Name: Optimized Method for Cultivation and Microbial Bioaugmentation of Typha latifolia (Cattail) (Video) | JoVE Journal
Uploaded: 2025-07-25T12:50:00.000+00:00
Duration: 9 min 14 s
Description: Learn reproducible methods for seed germination and microbial bioaugmentation of Typha latifolia. This protocol enables standardized lab cultivation and supports plant-microbe interaction research in wetland ecosystems.

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5 minutes

AZ was funded by a Natural Sciences and Engineering Research Council of Canada (NSERC) CGS-M Graduate Award. Research on CWTS in the Muench lab is supported by Genome Canada through a Large Scale Applied Research Project (LSARP) grant (#18207) in partnership with Genome Alberta and Genome Quebec.

1. Seed scarification
<ol>
	<li>Cattail seeds were sourced from a wetland in Calgary, AB, Canada, in fall 2023 (51.11312&#176; N, 114.39381&#176; W). Collect cattail inflorescence in the fall and store in a paper bag in the dark until use. If still on the stalk, collect the inflorescence in the spring, but seed recovery will be diminished.</li>
	<li>Using garden shears, cut the plant stem ~2 cm from the base of the inflorescence. Pull the seeds off the inflorescence and place into a laboratory blender until the volume of the blender is approximately one-quarter&#160;full. This is approximately 250 mL of seeds not compacted.</li>
	<li>Fill the blender with 500 mL of tap water. Maintain a 10 cm headspace in the blender. Blend at a medium-low speed for 20 s and immediately transfer to a large 1 L beaker. The resulting solution should be viscous.</li>
	<li>Transfer approximately 100 mL of this seed-water sludge back into the blender and fill the blender with 400-600 mL of fresh tap water. Blend on medium-high speed for 20 s and immediately transfer to a fresh 1 L beaker.</li>
	<li>Fill the beaker with tap water to 800 mL and let it sit for at least 60 s. Scarified and viable cattail seeds will sink to the bottom of the beaker, and the plume and beak (see Figure 1 for a description of cattail seed structure) will float to the top. Scoop off the sludge at the top of the beaker and slowly pour the water from the beaker without disturbing the seeds at the bottom. Transfer the seeds to a 100 mL beaker.</li>
	<li>Repeat the blending process on the remainder of the cattail sludge from step 1.5.</li>
	<li>Place the beaker containing the seeds on a stir plate and spin at medium speed for 1 h. After 1 h scoop off any plant material floating at the top of the beaker.</li>
	<li>Pour the seeds into a B&#252;chner funnel with filter paper attached to a vacuum to dry the seeds overnight. Dry seeds can be stored at -20 &#176;C in 15 mL conical polypropylene tubes.</li>
</ol>
2. Germination using non-sterile technique
<ol>
	<li>Prepare half-strength Murashige and Skoog (MS) media plates with 1% phytoagar12.</li>
	<li>Place approximately 1 mL of dry seeds into a 15 mL conical polypropylene tube and fill with 10 mL of tap water. Rotate on an orbital shaker at low speed for 24 h to induce germination.</li>
	<li>Remove excess water such that 3 mL remains in the 15 mL conical polypropylene tube.</li>
	<li>Cut the tip of a 1000 &#181;L plastic pipette tip to increase the bore size and pipette the seed solution vigorously to suspend seeds within the pipette tip.</li>
	<li>Place seeds and water on the half-strength MS agar plates. Swirl the plate to distribute the seeds evenly.</li>
	<li>Wrap the plates in laboratory sealing film and place them in a growth chamber with a 16 h/8 h light-dark cycle at 23 &#176;C and 70% humidity. Incubate the plates for 1 week (up to 2 weeks) to induce seed germination.</li>
</ol>
3. Germination using sterile technique
NOTE: Different seed sterilization protocols were tested and compared. The technique that produced the highest germination rates while producing fully sterilized seeds was modified from a previous protocol13 and is described here.
