John L. Gallagher

Halophytic crops for cultivation at seawater salinity

Several hundred halophytes from salt marshes and salt deserts of the world have been evaluated in our laboratory at various degrees of intensity, and a few have been selected for development as crops. The development of the cultivars and the basic biology of the plants is being studied in Delaware in the United States. Agronomic testing, feeding trials, and development of the best agronomic practices are taking place in the saline desert at the American University in Cairo research station in Sadat City. Our present efforts focus primarily on three forages, one grain, and one vegetable.Sporobolus virginicus cultivars for both hay and pasture are being tested. ADistichlis spicata cultivar for hay has been identified, andSpartina patens is being evaluated as a hay as well. Although we do not yet have the data for a full years growth in Egypt, forage yields of these various cultivars, when harvested as hay crops, range to 6.9 or more tons per acre, depending on the salinity and other environmental conditions, and the crude protein content as indicated by the nitrogen content ranges from 6 to 10%. Cultivars having the most useful agronomic qualities have been identified and are being increased in quantity. The grain cropKosteletzkya virginica is a perennial, producing a seed which resembles millet; its whole seeds contain approximately 25% protein and 15% oil. The yields of one of our better cultivars are about 1460 kg/ha (1300 lb/acre) when grown under 25‰ salinity. The vegetableAtriplex triangularis (similar to spinach) has been under mass selection for four years; a cultivar has been identified and seed is now being increased for this species.

Root-secreted Allelochemical in the Noxious Weed Phragmites Australis Deploys a Reactive Oxygen Species Response and Microtubule Assembly Disruption to Execute Rhizotoxicity

Phragmites australis is considered the most invasive plant in marsh and wetland communities in the eastern United States. Although allelopathy has been considered as a possible displacing mechanism in P. australis, there has been minimal success in characterizing the responsible allelochemical. We tested the occurrence of root-derived allelopathy in the invasiveness of P. australis. To this end, root exudates of two P. australis genotypes, BB (native) and P38 (an exotic) were tested for phytotoxicity on different plant species. The treatment of the susceptible plants with P. australis root exudates resulted in acute rhizotoxicity. It is interesting to note that the root exudates of P38 were more effective in causing root death in susceptible plants compared to the native BB exudates. The active ingredient in the P. australis exudates was identified as 3,4,5-trihydroxybenzoic acid (gallic acid). We tested the phytotoxic efficacy of gallic acid on various plant systems, including the model plant Arabidopsis thaliana. Most tested plants succumbed to the gallic acid treatment with the exception of P. australis itself. Mechanistically, gallic acid treatment generated elevated levels of reactive oxygen species (ROS) in the treated plant roots. Furthermore, the triggered ROS mediated the disruption of the root architecture of the susceptible plants by damaging the microtubule assembly. The study also highlights the persistence of the exuded gallic acid in P. australis’s rhizosphere and its inhibitory effects against A. thaliana in the soil. In addition, gallic acid demonstrated an inhibitory effect on Spartina alterniflora, one of the salt marsh species it successfully invades.

Persistent Differences in Two Forms of Spartina Alterniflora: A Common Garden Experiment

Tall- and short-form Spartina alterniflora plants were transplanted from the Delaware marsh to common garden plots during the summer of 1978 and irrigated with saltwater three times a week during that and each subsequent growing season. Nine years later plant biomass, culm height, density, and diameter and flowering frequency remained distinct. Underground regrowth reserves and root profiles likewise remained distinct for the two forms. Since the two growth forms had been living in the same environment for 9 yr and had retained many morphological and physiological differences, some genetic control of the morphology and physiology of the two growth forms is highly probable.

Rhizome growth dynamics of native and exotic haplotypes ofPhragmites australis (Common reed)

