Anne-Marie Stomp

In vitro selection of duckweed geographical isolates for potential use in swine lagoon effluent renovation

Plant-based systems for nutrient sequestration into valuable biomass have the potential to help avoid the environmental problems associated with the disposal of large volumes of animal waste. The objective of this study was to select superior duckweed (Lemnaceae) genotypes for the utilization of nutrients in animal wastes. A two-step protocol was used to select promising duckweed geographic isolates to be grown on swine lagoon effluent. Forty-one geographic isolates from the worldwide germplasm collection were used in an in vitro screening test, because they were noted to be fast-growing genotypes during routine collection maintenance. In vitro screening was accomplished by growing geographic isolates on a synthetic medium that approximated swine lagoon effluent in terms of nutrient profile, total ionic strength, pH, and buffering capacity. Large differences among geographic isolates were observed for wet weight gain during the 11-day growing period, percent dry weight, and percent protein in dry biomass. Total protein production per culture jar differed 28-fold between the most disparate of the 41 geographic isolates and was the variable used for selection of superior geographic isolates. The challenge of eight of the 41 geographic isolates with full-strength swine lagoon effluent in the greenhouse led to the selection of three that are promising as genotypes to be grown on lagoon effluent.

The duckweeds: A valuable plant for biomanufacturing

Inherent characteristics of duckweed, including fast, clonal growth, small size and simple growth habit, argue for their use as a biomanufacturing platform for proteins, polymers and small molecules. This review addresses five areas relevant to commercialization of the duckweed platform: (1) the characteristics of wild-type duckweed and general cultural requirements; (2) the genetics and biochemistry of the plants and recent scientific developments that provide the technology necessary to genetically modify duckweed; (3) the advantages provided by inherent duckweed characteristics and genetic engineering technology relative to bioproduction; (4) recent progress towards commercialization of duckweed-based products and (5) the major research needs for further R&D.

Predicting Shine–Dalgarno Sequence Locations Exposes Genome Annotation Errors

In prokaryotes, Shine–Dalgarno (SD) sequences, nucleotides upstream from start codons on messenger RNAs (mRNAs) that are complementary to ribosomal RNA (rRNA), facilitate the initiation of protein synthesis. The location of SD sequences relative to start codons and the stability of the hybridization between the mRNA and the rRNA correlate with the rate of synthesis. Thus, accurate characterization of SD sequences enhances our understanding of how an organisms transcriptome relates to its cellular proteome. We implemented the Individual Nearest Neighbor Hydrogen Bond model for oligo–oligo hybridization and created a new metric, relative spacing (RS), to identify both the location and the hybridization potential of SD sequences by simulating the binding between mRNAs and single-stranded 16S rRNA 3′ tails. In 18 prokaryote genomes, we identified 2,420 genes out of 58,550 where the strongest binding in the translation initiation region included the start codon, deviating from the expected location for the SD sequence of five to ten bases upstream. We designated these as RS+1 genes. Additional analysis uncovered an unusual bias of the start codon in that the majority of the RS+1 genes used GUG, not AUG. Furthermore, of the 624 RS+1 genes whose SD sequence was associated with a free energy release of less than −8.4 kcal/mol (strong RS+1 genes), 384 were within 12 nucleotides upstream of in-frame initiation codons. The most likely explanation for the unexpected location of the SD sequence for these 384 genes is mis-annotation of the start codon. In this way, the new RS metric provides an improved method for gene sequence annotation. The remaining strong RS+1 genes appear to have their SD sequences in an unexpected location that includes the start codon. Thus, our RS metric provides a new way to explore the role of rRNA–mRNA nucleotide hybridization in translation initiation.

Genetic transformation of duckweed Lemna Gibba and Lemna Minor

SummaryWe developed efficient genetic transformation protocols for two species of duckweed, Lemna gibba (G3) and Lemna minor (8627 and 8744), using Agrobacterium-mediated gene transfer. Partially differentiated nodules were co-cultivated with Agrobacterium tumefaciens harboring a binary vector containing β-glucuronidase and nptII expression cassettes. Transformed cells were selected and allowed to grow into nodules in the presence of kanamycin. Transgenic duckweed fronds were regenerated from selected nodules. We demonstrated that transgenic duckweed could be regenerated within 3 mo. after Agrobacterium-mediated transformation of nodules. Furthermore, we developed a method for transforming L. minor 8627 in 6 wk. These transformation protocols will facilitate genetic engineering of duckweed, ideal plants for bioremediation and large-scale industrial production of biomass and recombinant proteins.

Agrobacterium-mediated DNA transfer in sugar pine

DNA transfer using Agrobacterium tumefaciens has been demonstrated in sugar pine, Pinus lambertiana Dougl. Shoots derived from cytokinin-treated cotyledons formed galls after inoculation with A. tumefaciens strains containing the plasmid pTiBo542. A selectable marker, neomycin phosphotransferase II, conferring resistance to kanamycin, was transferred into sugar pine using a binary armed vector system. Callus proliferated from the galls grew without hormones and in some cases, kanamycin-resistant callus could be cultured. Southern blots provided evidence of physical transfer of T-DNA and the nptII gene. Expression of the nptII gene under control of the nos promoter was demonstrated by neomycin phosphotransferase assays. Several aspects of DNA transfer were similar to those previously observed in angiosperms transformed by A. tumefaciens. This is the first evidence for DNA transfer by Agrobacterium in this species and the first physical evidence for transfer in any pine. These results bring us closer to genetic engineering in this commercially important genus of forest trees.