<ol>
	<li>Prepare half-strength MS media plates with 1% phytoagar12.</li>
	<li>Place a volume of approximately 1 mL of seeds into a 15 mL conical polypropylene tube and fill with 10 mL of sterile ddH2O. Place on an orbital shaker at medium-high speed for 10 min.</li>
	<li>Remove the water and add 5 mL of a 0.1% Polysorbate 20 solution (prepared in sterile ddH2O) to the 15 mL conical polypropylene tube. Place on an orbital shaker at medium-high speed for 10 min.</li>
	<li>Remove the solution and add 5 mL of a 30% commercial bleach and 0.025% Polysorbate 20 solution (prepared in sterile ddH2O) to the 15 mL conical polypropylene tube. Shake at a medium-high speed for 30 min.</li>
	<li>Remove the solution and replace it with sterile ddH2O. Shake on medium-high speed for 5 min. Repeat for a total of 3 water rinses.</li>
	<li>Fill the 15 mL conical polypropylene tube with sterile ddH2O and rotate at a low speed for 24 h to induce germination. After 24 h, remove the excess water so that 3 mL remains in the plastic tube.</li>
	<li>Aseptically cut the tip of a 1000 &#181;L plastic pipette tip and vigorously pipette to suspend seeds within the tip.</li>
	<li>Plate seeds and liquid on half-strength MS agar plates. Swirl the plate to evenly distribute seeds.</li>
	<li>Wrap plates in laboratory sealing film and place in a growth chamber with a 16 h/8 h light-dark cycle at 23 &#176;C and 70% humidity. Leave plates for 1 week (up to 2 weeks) to allow for germination to occur.</li>
</ol>
4. Seed inoculation with selected bacteria
<ol>
	<li>To inoculate cattail seed, first follow the sterile seed germination protocol above until step 3.6 to produce sterile seeds. Remove the final water rinse from the 15 mL conical polypropylene tube as described in step 3.6 above.</li>
	<li>Using overnight-grown microbial cultures inoculated from a single isolate or community, measure the OD600 and normalize to a 1.0 absorbance value by diluting in culture medium. Incubate cultures for a longer period of time if an OD600 of 1.0 is not achieved. 
	NOTE: Here, cultures were established from a Luteimonas sp. isolated from plant roots in northern Alberta and conjugated to express a DsRed fluorescent protein for microscopic visualization.</li>
	<li>Spin 1 mL of culture at 9300 x g for 2 min. Remove the supernatant and resuspend the pellet in 1 mL of 1x Phosphate buffered saline. Spin at 9300 x g for 2 min.</li>
	<li>Remove the supernatant and resuspend the bacterial pellet in 1 mL of sterile ddH2O.</li>
	<li>In a 15 mL conical polypropylene tube, shake approximately 1 mL of seeds at a low speed for 24 h in 10 mL of a 1:10 dilution of the prepared inoculum with 0.025% organosilicone surfactant.</li>
	<li>Follow the remainder of the sterile seed germination protocol starting from step 3.8.</li>
</ol>
5. Non-sterile Cattail growth on soil
<ol>
	<li>Fill pots with low-organic-matter soil of choice, leaving 1 cm of space from the top of the pot. Pre-moisten the soil with tap water and poke 1 cm holes into the soil.</li>
	<li>Using tweezers, gently remove the 1-week-germinated seedlings from the MS agar plates grown as described in step 3 above, taking care not to damage the roots. Gently place a seedling into each hole in the soil and cover it with soil.</li>
	<li>Place the pots in a tray and fill them with tap water to the soil level. For every 10 pots, add 100 mL of 0.5% 20/20/20 NPK fertilizer.</li>
	<li>To each set of ten pots, add fertilizer as follows: week 2 - 100 mL of 0.5% fertilizer, week 3 - 100 mL of 1.0% fertilizer, week 4 - 100 mL of 1.0% fertilizer.</li>
	<li>After 4 weeks, stop weekly fertilization and fertilize only once monthly with 100 mL of 1.0% fertilizer solution. At 4 weeks, transplant into larger pots, if needed. The plants are less sensitive to soil type at this point, so use any mix.</li>
