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0 Halophyte Biotechnology Center, College of Marine Studies, University of Delaware, 700 Pilottown Road, Lewes, Delaware 19958 USA
Received for publication December 17, 1998. Accepted for publication May 4, 1999.
ABSTRACT
The salt marsh grass Distichlis spicata was regenerated from tissue culture and propagated in a greenhouse. Selected regenerants, along with selections from six wild populations, were grown for two years in a common garden flood-irrigated thrice weekly with tidal creek water. Selected wild and regenerated plants were also planted in a created salt marsh. Significant differences among regenerant and wild population selections were found in several functionally important salt marsh plant characteristics, including potential detritus production, belowground organic matter production, canopy structure, and decomposition rate. A combination of characteristics not found in the wild populations was evident in a regenerated line that exhibited both a high detritus production potential and a high decomposition rate. The amount of variation that occurred among regenerants from one parental line via somaclonal variation was similar to that which occurred among the wild population selections. Results of this study suggest that tissue culture may provide a means of producing marsh grasses with specific characteristics for directing the functional development of newly created salt marshes.
Key Words: Distichlis spicata • marsh creation • marsh restoration • Poaceae • salt marsh • somaclonal variation • tissue culture
Distichlis spicata L. is a dioecious salt marsh grass that occurs along the east (Nova Scotia to Florida), west (British Columbia to Mexico), and Gulf (Florida to Texas) coasts of North America, as well as at saline sites in the interior of the country (Hitchcock, 1971
). It is an important component of many salt marsh ecosystems and grows in the mid-to-high intertidal zone, often at relatively high salinities. Distichlis spicata has been shown to be an important colonizer in marshes recovering from disturbance caused by major storms (Allison, 1995, 1996
). Distichlis spicata marshes provide valuable habitats for birds. Weller (1994)
reports that most birds using an estuarine marsh in Texas favored areas of D. spicata and Scirpus olneyi for feeding and resting. Likewise, for the cinnamon teal, D. spicata habitats were shown to be rich feeding grounds (Thorn and Zwank, 1993
).
Different "varieties" of D. spicata have been studied for use as forage in areas of the world where the soil or water is too saline to grow traditional crops (Gallagher, 1985
; Gallagher and Seliskar, 1993
). In tissue culture experiments, Warren and Gould (1982)
demonstrated that much of the salt tolerance of this species is cellularly based. In the ecological literature, intraspecific variation in clonal morphology of D. spicata has been reported. Such differences have been shown to result in different growth rates, rhizome morphology, and response to disturbance (Brewer and Bertness, 1996
). Two ecotypes of D. spicata responded differently to chloride and sulfate salinity and selenium soil contamination (Enberg and Wu, 1995
). Intraspecific variation in this species can be quite useful. Seliskar (1995)
found that various genotypes of Spartina alterniflora and Spartina patens affected marsh functions differently. For example, rooting depth was dependent on genotype as was canopy height and many other morphological features. These different plants provide a range of characteristics from which can be chosen "varieties" to serve a particular purpose in, for example, a newly created marsh.
Variation, similar to that found in nature, can be generated through tissue culture via somaclonal variation (Larkin and Scowcroft, 1981
; McClintock, 1984
). Seliskar (1998)
found such variation in the marsh plant Sporobolus virginicus. Using tissue culture techniques and the somaclonal variation often exhibited in regenerated plants provides a means of producing "varieties" of D. spicata that may serve particular functions in various settings. Numerous examples of improving crop cultivars via somaclonal variation exist in the literature (Winicov, 1991
; Roylance, Hill, and Parrott, 1994
; Adkins, Kunanuvatchaidach, and Godwin, 1995
; Arnold, Flegmann, and Clarkson,1995
; Racchi et al., 1995
). Thus, this approach could also be used to produce non-crop plants with characteristics valuable for other purposes, such as marsh creation and restoration, practices used to prevent further loss of valuable wetland ecosystems.
The purpose of the present study was twofold: (1) to determine whether or not we could find evidence of somaclonal variation in attributes important to marsh ecosystem function occurring during the tissue culturing of D. spicata, and (2) to compare selections produced through tissue culture with selections of D. spicata collected from wild populations for characteristics with the potential to affect marsh function in created or restored salt marshes.
MATERIALS AND METHODS
Wild populations
Distichlis spicata plants were collected from natural marshes in California, Utah, Massachusetts, Virginia, South Carolina, and Georgia by digging 15 cm diameter clumps of these rhizomatous plants. They were then grown in pots in a greenhouse where they were propagated vegetatively.
