. Then shake the tube at medium high speed for five minutes. After the third rinse, rotate the tube at low speed for 24 hours to induce germination.

The next day, remove the excess water and ensure three milliliters of liquid remain in the tube. Now aseptically, cut the tip of a 1000 microliter plastic pipette tip. Use the cut pipette tip to vigorously pipette and suspend the seeds within the tip.

Plate the seeds and liquid onto one half strength Murashige and Skoog agar plates. Gently swirl the plate to evenly distribute the seeds. Wrap the plates with laboratory sealing film, then place them in a growth chamber with a 16 hour light and 8 hour dark cycle at 23 degrees Celsius and 70%humidity.

To inoculate cattail seeds, sterilize seeds with polysorbate 20 bleach and double distilled water as demonstrated. Measure the optical density of an overnight culture of Luteimonas isolate at 600 nanometers. Normalize the culture to an absorbance value of 1.0 by diluting in culture medium.

Now centrifuge one milliliter of the culture at 9, 300 G For two minutes. Remove the supernatant and resuspend the pellet in one milliliter of PBS. After centrifuging and removing the supernatant again, resuspend the bacterial pellet in one milliliter of sterile double distilled water.

Use sterilized seeds, then dilute the inoculum at a one to 10 dilution with 0.025%organosilicone surfactant in the same tube. Shake the suspension at low speed for 24 hours before seed germination. Start by filling a sterile plant growth chamber unit with a soil of choice pre-wetted with tap water.

Cover the Luer lock tip with aluminum foil and autoclave the unit on a liquid cycle for 20 minutes. Next, under sterile conditions, attaches a 0.2 micrometer filter unit to the Luer lock connector on the growth chamber. Open the chamber and add 500 microliters of filter sterilized 1%20 by 20 by 20 fertilizer to the soil.

Mix the soil with a sterile spatula while simultaneously adding sterile double distilled water to maintain hydration without oversaturating the soil. Now, use a sterile razor blade to cut Murashige and Skoog agar from plates containing one weak old seedlings into quarters. With a sterile spatula, transfer one agar section with seedling onto the prepared soil in the growth chamber.

Transfer the growth chamber unit into a plant growth incubator set to a 16 hour light and 8 hour dark cycle at 23 degrees Celsius and 70%humidity. Complete scarification of Typha seeds resulted in visibly separated seed components, whereas incomplete scarification left the beak attached and non-scarified seeds remained intact. The sterile hydroponic system has been shown to support the growth of cattail and Juncus species, imaged here as Juncus, which was maintained within the sterile system for up to one year.

The highest seed germination rate of 20.8%was observed using the 30%bleach and polysorbate 20 method, which was significantly greater than all other sterilization treatments except for the 1 hour chlorine gas method which was not able to achieve complete seed sterilization. At seven days post-scarification, germinating Typha seedlings developed visible radicals and shoot tissue in the non-sterile Petri plate environment. Following transplant to soil, a subset of Typha's seedlings established successfully and produced new shoot tissue within one to two weeks.

After inoculation with Luteimonas species carrying a DsRed plasmid, red fluorescent bacterial colonization was observed throughout the Typha seedling roots at 16 days post-inoculation.