	<li>Continue to transplant cattails as they outgrow their container. Remove dead foliage with shears. Fertilize cattails once a month.</li>
	<li>For experiments, transplant seedlings into a small pot that, when placed into the bottom of an empty pipette tip box, is anchored by the sides of the box. This allows proper flooding of the soil, thereby mimicking wetland conditions. If conducting experiments on older cattails modify similarly so that the pot can be nearly fully submerged in water.</li>
</ol>
6. Sterile seedling growth
<ol>
	<li>Seedling growth chamber design 
	NOTE: Assembly of each seedling growth chamber unit requires two culture boxes, one syringe with a luer lock tip, a disposable 0.2 &#181;m sterile filter, and heat-resistant silicone. See Figure 2 for a visual representation of the chamber setup.
	<ol>
		<li>Remove the lids from the culture boxes and use a handheld drill to create 3.5 cm diameter holes in the center of the lids. Use heat-resistant silicone to glue the tops of the two lids together.</li>
		<li>Drill a hole the size of the syringe tip (~ 1 cm diameter) in the bottom corner of one of the culture boxes. Cut the tip off a syringe and discard the body of the syringe.</li>
		<li>Use heat-resistant silicone to fix the tip of the syringe into the culture box with the Luer lock thread on the outside of the box.</li>
		<li>Allow silicone to cure at least 24 h before use.</li>
	</ol>
	</li>
	<li>Sterile growth in soil
	<ol>
		<li>Follow the sterile seed germination protocol described in step 3 to obtain sterile seeds.</li>
		<li>Fill the sterile growth chamber unit with soil of choice, wetted with tap water.</li>
		<li>Autoclave on a liquid 20 min cycle, covering the lure lock tip with aluminum foil. After 24 h, autoclave the system again on a liquid 20 min cycle. Perform all subsequent steps in a laminar flow hood.</li>
		<li>Attach a 0.2 &#181;m filter unit to the Luer lock attachment on the growth chamber (Figure 2). Open the growth chamber and add 500 &#181;L of filter-sterilized 1% 20/20/20 fertilizer to the soil. Mix the soil with a sterile spatula. While mixing the soil, add sterile ddH2O to ensure it is adequately hydrated without oversaturation.</li>
		<li>Use a sterile razor blade to cut the MS agar from plates containing 1-week-old seedlings into quarters. Use a sterile spatula to lay the agar piece onto the soil.</li>
		<li>Place the growth chamber unit into a plant growth incubator on a 16 h/8 h day-night cycle at 23 &#176;C and 70% humidity.</li>
	</ol>
	</li>
	<li>Sterile growth in hydroponic solution
	<ol>
		<li>Follow the sterile germination protocol described in step 3 to obtain sterile seeds.</li>
		<li>Autoclave a pipette tip box and half-strength MS agar on a liquid 20 min cycle. In a laminar flow hood, wrap the bottom of the tip insert with sterile laboratory sealing film. Fill each pipette tip opening with agar almost to the top.</li>
		<li>Add the tip insert back to the tip box filling the bottom of the box with half-strength Hoagland&#39;s hydroponic growth solution (0.5 mM NH4H2PO4, 3 mM KNO3, 2 mM Ca(NO3)2, 1mM MgSO4, 0.023 mM H3BO3, 0.005 mM MnCl2&#183;4H2O, 0.0004 mM ZnSO4&#183;7H2O, 0.0002 mM CuSO4&#183;5H2O, 0.00007 mM H2MoO4&#183;1H2O, and 0.045 mM FeSO4&#183;7H2O, adjusted to pH 7.0).</li>
		<li>Carefully remove seedlings from the MS agar plates and place them on the MS agar on top of the agar in the pipette opening in the tip box insert.</li>
		<li>Close the tip box lid and place in a growth chamber on a 16 h/8 h day-night cycle at 23 &#176;C and 70% humidity.</li>
		<li>When plants are too large for the tip box, transplant them to the culture growth chamber described in step 5.1. To transfer, remove the entire agar plug with the plant and move it into a foam stopper with a radial cut. The foam stopper can be placed between the two culture boxes, and the bottom culture box can be filled with half-strength Hoagland&#39;s.</li>
	</ol>
	</li>
</ol>

