Edmund O. Bauer

High frequency somatic embryogenesis from leaf tissue of Populus spp.

Abstract A high frequency somatic embryogenesis protocol was developed for biotechnological manipulations of hybrid poplar in which embryogenic callus was derived from non-embryonic explants. Somatic embryos of Populus alba × P. grandidentata (NC-5339) were differentiated directly on leaf explants and indirectly from embryogenic cell suspension callus initiated from leaf tissue. Leaf explants were induced to form embryogenic callus in darkness on Murashige and Skoog (MS) medium with 5 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 mg/l benzyladenine (BA), and 30 gg/l sucrose. The auxin to cytokinin ratio (5 mg/l 2,4-D to 0.05 mg/l zeatin) was optimized for globular-shaped embryo initiation from cell suspension callus. Heart-shaped embryo development occurred after removal of 2,4-D from the culture medium, and development progressed to embryo maturity on the same medium. Late stage torpedo-shaped embryos germinated following a 1-week subculture on MS medium containing 5 mg/l IAA and 0.05 mg/l BA, and germinated embryos were successfully transferred ex vitro to greenhouse growth conditions.

Clonal variation in survival and growth of hybrid poplar and willow in an in situ trial on soils heavily contaminated with petroleum hydrocarbons.

Species and hybrids between species belonging to the genera Populus (poplar) and Salix (willow) have been used successfully for phytoremediation of contaminated soils. Our objectives were to: 1) evaluate the potential for establishing genotypes of poplar and willow on soils heavily contaminated with petroleum hydrocarbons and 2) identify promising genotypes for potential use in future systems. We evaluated height, diameter, and volume after first year budset by testing 20 poplar clones and two willow clones. Unrooted cuttings, 20 cm long, were planted in randomized complete blocks at 0.91- × 0.91-m spacing at Gary, IN, USA (41.5°N, 87.3°W). Four commercial poplar clones (NM6, DN5, DN34, and DN182) were planted as 20- and 60-cm cuttings. Sixty-cm cuttings exhibited greater height and diameter than 20-cm cuttings; however, we recommend continued use and testing of different combinations of genotype and cutting length. We identified promising genotypes for potential use in future systems and we recommend allocating the majority of resources into commercial poplar clones, given their generalist growth performance. However, further utilization and selection of experimental clones is needed. Specific clones rather than genomic groups should be selected based on the geographic location and soil conditions of the site.

SELECTING AND UTILIZING POPULUS AND SALIX FOR LANDFILL COVERS: IMPLICATIONS FOR LEACHATE IRRIGATION

The success of using Populus and Salix for phytoremediation has prompted further use of leachate as a combination of irrigation and fertilization for the trees. A common protocol for such efforts has been to utilize a limited number of readily-available genotypes with decades of deployment in other applications, such as fiber or windbreaks. However, it may be possible to increase phytoremediation success with proper genotypic screening and selection, followed by the field establishment of clones that exhibited favorable potential for clean-up of specific contaminants. There is an overwhelming need for testing and subsequent deployment of diverse Populus and Salix genotypes, given current availability of clonal material and the inherent genetic variation among and within these genera. Therefore, we detail phyto-recurrent selection, a method that consists of revising and combining crop and tree improvement protocols to meet the objective of utilizing superior Populus and Salix clones for remediation applications. Although such information is lacking for environmental clean-up technologies, centuries of plant selection success in agronomy, horticulture, and forestry validate the need for similar approaches in phytoremediation. We bridge the gap between these disciplines by describing project development, clone selection, tree establishment, and evaluation of success metrics in the context of their importance to utilizing trees for phytoremediation.

Evaluation of Populus and Salix continuously irrigated with landfill leachate I. Genotype-specific elemental phytoremediation

There is a need for the identification and selection of specific tree genotypes that can sequester elements from contaminated soils, with elevated rates of uptake. We irrigated Populus (DN17, DN182, DN34, NM2, NM6) and Salix (94003, 94012, S287, S566, SX61) genotypes planted in large soil-filled containers with landfill leachate or municipal water and tested for differences in inorganic element concentrations (P, K, Ca, Mg, S, Zn, B, Mn, Fe, Cu, Al, Na, and Cl) in the leaves, stems, and roots. Trees were irrigated with leachate or water during the final 12 wk of the 18-wk study. Genotype-specific uptake existed. For genera, tissue concentrations exhibited four responses. First, Populus had the greatest uptake of P, K, S, Cu, and Cl. Second, Salix exhibited the greatest uptake of Zn, B, Fe, and Al. Third, Salix had greater concentrations of Ca and Mg in leaves, while Populus had greater concentrations in stems and roots. Fourth, Populus had greater concentrations of Mn and Na in leaves and stems, while Salix had greater concentrations in roots. Populus deltoides × P. nigra clones exhibited better overall phytoremediation than the P. nigra × P. maximowiczii genotypes tested. Phytoremediation for S. purpurea clones 94003 and 94012 was generally less than for other Salix genotypes. Overall, concentrations of elements in the leaves, stems, and roots corroborated those in the plant–sciences literature. Uptake was dependent upon the specific genotype for most elements. Our results corroborated the need for further testing and selecting of specific clones for various phytoremediation needs, while providing a baseline for future researchers developing additional studies and resource managers conducting on-site remediation.

Evaluation of Populus and Salix Continuously Irrigated with Landfill Leachate II. Soils and Early Tree Development

Soil contaminant levels and early tree growth data are helpful for assessing phytoremediation systems. Populus (DN17, DN182, DN34, NM2, and NM6) and Salix (94003, 94012, S287, S566, and SX61) genotypes were irrigated with landfill leachate or municipal water and tested for differences in 1) element concentrations (P, K, Ca, Mg, S, Zn, B, Mn, Fe, Cu, Al, and Na) of a topsoil layer and a layer of sand in tanks with a cover crop of trees or no trees and 2) height, diameter, volume, and dry mass of leaves, stems, and roots. Trees were irrigated with leachate or water during the final 12 wk of the 18-wk study. Differences in most soil element concentrations were negligible (P > 0.05) for irrigation treatments and cover main effects. Phosphorous, K, Mg, S, Zn, Mn, Fe, and Al concentrations were greater in topsoil than sand (P = 0.0011 for Mg; P < 0.0001 for others). There was broad variation between genera and among clones for all growth traits. The treatment × clone interaction governed height, volume, and root dry mass, with (94012, SX61), (NM2, S566, SX61), and (S287, S566) exhibiting the greatest levels, respectively, following leachate application. Given the broad amount of variability among and within these genera, there is great potential for the identification and selection of specific genotypes with a combination of elevated phytoremediation capabilities and tree yield. From a practical standpoint, these results may be used as a baseline for the development of future remediation systems.