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

Summary

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.

Abstract

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.

Introduction

As a cost and energy-efficient technology, constructed wetland treatment systems (CWTS) have gained significant popularity for the remediation of environmental contaminants^1,2,3. 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 potential concern in wastewaters^2,4,5. 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 species^6,7. 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 conditions^9,10.

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.

Protocol

1. Seed scarification

1. Cattail seeds were sourced from a wetland in Calgary, AB, Canada, in fall 2023 (51.11312° N, 114.39381° 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.
2. 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 full. This is approximately 250 mL of seeds not compacted.
3. 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.
4. 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.
5. 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.
6. Repeat the blending process on the remainder of the cattail sludge from step 1.5.
7. 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.
8. Pour the seeds into a Büchner funnel with filter paper attached to a vacuum to dry the seeds overnight. Dry seeds can be stored at -20 °C in 15 mL conical polypropylene tubes.

2. Germination using non-sterile technique

1. Prepare half-strength Murashige and Skoog (MS) media plates with 1% phytoagar¹².
2. 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.
3. Remove excess water such that 3 mL remains in the 15 mL conical polypropylene tube.
4. Cut the tip of a 1000 µL plastic pipette tip to increase the bore size and pipette the seed solution vigorously to suspend seeds within the pipette tip.
5. Place seeds and water on the half-strength MS agar plates. Swirl the plate to distribute the seeds evenly.
6. 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 °C and 70% humidity. Incubate the plates for 1 week (up to 2 weeks) to induce seed germination.

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.

1. Prepare half-strength MS media plates with 1% phytoagar¹².
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. Aseptically cut the tip of a 1000 µL plastic pipette tip and vigorously pipette to suspend seeds within the tip.
8. Plate seeds and liquid on half-strength MS agar plates. Swirl the plate to evenly distribute seeds.
9. Wrap plates in laboratory sealing film and place in a growth chamber with a 16 h/8 h light-dark cycle at 23 °C and 70% humidity. Leave plates for 1 week (up to 2 weeks) to allow for germination to occur.

4. Seed inoculation with selected bacteria

1. 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.
2. 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.
3. 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.
4. Remove the supernatant and resuspend the bacterial pellet in 1 mL of sterile ddH2O.
5. 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.
6. Follow the remainder of the sterile seed germination protocol starting from step 3.8.

5. Non-sterile Cattail growth on soil

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. Continue to transplant cattails as they outgrow their container. Remove dead foliage with shears. Fertilize cattails once a month.
7. 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.

6. Sterile seedling growth

1. 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 µm sterile filter, and heat-resistant silicone. See Figure 2 for a visual representation of the chamber setup.
   1. 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.
   2. 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.
   3. 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.
   4. Allow silicone to cure at least 24 h before use.
2. Sterile growth in soil
   1. Follow the sterile seed germination protocol described in step 3 to obtain sterile seeds.
   2. Fill the sterile growth chamber unit with soil of choice, wetted with tap water.
   3. 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.
   4. Attach a 0.2 µm filter unit to the Luer lock attachment on the growth chamber (Figure 2). Open the growth chamber and add 500 µL 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.
   5. 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.
   6. Place the growth chamber unit into a plant growth incubator on a 16 h/8 h day-night cycle at 23 °C and 70% humidity.
3. Sterile growth in hydroponic solution
   1. Follow the sterile germination protocol described in step 3 to obtain sterile seeds.
   2. 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.
   3. Add the tip insert back to the tip box filling the bottom of the box with half-strength Hoagland'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 MnCl₂·4H₂O, 0.0004 mM ZnSO₄·7H₂O, 0.0002 mM CuSO₄·5H₂O, 0.00007 mM H₂MoO₄·1H₂O, and 0.045 mM FeSO₄·7H₂O, adjusted to pH 7.0).
   4. 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.
   5. Close the tip box lid and place in a growth chamber on a 16 h/8 h day-night cycle at 23 °C and 70% humidity.
   6. 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's.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.2 µm sterile filter	VWR	514-4126
3′,5′-Dimethoxy-4′-hydroxyacetophenone	PhytoTech Labs	S7777
Culture boxes with lids (77 mm × 77 mm × 97 mm)	Millipore Sigma	V8505
Heat-resistant silicone sealant hi-temperature	Imperial Manufacturing Group	KK0205
Laboratory sealing film	Millipore Sigma	P7793
Murashige and Skoog media	PhytoTech Labs	M401
Organosilicone surfactant	PhytoTech Labs	S7777
Phytoagar	GoldBiotechnology	P1003
Polysorbate 20	Roche	11332465001