<ol><li>Ciria, M. P., Solano, M. L., Soriano, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+Macrophyte+Typha+latifolia+in+a+Constructed+Wetland+for+Wastewater+Treatment+and+Assessment+of+Its+Potential+as+a+Biomass+Fuel%5BTitle%5D)+AND+%22Biosys+Eng%22%5BJournal%5D)">Role of Macrophyte Typha latifolia in a Constructed Wetland for Wastewater Treatment and Assessment of Its Potential as a Biomass Fuel.</a> Biosys Eng. 92 (4), 535-544 (2005).</li>
<li>Shelef, O., Gross, A., Rachmilevitch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+Plants+in+a+Constructed+Wetland%3A+Current+and+New+Perspectives%5BTitle%5D)+AND+%22Water%22%5BJournal%5D)">Role of Plants in a Constructed Wetland: Current and New Perspectives.</a> Water. 5 (2), 405-419 (2013).</li>
<li>Parde, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+review+of+constructed+wetland+on+type%2C+treatment+and+technology+of+wastewater%5BTitle%5D)+AND+%22Environ+Tech+Innovation%22%5BJournal%5D)">A review of constructed wetland on type, treatment and technology of wastewater.</a> Environ Tech Innovation. 21, 101261(2021).</li>
<li>Abdullah, S. R. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant-assisted+remediation+of+hydrocarbons+in+water+and+soil%3A+Application%2C+mechanisms%2C+challenges+and+opportunities%5BTitle%5D)+AND+%22Chemosphere%22%5BJournal%5D)">Plant-assisted remediation of hydrocarbons in water and soil: Application, mechanisms, challenges and opportunities.</a> Chemosphere. 247, 125932(2020).</li>
<li>Noor, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heavy+metal+and+metalloid+toxicity+in+horticultural+plants%3A+Tolerance+mechanism+and+remediation+strategies%5BTitle%5D)+AND+%22Chemosphere%22%5BJournal%5D)">Heavy metal and metalloid toxicity in horticultural plants: Tolerance mechanism and remediation strategies.</a> Chemosphere. 303, 135196(2022).</li>
<li>Vymazal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plants+used+in+constructed+wetlands+with+horizontal+subsurface+flow%3A+A+review%5BTitle%5D)+AND+%22Hydrobiologia%22%5BJournal%5D)">Plants used in constructed wetlands with horizontal subsurface flow: A review.</a> Hydrobiologia. 674 (1), 133-156 (2011).</li>
<li>Sandoval, L., Zamora-Castro, S. A., Vidal-Álvarez, M., Marín-Muñiz, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+Wetland+Plants+and+Use+of+Ornamental+Flowering+Plants+in+Constructed+Wetlands+for+Wastewater+Treatment%3A+A+Review%5BTitle%5D)+AND+%22Appl+Sci%22%5BJournal%5D)">Role of Wetland Plants and Use of Ornamental Flowering Plants in Constructed Wetlands for Wastewater Treatment: A Review.</a> Appl Sci. 9 (4), 685(2019).</li>
<li>Wu, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Constructed+wetlands+for+pollution+control%5BTitle%5D)+AND+%22Nat+Rev+Earth+Environ%22%5BJournal%5D)">Constructed wetlands for pollution control.</a> Nat Rev Earth Environ. 4, 218-234 (2023).</li>
<li>Yang, Y., Shen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phytoremediation+of+cadmium-contaminated+wetland+soil+with+Typha+latifolia+L.+and+the+underlying+mechanisms+involved+in+the+heavy-metal+uptake+and+removal%5BTitle%5D)+AND+%22Environ+Sci+Pollution+Res%22%5BJournal%5D)">Phytoremediation of cadmium-contaminated wetland soil with Typha latifolia L. and the underlying mechanisms involved in the heavy-metal uptake and removal.</a> Environ Sci Pollution Res. 27 (5), 4905-4916 (2020).</li>