Phragmites australis (common reed), a clonal grass, has expanded from a minor component of the mid-Atlantic wetlands to a dominant species. It has been suggested that invasive populations ofPhragmites are an exotic haplotype responsible for the dramatic increase in the distribution of the species. We used field observations and measurements and a greenhouse assay to compare native (haplotype F) and exotic (haplotype M) populations, growing adjacent to one another in a brackish marsh near Odessa, Delaware. In the marsh, shoots of the exotic strain emerged from the rhizomes earlier than those of the native and by March there was an order of magnitude more new shoots of the exotic strain than the native. In August, the exotic strain was 30% taller than the native, had twice the leaf biomass, and twice the total biomass. Nine of ten morphological and biomass characteristics measured differed significantly between the native and exotic strains. A greenhouse assay was conducted by planting rhizomes collected in March in shallow trays and growing them for 70 d followed by shoot harvest (Harvest 1). Rhizomes were measured, replanted, and grown for 35 d after which time they were measured and shoots were harvested (Harvest 2). At Harvest 1, shoot height was approximately 80% greater in the exotic strain, shoot biomass was three times higher, aboveground to belowground biomass ratio was twice as high, and rhizome internode length was 50% greater in the exotic strain than the native. These traits, in addition to number of shoots, were also greater in the exotic strain at Harvest 2. The number of rhizome buds at Harvest 1 was three times greater in the native than in the exotic strain. The greater number of rhizome buds in the native would seem to be an advantage, but it did not result in more shoot production. Buds were maintained in an inactive state that does not allow this strain to compete well in a wetland environment inhabited by a more efficient spreader. The earlier emergence of new shoots from the rhizomes, the greater aboveground structure, the greater rhizome internode length, and the quick transition of rhizome buds to shoot or rhizome explain in part the exotic strains advantage over the native and the mechanisms for its invasive nature.

Morphological and physiological responses to increased salinity in marsh and dune ecotypes ofSporobolus virginicus (L.) Kunth

SummarySporobolus virginicus (L.) Kunth is a halophytic grass native to tropical and warm temperate coasts throughout the world. A rhizomatous perennial with erect culms,S. virginicus occurs as two genetically distinct growth forms, which are designated by their characteristic habitats as “marsh” and “dune”. What accounts for the specific distribution and maintenance of two separate ecotypes ofS. virginicus is not known. The present study examined the effects of seawater salinity on several morphological and physiological responses of hydroponically cultivated marsh and dune plants to determine whether differential tolerance to substrate salinity might contribute to the observed pattern of habitation. Both marsh and dune form plants survived prolonged exposure to full-strength seawater and reproduced vegetatively via culms and rhizomes. Salinity-induced reductions in culm height, internode length, and leaf size led to a miniaturization of marsh and dune plants. Sodium ion levels were low (<1.0 mmol/g dry weight) in various organs of salinized plants irrespective of ecotype, and potassium ion content increased in all salt-challenged plants, as did quarternary ammonium compounds and proline. Significant differences, however, between marsh and dune plants with respect to the effects of salinity on resource allocation, flowering phenology, and protein composition suggested that external salt concentration has a role in determining ecotype distribution.

The response of plasma membrane lipid composition in callus of the halophyte Spartina patens (Poaceae) to salinity stress

Callus cultures of the salt marsh grass Spartina patens were examined to determine changes and consistencies in membrane lipid composition in response to salt. Major membrane lipid classes remained stable at all salinity levels (0, 170, 340 mmol/L). However, the membrane protein to lipid ratio decreased significantly in response to elevated NaCl. Callus plasma membrane (PM) consisted predominantly of sterols, about 60% (mol%) of the total lipids. Glycolipid was the second largest lipid class, making up about 20% (mol%) of the total. With increasing salinity, the relative percentage of sitosterol decreased, while that of campesterol increased. The phospholipid species detected were phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI). When callus was grown at 340 mmol/L NaCl, PC increased significantly. PI and PS were also significantly elevated in salinity treatments. Only 24-32% of the PM fatty acids were common plant membrane fatty acids, C16, C18, C20, and C22, while over 60% were the less common fatty acids, C11 and C14. Membrane fluidity remained stable in response to growth medium salinity. The findings on membrane responses to salinity will facilitate a better understanding of this halophytes tactics for salt tolerance.

Exploiting wild population diversity and somaclonal variation in the salt marsh grass Distichlis spicata (Poaceae) for marsh creation and restoration

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.

Osmotic potential and turgor maintenance in Spartina alterniflora Loisel.