Genetic improvement of tree species for remediation of hazardous wastes

SummaryPhytoremediation, using plants to clean up toxic wastes, is an idea that is attracting increasing attention from scientists, remediation engineers, and environmental professionals in government, industry, and universities. In situ remediation using plants has the potential to be less expensive than current technologies and to simultaneously initiate both detoxification of hazardous waste and site restoration. The perennial habit, extensive root mass, and large transpirational rates give trees advantages over other plants for use in remediation. Trees are already used for wastewater clean-up, for site stabilization, and as barriers to subsurface flow of contaminated groundwater. Clonal propagation and the genetic tools of both classical breeding and genetic engineering exist for a number of both angiosperm and gymnosperm species, opening the door to creation of tree “remediation” cultivars. Work is underway to screen tree species for their ability to tolerate, take up, translocate, sequester, and degrade organic compounds and heavy metal ions. Both an indirect approach to remediation, through enhancing rhizosphere degradation of pollutants by engineering larger root masses in trees usingAgrobacterium rhizogenes, and a direct approach to remediation, through transformation of trees with bacterial genes known to initiate the mineralization of halogenated phenolic compounds and trichloroethylene are reviewed.

Transient expression from microprojectile-mediated DNA transfer in pinus taeda.

SummaryTransfer of plasmid DNA to Pinus taeda L. (loblolly pine) cotyledon cells by microprojectile bombardment has been demonstrated using beta-glucuronidase (GUS). GUS histochemical staining indicated active enzyme in localized centers (blue spots) 24 hours after bombardment. GUS expression declined during subsequent culture, but remained detectable in meristematic tissue 62 days post-bombardment, however, transgenic shoots were not recovered. Localized GUS expression events resulted predominantly from single-cell events containing one microprojectile. The staining pattern was complex, with indigo found both in the central target cell and in adjacent cells. Cellular damage sustained by GUS-positive cells ranged from undetectable to sufficiently extensive to cause cell death. Microprojectile bombardment provides a useful method to assay transient gene expression in loblolly pine and has potential for the production of transgenic plants in pine.

Combining constructed wetlands and aquatic and soil filters for reclamation and reuse of water

Abstract Reclamation and reuse of water and nutrients at their source provide the opportunity to use simple, less costly technologies and lessens potentials for catastrophic effects due to centralized treatment system failures. The combination of multiple treatment environments within constructed wetlands can provide water quality suitable for reuse. A current project in rural Chatham County, NC, uses simple, aesthetically pleasing treatment components constructed both outdoors and indoors to reclaim domestic sewage for toilet flushing, landscape irrigation and aesthetic water features. A courtyard containing constructed wetlands and a solarium with modular soil filter components and aquatic chambers are designed to treat sewage from within a small business facility and to provide recreational space for its 60 employees. The combination of vertical flow and horizontal flow constructed wetlands with fill and draw controls provides the necessary environments for nitrification–denitrification, removal of organic materials and phosphorus adsorption reactions. The system is designed to treat and reuse 4500 l day −1 (1200 gal day −1 ) of domestic sewage from the business. Some of the plants used are selectively bred or genetically engineered to maximize their water reclamation potential. Utilization of simple treatment and reuse technology has permitted the business owner to renovate an abandoned and deteriorating school building into a home for two thriving and internationally based businesses and to protect the water quality of a nearby reservoir.

Effects of medium components and light on callus induction, growth, and frond regeneration in Lemna gibba (Duckweed)

SummaryBasal media, plant growth regulator type and concentration, sucrose, and light were examined for their effects on duckweed (Lemna gibba) frond proliferation, callus induction and growth, and frond regeneration. Murashige and Skoog medium proved best for callus induction and growth, while Schenk and Hildebrandt medium proved best for frond proliferation. The ability of auxin to induce callus was associated with the relative strength of the four auxins tested, with 20 or 50 µM 2,4-dichlorophenoxyacetic acid giving the highest frequency (10%) of fronds producing callus. Auxin combinations did not improve callus induction frequency. Auxin in combination with other plant growth regulators was needed for long-term callus growth; the two superior plant growth regulator combinations were 10 µM naphthaleneacetic acid, 10 µM gibberellic acid, and 2 µM benzyladenine with either 1 or 20 µM 2,4-dichlorophenoxyacetic acid. Three percent sucrose was best for callus induction and growth. Callus induction and growth required light. Callus that proliferated from each frond’s meristematic zone contained a mixture of dedifferentiated and somewhat organized cell masses. Continual callus selection was required to produce mostly dedifferentiated, slow-growing callus cell lines. Frond regeneration occurred on Schenk and Hildebrandt medium without plant growth regulators but was promoted by 1 µM benzyladenine. Callus maintained its ability to regenerate fronds for at least 10 mo. Regenerated fronds showed a slower growth rate than normal fronds and a low percentage of abnormal morphologies that reverted to normal after one or two subcultures.

The production of duckweed as a source of biofuels

Duckweed is a promising feedstock for the production of biofuels. Advantageous characteristics include rapid, clonal growth as small free-floating plants on nutrient-rich water; global adaptability across a broad range of climates; naturally high protein content; and inducible high starch content with low or no lignin, which enables other value-added products. The objective of this article is to review the published research on duckweed cultivation in nutrient-rich wastewaters, starch enrichment in duckweed plants and conversion of high-starch duckweed to biofuels. Duckweed yields of 39.1–105.9 t ha-1 year-1 have been achieved using wastewater as the nutrient source, which are much higher than the yields of most other potential energy crops. Duckweed starch contents of 31.0–45.8% dry weight have been achieved after it has been subjected to nutrient starvation for 5–10 days, and up to 94.7% of the starch could be converted to ethanol using the existing technologies for corn starch conversion. Future research objectives include selecting high-performance duckweed strains, improving starch enrichment and conversion, and developing technologies for large-scale operations.