<li>Leto, C., Tuttolomondo, T., La Bella, S., Leone, R., Licata, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+plant+species+in+a+horizontal+subsurface+flow+constructed+wetland+-+phytoremediation+of+treated+urban+wastewater+with+Cyperus+alternifolius+L.+and+Typha+latifolia+L.+in+the+West+of+Sicily+%28Italy%29%5BTitle%5D)+AND+%22Ecol+Eng%22%5BJournal%5D)">Effects of plant species in a horizontal subsurface flow constructed wetland - phytoremediation of treated urban wastewater with Cyperus alternifolius L. and Typha latifolia L. in the West of Sicily (Italy).</a> Ecol Eng. 61, 282-291 (2013).</li>
<li>Sesin, V., Davy, C. M., Freeland, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Review+of+Typha+spp.+%28cattails%29+as+toxicity+test+species+for+the+risk+assessment+of+environmental+contaminants+on+emergent+macrophytes%5BTitle%5D)+AND+%22Environ+Pollution%22%5BJournal%5D)">Review of Typha spp. (cattails) as toxicity test species for the risk assessment of environmental contaminants on emergent macrophytes.</a> Environ Pollution. 284, 117105(2021).</li>
<li>Murashige, T., Skoog, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Revised+Medium+for+Rapid+Growth+and+Bio+Assays+with+Tobacco+Tissue+Cultures%5BTitle%5D)+AND+%22Physiol+Plantarum%22%5BJournal%5D)">A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures.</a> Physiol Plantarum. 15 (3), 473-497 (1962).</li>
<li>Rogers, S., Beech, J., Sarma, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shoot+regeneration+and+plant+acclimatization+of+the+wetland+monocot+cattail+%28Typha+latifolia%29%5BTitle%5D)+AND+%22Plant+Cell+Rep%22%5BJournal%5D)">Shoot regeneration and plant acclimatization of the wetland monocot cattail (Typha latifolia).</a> Plant Cell Rep. 18 (1), 71-75 (1998).</li>
<li>Tangen, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Distributions+of+native+and+invasive+Typha+%28cattail%29+throughout+the+Prairie+Pothole+Region+of+North+America%5BTitle%5D)+AND+%22Wetlands+Ecol+Management%22%5BJournal%5D)">Distributions of native and invasive Typha (cattail) throughout the Prairie Pothole Region of North America.</a> Wetlands Ecol Management. 30 (1), 1-17 (2022).</li>
<li>Pieper, S. J., Nicholls, A. A., Freeland, J. R., Dorken, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Asymmetric+Hybridization+in+Cattails+%28Typha+spp.%29+and+Its+Implications+for+the+Evolutionary+Maintenance+of+Native+Typha+latifolia%5BTitle%5D)+AND+%22J+Heredity%22%5BJournal%5D)">Asymmetric Hybridization in Cattails (Typha spp.) and Its Implications for the Evolutionary Maintenance of Native Typha latifolia.</a> J Heredity. 108 (5), 479-487 (2017).</li>
<li>Lansdown, R. V. Typha latifolia. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLansdown%2C+RV+%22The+IUCN+Red+List+of+Threatened+Species+2017%22">The IUCN Red List of Threatened Species 2017</a>. , (2017).</li>
<li>Bedish, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cattail+Moisture+Requirements+and+their+Significance+to+Marsh+Management%5BTitle%5D)+AND+%22Am+Midland+Naturalist%22%5BJournal%5D)">Cattail Moisture Requirements and their Significance to Marsh Management.</a> Am Midland Naturalist. 78 (2), 288(1967).</li>
</ol>