Tissue culture-generated plants
Regenerants of D. spicata used in this study were vegetatively propagated in the greenhouse from regenerants originally developed by Straub (1990)
. Regenerants R45 and R99 were derived from callus produced from one seed obtained by hand pollinating female plants from a Georgia population with pollen from male plants of another Georgia population. Similarly, R03 was derived from callus from one seed of another cross and R23, R22, and R71 from callus from one seed of a third cross. Thus we could investigate for somaclonal variation between R44 and R99 and among R23, R22, and R71. Original cultures were established using mature seeds as explant tissue, with callus grown on medium containing Murishige and Skoog (1962) salts plus 0.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 mg/L 1-naphthaleneacetic acid, 0.5 mg/L benzyladenine (BA), 30 g/L sucrose, and 5% coconut water. Plant regeneration was initiated by transferring callus to MSB medium (MS salts plus 1.0 mg/L BA), then from MSB medium to medium containing N6 (Chu et al., 1975
) salts plus 1 mg/L 2,4-D and 20 g/L sucrose, and then back to MSB medium (Straub, Decker, and Gallagher, 1989
).
Field studies
Plants were planted in 8-L plastic buckets modified with 25 evenly distributed drainage holes on sides and bottom, and an 8 x 2 cm slit 3 cm down from the top rim to allow for water to flow into and out of the bucket, similar to the units described by Gallagher and Wolf (1980)
. The buckets were filled to a level even with the bottom of the slit with field soil, which had been sieved to remove root material, and each was planted with four plants with attached rhizome sections of equal size. There were six replicate buckets for each plant selection. Buckets were set out during early June in a simulated marsh field plot such that the bottom of the slit was level with the soil surface. They were flood-irrigated thrice weekly during the growing season with saltwater pumped from a nearby tidal creek (salinity ~25 g/L) and harvested in early November of the second year. The buckets were taken to the greenhouse where the shoots were cut off at the soil surface and placed in plastic bags and stored in a refrigerator until morphological and biomass measurements could be made. Morphological measurements made later on the shoots included plant height, stem density, leaf length and width, internode length, and stem diameter. Using a sieve, the roots and rhizomes were washed free of soil. Roots and rhizomes were separated, dried at 60°C to constant mass, and weighed. Root :shoot ratios and root : rhizome ratios were determined.
In addition to the field study described above, one of the regenerants from the above study (R99) was also planted in a newly created marsh and compared to one other regenerant (R98) and plants from four wild populations in the United States (California, Utah, Delaware, and Georgia), three of which were also planted in the field plot experiment described above. This 0.2-ha created marsh was connected via tidal creeks to the adjacent natural Canary Creek marsh in Lewes, Delaware, USA. In the spring of the year following planting, attached dead leaf and shoot material were collected. Respiration rate of the microbial community associated with the dead plant material was determined by measuring oxygen uptake (Digital Oxygen Meter, YSI Model 58, Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio, USA) according to the methods of Gallagher and Pfeiffer (1977)
.
RESULTS
Variation was evident in shoot biomass among wild populations with the South Carolina (SC) and Georgia (GA) populations exhibiting the highest values and those from California (CA), Utah (UT), and Massachusetts (MA) the lowest, with Virginia (VA) exhibiting an intermediate value (Fig. 1). Biomass varied between 370 and 750 g dry mass/m2, a difference of 380 g/m2. Among the regenerants, there was significant variation between R45 and R99 (two plants regenerated from the same parent callus), a difference of ~370 g/m2, likely indicating somaclonal variation. This amount of variation is similar to that seen among the six wild populations. There was similar variation between R23 and R71, which originated from the same parent callus.
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Due to their high productivity and their three-dimensional physical structure, vascular plants are important regulators of salt marsh ecosystems. Food webs in salt marshes are largely detritus based, and the shoot biomass of the vascular plants is the primary detritus source. Therefore variation in the amount of biomass produced can significantly affect marsh processes. In the present study, the greatest quantity of potential detritus was produced by two of the regenerants, R99 and R03 (Fig. 1). Therefore, these selections produced through tissue culture, offer higher aboveground biomass production than any of the wild populations. Thus, these would be the best plants to use when selecting "varieties" of D. spicata for planting in a new marsh where plant density is low and there is little aboveground biomass to fuel detritus formation, and therefore the aerial and aquatic processes of the marsh. Also, R99 combined its high biomass with a high decomposition rate (Table 2), indicating that this selection may be more readily available to consumers.
The production of belowground organic matter is especially important in a newly excavated marsh where the topsoil has been removed to bring the elevation down to the appropriate level, leaving little organic matter in the remaining soil. Those plants that can direct the most organic matter belowground will be the best for fueling the soil microbial and invertebrate populations. Minello and Zimmerman (1992)
have demonstrated that the amount of organic material in the soil in transplanted marshes is positively correlated with the densities of decapod crustaceans and infauna. Once again, regenerants R99 and R03 appear to be the best candidates for the job, however the wild populations from along the east coast also put substantial amounts of organic matter belowground (Fig. 2).