SummaryThe dependence of leaf water potential (Ψ), osmotic potential (π) and turgor pressure (P) on relative water content (RWC) was determined for leaves of tall and short growth forms of Spartina alterniflora Loisel. from a site on Canary Creek marsh in Lewes, Delaware. Tall plants (ca. 1.5 m) occured along a drainage ditch where interstitial water salinity was approximately 20‰, and short plants (ca. 0.2 m) were 13 m away near a pan and exposed to 80‰ salinity during the most stressful period. Leaves were collected at dawn and pressure-volume measurements were made as they desiccated in the laboratory. Pressure equilibrium was used to measure Ψ, RWC was determined from weight loss and dry weight, π was determined from the pressure volume curve, and P was calculated as the difference between Ψ and π. Physical properties of the bulk leaf tissue that have a role in regulating water balance of the two growth forms were estimated: relative water content of apoplastic water (RWCa) relative water content at zero turgor (RWC0), the bulk modulus of elasticity (E), and water capacity (Cw). There were no detectable temporal trends in any of the parameters measured from Nune through September and no significant differences between the two growth forms when compared on the basis of RWCa, RWC0, E, and Cw. There was a clear difference between the two growth forms with respect to π; at RWC0, π was-4.5±0.40 MPa for short form plants and-3.3±0.40 MPa for tall form.Turgor pressure of plants in the field (P′) was lower in leaves from short form than for the tall form plants with average difference of about 0.4 MPa. In July, P′ in short form leaves dropped to zero by mid-morning as expected for leaves experiencing water stress.These results show that S. alterniflora is capable of reducing osmotic potential in response to increased salinity and that turgor pressure was lower in short growth form than in tall forms.

Tissue culture and plant regeneration of spartina alterniflora: Implications for wetland restoration

A tissue culture and plant regeneration protocol for the salt marsh grass, Spartina alterniflora, has been developed. Callus was efficiently induced on Murashige and Skoog (MS) medium supplemented with 1 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1 mg L−1 indole-3-acetic acid (IAA). Callus initiation from 6-day-old seedlings was faster and occurred with a greater frequency than callus initiation from coleoptile-covered segments from the same age seedlings. However, only the coleoptile-covered segments produced regenerable callus, which was maintained on MS medium supplemented with 1 mg L−1 2,4-D and 1 mg L−1 naphthaleneacetic acid (NAA). The regenerable callus differentiated into shoots upon transfer to shoot regeneration medium. A high frequency of shoot regeneration was obtained when the medium contained 3 mg L−1 6-benzylaminopurine (BA) or Thidiazuron (TDZ), with or without the addition of 0.2 mg L−1 IAA. Regenerated shoots were transferred to root regeneration medium, the optimal of which was determined to be half-strength MS medium supplemented with 1 mg L−1 indole-3-butyric acid (IBA). TDZ in the shoot regeneration medium inhibited root formation in the root regeneration medium, making BA rather than TDZ the optimal hormone for the shoot regeneration medium. The mode of plant regeneration was organogenesis. Upon transfer to soil, the most successful growth of plants occurred in a mixture of commercially available potting soil and natural marsh mud. The development of a tissue culture and regeneration protocol for S. alterniflora provides the possibility of selecting lines of this species, via somaclonal variation, with characteristics desirable for wetland creation and restoration.

Plant regeneration from callus cultures of salt marsh hay, Spartina patens, and its cellular-based salt tolerance

Salt marsh hay, Spartina patens (Ait.) Muhl. (Poaceae), is a perennial salt-tolerant grass common in salt marshes and sand dunes of the Atlantic and Gulf coasts of the USA, and grows vigorously at coastal seawater salinity. To study the salt tolerance mechanisms that operate in S. patens at the cellular level, a tissue culture and regeneration protocol for this species was developed. Callus was initiated from seedling mesocotyl on ADM medium (Murashige and Skoog (MS) salts + 3% sucrose + 1 mg l−1 indoleacetic acid (IAA) and 1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D)). Regenerable callus was selected from the several morphotypes that developed and was maintained on BND medium (MS salts + 3% sucrose + 0.5 mg l−1 6-benzylaminopurine (BAP), 1 mg l−1 1-naphthaleneacetic acid (NAA), 0.5 mg l−1 2,4-D, and 50 ml l−1 coconut water (CW)). Shoots formed from 90% of the cultures grown on shoot regeneration medium containing BAP and IAA. Roots formed from shoots when they were transferred to root regeneration medium containing indole-3-butyric acid (IBA) and activated charcoal or reduced strength MS medium. Plants regenerated via organogenesis have flowered and set viable seeds in a saltwater-irrigated field plot. Dry weight accumulation of unadapted callus at 510 mM NaCl is similar to that at 0 mM NaCl (control), indicating that S. patens has strong salt tolerance at the cellular level.