The authors have nothing to disclose.

The protocol presented here provides a detailed guide for the growth of cattail species from seed for laboratory applications. While cattail can be easily propagated from rhizome cuttings, starting plants from seed allows for genetic variation within a sample set, ensures plants are at equal growth stages, and provides the opportunity for early colonization for microbial bioaugmentation experiments. It is important to correctly identify the species being collected, as propagation of more invasive cattail species may prom...

As a cost and energy-efficient technology, constructed wetland treatment systems (CWTS) have gained significant popularity for the remediation of environmental contaminants1,2,3. CWTS uses physical, chemical, and biological processes to remove, transform, or stabilize contaminants. While the physical and chemical processes involved in chemical turnover have been well characterized since the genesis of CWTS, the impact of vegetation remained largely enigmatic2. In recent years, there has been an increased focus on understanding the mechanisms by which plants transform organic and inorganic contaminants of&#160;potential concern in wastewaters2,4,5. However, mechanistic studies such as these rely on the ability to cultivate large numbers of macrophytes and favor growth from seed to ensure each plant is at the same stage of growth and development.
CWTS in North America are frequently established using vegetation native to natural wetlands in the region, such as Typha, Scirpus, Juncus, and Phragmites species6,7. The choice of vegetation also depends on the constructed wetland being employed, which can vary in depth, water flow, substrate sourcing, and water sourcing (with or without recirculation)2. Finally, climate also influences wetland vegetation, with cooler climates favoring submerged plants due to their increased adaptability8. Typha species are of particular interest in deep, surface-flow constructed wetlands due to their ability to rapidly colonize an environment and to adapt to diverse environmental conditions9,10.
A recent review of results reported that, of 87 phytotoxicity tests using Typha species, only 15 studies started the test plants from seed, and of those, only one study examined growth in mature plants11. This underrepresentation of cattail experimentation from seed indicates a gap in the literature concerning protocols aimed at describing the cultivation of cattail for laboratory studies. The protocols outlined here aim to bridge this gap by presenting an in-depth protocol for the growth of Typha latifolia from seed to mature plant under sterile and non-sterile conditions. Additionally, this paper discusses a technique for bacterial bioaugmentation of cattail species at the seed stage where early inoculation can help maintain the long-term persistence of introduced rhizo- and endo-phytic microbes.

<table><tbody><tr><td>0.2 &amp;micro;m sterile filter</td><td>VWR</td><td>514-4126</td><td></td></tr><tr><td>3&amp;prime;,5&amp;prime;-Dimethoxy-4&amp;prime;-hydroxyacetophenone</td><td>PhytoTech Labs</td><td>S7777</td><td></td></tr><tr><td>Culture boxes with lids (77 mm &amp;times; 77 mm &amp;times; 97 mm)</td><td>Millipore Sigma&amp;nbsp;</td><td>V8505</td><td></td></tr><tr><td>Heat-resistant silicone sealant hi-temperature</td><td>Imperial Manufacturing Group</td><td>KK0205</td><td></td></tr><tr><td>Laboratory sealing film</td><td>Millipore Sigma&amp;nbsp;</td><td>P7793</td><td></td></tr><tr><td>Murashige and Skoog media&amp;nbsp;</td><td>PhytoTech Labs</td><td>M401</td><td></td></tr><tr><td>Organosilicone surfactant</td><td>PhytoTech Labs</td><td>S7777</td><td></td></tr><tr><td>Phytoagar&amp;nbsp;</td><td>GoldBiotechnology</td><td>P1003&amp;nbsp;</td><td></td></tr><tr><td>Polysorbate 20</td><td>Roche</td><td>11332465001</td><td></td></tr></tbody></table>

optimized method for cultivation and microbial bioaugmentation of typha latifolia (cattail)

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

Typha latifolia, more commonly known as the broadleaf cattail or the common bulrush, has a globally reaching range and dominates wetland ecosystems in North America. While different species of cattail are often considered invasive in North America, T. latifolia is considered the native species to the region and is found throughout the entire continent as the dominant Typha species. Historically, Typha has served various functions, from food sources to building materials. More recently, T. latifolia has emerged as a prominent species to aid bioremediation efforts. With increasing interest in the development of constructed wetland treatment systems (CWTS) for contaminant remediation, reproducible techniques to cultivate cattail in a laboratory environment are necessary. The work presented here examined and tested various growth parameters for the successful cultivation of T. latifolia from seed. Successful germination of Typha species involves scarification (rupture of the seed coat), which was achieved using mechanical techniques for large-scale production. Early seed establishment was shown to favor low nutrient growth conditions for the first week, followed by the introduction of fertilizer in subsequent weeks to enhance post-transplant survival. For microbial bioaugmentation of the plant system, results showed that soaking the seeds in inoculum leads to more extensive colonization of the root tissue and long-term bacterial persistence. An optimized seed sterilization technique using a combination of bleach and detergent was used to improve microorganism colonization success. The growth vessels, both sterile and non-sterile, designed in this study support the long-term growth of T. latifolia under various conditions.