The size of the network of roots and rhizomes is also critical where erosion is a concern, as is the case in many coastal habitats, and the same selections just mentioned would be the best for protecting against erosion as well. Allocation of photosynthate to above- and belowground portions of the plant is indicated by the root : shoot ratio, with a value >1.0 indicating more roots and rhizomes than shoots and a value <1.0 indicating that more resources were directed to shoots than to belowground parts. There was less variation among the regenerants in this characteristic than among the wild populations (Table 1). One might want to select for plants with a high root :shoot ratio in potential erosion sites. The MA selection appears to be the best candidate for this purpose. However, the actual amount of biomass belowground is not significantly more than in the other three east coast selections and the shoot biomass is highest in the SC and GA plants, therefore it may be advantageous to choose one of these selections that can enhance both above- and belowground processes. For the same reason, R99 and R03 would be the best among the regenerants and are probably better choices than the wild populations because both their above- and belowground biomass exceeds that of the east coast selections.
Root : rhizome ratio was significantly greater in the regenerant R22 than in any of the other regenerant or wild selections. This may indicate that R22 would be a good candidate for quickly forming a root network since it does not put a lot of resources into rhizome storage and may be a good plant for quickly stabilizing a potentially erosional site. Another aspect of belowground growth is how it affects the competitive ability of the plant. For example, Barber (1982)
suggested that the dense rhizomes of Spartina patens limit the spread of Spartina alterniflora. Such characteristics could be considered when selecting plants for a particular marsh site and potential interactions with other species are taken into consideration.
The architecture of the vegetation canopy is a significant feature of the salt marsh habitat. For fish and other fauna, canopy structure determines the quality of the habitat for nursery and feeding grounds and for protection from predation. Canopy height, for example, has been shown to be critical for bird nesting in the salt marsh (Zedler, 1993
). Canopy height may also play a role in protecting young fish from avian predators. However, in some constructed marshes the canopy height typical of natural marshes has not been obtained. For example, Gibson, Zedler, and Langis (1994)
demonstrated that nitrogen amendments did not result in the desired tall canopies for Spartina foliosa in created marshes in San Diego Bay, California, USA. Canopy height may be a characteristic that would be best selected for by tissue culture or some other genetic means rather than by trying to obtain the desired canopy height with added nutrients, which has been shown to not always work. In the present study, the tallest canopies of D. spicata were developed by the regenerant plants (Fig. 3).
Minello and Zimmerman (1983)
have determined that stem density is a significant factor in protecting young fish from predators. In this study, both wild populations and regenerants offer plants that provide a high stem density (Fig. 4). If a low stem density is desirable for a particular site, R23 offers the best option.
Canopy structure also determines the amount of light reaching the soil surface and the edaphic algal community. With this factor, not only stem density and height play a role, but so does leaf structure, i.e., length and width, along with stem diameter and internode length. For example, the UT selection has the narrowest leaf width, a relatively short leaf length, and a long internode length, all of which may allow more light to reach the soil surface (Table 1). Stem diameter, which can affect the stability and uprightness of the canopy, was greatest in the R03 plants. Four of the six regenerants had thicker stems than did the wild population plants.
We found that the amount of variation in many characteristics measured that occurred among regenerants from one parental line via somaclonal variation was similar to that which occurred among all the wild population selections, suggesting that tissue culture can be a valuable tool in developing plants with special characteristics for use in wetland creation and restoration. In comparison with the characteristics of plants from wild populations, we found that some of the plants we regenerated had undergone somaclonal variation, which resulted in some new plants with attributes not found in the wild populations tested. For example, canopy height was found to be greatest in a couple of the regenerant plants and the lowest stem density was produced by one of the regenerants. The plant selection having the highest percentage of flowering stems was also a regenerant, as was the one having the greatest root : rhizome ratio. However, perhaps more significant is the potential to produce plants with a combination of characteristics not found in wild populations. Plants with the ability to produce the greatest amount of shoot biomass, and therefore potential detritus to feed the detritus-based marsh ecosystem, were two of the regenerants and one of these two regenerants (R99) was tested in the created marsh and exhibited a higher decomposition rate (Table 2) than three out of four of the wild populations. And, of all the plants tested, only the regenerant R99 had the combination of both high biomass and rapid decomposition rate, thus having the ability to put a lot of biomass that breaks down quickly into the ecosystem. Regenerants (R99 and R03) also exhibited a combination of high stem density and high shoot biomass not offered by the wild populations.
FOOTNOTES
1 The authors thank Mike Gross, Xianggan Li, Laurie Mutz, Divakar Rao, Ron Smith, Tonny Wijte, and Jinglan Wu for their assistance with various aspects of the field work and data collection. Support for this research came from the Coastal Ocean Program Office of the National Oceanic and Atmospheric Administration through Grant Number NA90AA-D-SG457 to the University of Delaware Sea Grant Program. The original genotypes used for this project were developed under Grant Number NA83AA-D-00017, Project R/B-22 of the University of Delaware Sea Grant Program. 
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= 0.05 as determined by one-way ANOVA and the Fisher's LSD test
