Stable transformation of ferns using spores as targets: Pteris vittata and Ceratopteris thalictroides.

Ferns (Pteridophyta) are very important members of the plant kingdom that lag behind other taxa with regards to our understanding of their genetics, genomics, and molecular biology. We report here, to our knowledge, the first instance of stable transformation of fern with recovery of transgenic sporophytes. Spores of the arsenic hyperaccumulating fern Pteris vittata and tetraploid 'C-fern Express' (Ceratopteris thalictroides) were stably transformed by Agrobacterium tumefaciens with constructs containing the P. vittata actin promoter driving a GUSPlus reporter gene. Reporter gene expression assays were performed on multiple tissues and growth stages of gametophytes and sporophytes. Southern-blot analysis confirmed stable transgene integration in recovered sporophytes and also confirmed that no plasmid from A. tumefaciens was present in the sporophyte tissues. We recovered seven independent transformants of P. vittata and four independent C. thalictroides transgenics. Inheritance analyses using ß-glucuronidase (GUS) histochemical staining revealed that the GUS transgene was stably expressed in second generation C. thalictroides sporophytic tissues. In an independent experiment, the gusA gene that was driven by the 2× Cauliflower mosaic virus 35S promoter was bombarded into P. vittata spores using biolistics, in which putatively stable transgenic gametophytes were recovered. Transformation procedures required no tissue culture or selectable marker genes. However, we did attempt to use hygromycin selection, which was ineffective for recovering transgenic ferns. This simple stable transformation method should help facilitate functional genomics studies in ferns.

Sensory analysis of calcium-biofortified lettuce.

Vegetables represent an attractive means of providing increased calcium nutrition to the public. In this study, it was demonstrated that lettuce expressing the deregulated Arabidopsis H(+)/Ca(2+) transporter sCAX1 (cation exchanger 1) contained 25%-32% more calcium than controls. These biofortified lettuce lines were fertile and demonstrated robust growth in glasshouse growth conditions. Using a panel of highly trained descriptive panellists, biofortified lettuce plants were evaluated and no significant differences were detected in flavour, bitterness or crispness when compared with controls. Sensory analysis studies are critical if claims are to be made regarding the efficacy of biofortified foods, and may be an important component in the public acceptance of genetically modified foods.

Pilot-scale demonstration of phytofiltration for treatment of arsenic in New Mexico drinking water.

Arsenic contamination of drinking water poses serious health risks to millions of people worldwide. To reduce such risks, the United States Environmental Protection Agency recently lowered the Maximum Contaminant Level for arsenic in drinking water from 50 to 10 microgL(-1). The majority of water systems requiring compliance are small systems that serve less than 10,000 people. Current technologies used to clean arsenic-contaminated water have significant drawbacks, particularly for small treatment systems. In this pilot-scale demonstration, we investigated the use of arsenic-hyperaccumulating ferns to remove arsenic from drinking water using a continuous flow phytofiltration system. Over the course of a 3-month demonstration period, the system consistently produced water having an arsenic concentration less than the detection limit of 2 microgL(-1), at flow rates as high as 1900 L day(-1) for a total treated water volume of approximately 60,000 L. Our results demonstrate that phytofiltration provides the basis for a solar-powered hydroponic technique to enable small-scale cleanup of arsenic-contaminated drinking water.

Phytofiltration of arsenic from drinking water using arsenic-hyperaccumulating ferns.

Arsenic contamination of drinking water poses serious health risks to millions of people worldwide. Current technologies used to clean arsenic-contaminated water have significant drawbacks, such as high cost and generation of large volumes of toxic waste. In this study, we investigated the potential of using recently identified arsenic-hyperaccumulating ferns to remove arsenic from drinking water. Hydroponically cultivated, two arsenic-hyperaccumulating fern species (Pteris vittata and Pteris cretica cv. Mayii) and a nonaccumulating fern species (Nephrolepis exaltata) were suspended in water containing 73As-labeled arsenic with initial arsenic concentrations ranging from 20 to 500 microg L(-1). The efficiency of arsenic phytofiltration by these fern species was determined by continuously monitoring the depletion of 73As-labeled arsenic concentration in the water. With an initial water arsenic concentration of 200 microg L(-1), P. vittata reduced the arsenic concentration by 98.6% to 2.8 microg L(-1) in 24 h. When the initial water arsenic was 20 microg L(-1), P. vittata reduced the arsenic concentration to 7.2 microg L(-1) in 6 h and to 0.4 microg L(-1) in 24 h. At similar plant ages, both P. vittata and P. cretica had similar arsenic phytofiltration efficiency and were able to rapidly remove arsenic from water to achieve arsenic levels below the new drinking water limit of 10 microg L(-1). However, N. exaltata failed to reduce water arsenic to achieve the limit under the same experimental conditions. The significantly higher efficiency of arsenic phytofiltration by arsenic-hyperaccumulating fern species is associated with their ability to rapidly translocate absorbed arsenic from roots to shoots. The nonaccumulating fern N. exaltata was unable to translocate the absorbed arsenic to the shoots. Our results demonstrate that the arsenic-phytofiltration technique may provide the basis for a solar-powered hydroponic technique that enables small-scale cleanup of arsenic-contaminated drinking water.

Mechanisms of arsenic hyperaccumulation in Pteris species: root As influx and translocation.

Several species of fern from the Pteris genus are able to accumulate extremely high concentrations of arsenic (As) in the fronds. We have conducted short-term unidirectional As influx and translocation experiments with 73As-radiolabeled arsenate, and found that the concentration-dependent influx of arsenate into roots was significantly larger in two of these As-hyperaccumulating species, Pteris vittata (L.) and Pteris cretica cv. Mayii (L.), than in Nephrolepis exaltata (L.), a non-accumulating fern. The arsenate influx could be described by Michaelis-Menten kinetics and the kinetic parameter Km was found to be lower in the Pteris species, indicating higher affinity of the transport protein for arsenate. Quantitative analysis of kinetic parameters showed that phosphate inhibited arsenate influx in a directly competitive manner, consistent with the hypothesis that arsenate enters plant roots on a phosphate-transport protein. The significantly augmented translocation of arsenic to the shoots that was seen in these As hyperaccumulator species is proposed to be due to a combination of the increased root influx and also decreased sequestration of As in the roots, as a larger fraction of As could be extracted from roots of the Pteris species than from roots of N. exaltata. This leaves a larger pool of mobile As available for translocation to the shoot, probably predominantly as arsenite.