Figure 1 shows a representation of a Typha seed that has gone through the scarification process, along with an incompletely scarified seed with the beak removed, and a non-scarified seed. Ensure that the blending time is sufficient to produce primarily fully scarified seeds.
After scarifying and drying the seeds, the viability of the seeds should be tested using the non-sterile germination technique. At the end of 7 days, a minimum of 20% of the seeds sho...

Typha latifolia, which primarily propagates asexually through rhizomes, poses collection challenges due to its extensive root system. This paper presents a method for growing T. latifolia from seed, facilitating easier lab cultivation and offering the potential for sterile plant growth and early microbial bioaugmentation.

Typha latifolia, more commonly known as the broadleaf cattail or the common bulrush, has a globally reaching range and dominates wetland ecosystems in ...

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.

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Research

JoVE Journal

Environment

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Here, the HDBR is successfully applied in a photobioreactor (PBR) configuration for the study of nitrogen metabolism by a mixed high density algal community.

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

The described hydroponic cocultivation system supports intact plants with metal mesh screens and cocultivates them with bacteria. Plant tissue, bacteria, and secreted molecules can then be separately harvested for downstream analyses, simultaneously allowing for the molecular responses of both plant hosts and interacting microbes or microbiomes to be investigated.

A Hydroponic Co-cultivation System for Simultaneous and Systematic Analysis of Plant/Microbe Molecular Interactions and Signaling

On-site microbial enrichment or in situ cultivation techniques can facilitate the isolation of difficult-to-culture microbial taxa, especially from low-biomass or geochemically extreme environments. Here, we describe an electrochemical set-up without using an external power source to enrich microbial strains that are capable of extracellular electron transport (EET).

Self-standing Electrochemical Set-up to Enrich Anode-respiring Bacteria On-site

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

A protocol is described to utilize the carbon dioxide in natural gas power plant flue gas to cultivate microalgae in open raceway ponds. Flue gas injection is controlled with a pH sensor, and microalgae growth is monitored with real time measurements of optical density.

Coupling Carbon Capture from a Power Plant with Semi-automated Open Raceway Ponds for Microalgae Cultivation

This protocol describes how to use the Microbial Microdroplet Culture system (MMC) to conduct automated microbial cultivation and adaptive evolution. MMC can cultivate and sub-cultivate microorganisms automatically and continuously and monitor online their growth with relatively high throughput and good parallelization, reducing labor and reagent consumption.

Automated Microbial Cultivation and Adaptive Evolution using Microbial Microdroplet Culture System (MMC)

A high-throughput protocol for the surface sterilization of Arabidopsisthaliana (Arabidopsis) seeds is provided, optimizing the liquid handling steps with a simple suction device constructed with a vacuum pump. Hundreds of seed samples can be surface-sterilized in one day.

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

This protocol outlines the utilization of Agrobacterium tumefaciens-mediated transformation (AMT) for integrating gene(s) of interest into the nuclear genome of the green microalgae Chlorella vulgaris, leading to the production of stable transformants.

Agrobacterium tumefaciens-Mediated Genetic Engineering of Green Microalgae, Chlorella vulgaris

We present an efficient, optimized protocol for Agrobacterium-mediated transformation of an asexual amphibious Brassicaceae species, Rorippa aquatica, without resource-intensive sterile tissue culture procedures. Utilizing a visual transformation marker and shoot regeneration pipeline successfully generated near clonal transformants, thereby advancing the future studies of plant's adaptation to the underwater environment.

Efficient Regeneration-based Agrobacterium-Mediated Transformation of an Asexual Amphibious Brassicaceae Species, Rorippa aquatica

This protocol describes the stepwise method for adapting Euglena gracilis to cassava-based substrates and the subsequent evaluation of growth, viability, and biomass production across multiple cassava products under heterotrophic cultivation...

A Method for Adapting Euglena gracilis to Cassava-Based Substrates for Heterotrophic Cultivation

Learn reproducible methods for seed germination and microbial bioaugmentation of Typha latifolia. This protocol enables standardized lab cultivation and supports plant-microbe interaction research in wetland ecosystems.

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

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