Charles Kwit

Transgene introgression in crop relatives: molecular evidence and mitigation strategies

Incorporation of crop genes into wild and weedy relative populations (i.e. introgression) has long been of interest to ecologists and weed scientists. Potential negative outcomes that result from crop transgene introgression (e.g. extinction of native wild relative populations; invasive spread by wild or weedy hosts) have not been documented, and few examples of transgene introgression exist. However, molecular evidence of introgression from non-transgenic crops to their relatives continues to emerge, even for crops deemed low-risk candidates for transgene introgression. We posit that transgene introgression monitoring and mitigation strategies are warranted in cases in which transgenes are predicted to confer selective advantages and disadvantages to recipient hosts. The utility and consequences of such strategies are examined, and future directions provided.

FRUIT ABUNDANCE AND LOCAL DISTRIBUTION OF WINTERING HERMIT THRUSHES (CATHARUS GUTTATUS) AND YELLOW-RUMPED WARBLERS (DENDROICA CORONATA) IN SOUTH CAROLINA

Abstract We conducted winter censuses of two short-distance migrants, Hermit Thrushes (Catharus guttatus) and Yellow-rumped Warblers (Dendroica coronata), over seven years in five different habitats to determine whether their local abundances could be predicted by fruit pulp biomass. Sampled habitats were stands of upland and bottomland hardwood, loblolly pine (Pinus taeda), longleaf pine (P. palustris), and young (<10 years) longleaf pine. Hermit Thrush abundance, which was highest in bottomland hardwood habitats, was positively related to total dry mass of fruit pulp. Those results are consistent with the hypothesis that resource availability affects the local distribution of migrant passerines on their wintering grounds. Our results also indicate that bottomland hardwood habitats in the southeastern United States may be especially important to wintering Hermit Thrushes. Yellow-rumped Warbler abundance was correlated with ripe-fruit pulp dry mass of Myrica cerifera, a major source of winter food for that species. However, because M. cerifera pulp dry mass was confounded with habitat type, we could not distinguish the relative importance of fruit resources and habitat for Yellowrumped Warblers. Our results underscore the importance of fruit to wintering birds. However, the overall percentage of variation in winter bird abundance explained by differences in ripefruit biomass was modest, indicating that other factors are also important.

Gene flow matters in switchgrass (Panicum virgatum L.), a potential widespread biofuel feedstock

There currently exists a large push for the use, improvement, and expansion via landscape modification of dedicated biofuel crops (feedstocks) in the United States and in many parts of the world. Ecological concerns have been voiced because many biofuel feedstocks exhibit characteristics associated with invasiveness, and due to potential negative consequences of agronomic genes in native wild populations. Seed purity concerns for biofuel feedstock cultivars whose seeds would be harvested in agronomic fields also exist from the agribusiness sector. The common thread underlying these concerns, which have regulatory implications, is gene flow; thus detailed knowledge of gene flow in biofuel crop plants is important in the formulation of environmental risk management plans. Here, we synthesize the current state of knowledge of gene flow in an exemplary biofuel crop, switchgrass (Panicum virgatum L.), which is native to eastern North America and is currently experiencing conventional and technological advances in biomass yields and ethanol production. Surprisingly little is known regarding aspects of switchgrass pollen flow and seed dispersal, and whether native populations of conspecific or congeneric relatives will readily cross with current agronomic switchgrass cultivars. We pose that filling these important gaps will be required to confront the sustainability challenges of widespread planting of biofuel feedstocks.

Advances in biotechnology and genomics of switchgrass

Switchgrass (Panicum virgatum L.) is a C4 perennial warm season grass indigenous to the North American tallgrass prairie. A number of its natural and agronomic traits, including adaptation to a wide geographical distribution, low nutrient requirements and production costs, high water use efficiency, high biomass potential, ease of harvesting, and potential for carbon storage, make it an attractive dedicated biomass crop for biofuel production. We believe that genetic improvements using biotechnology will be important to realize the potential of the biomass and biofuel-related uses of switchgrass. Tissue culture techniques aimed at rapid propagation of switchgrass and genetic transformation protocols have been developed. Rapid progress in genome sequencing and bioinformatics has provided efficient strategies to identify, tag, clone and manipulate many economically-important genes, including those related to higher biomass, saccharification efficiency, and lignin biosynthesis. Application of the best genetic tools should render improved switchgrass that will be more economically and environmentally sustainable as a lignocellulosic bioenergy feedstock.

KIRTLAND'S WARBLERS IN ANTHROPOGENICALLY DISTURBED EARLY-SUCCESSIONAL HABITATS ON ELEUTHERA, THE BAHAMAS

Abstract. To characterize the nonbreeding habitat of Kirtlands Warbler (Dendroica kirtlandii) on Eleuthera, The Bahamas, we quantified the habitat at sites where we captured the warblers and compared these traits with those of random sites and sites of tall coppice. On the basis of a chronosequence of satellite imagery, 153 capture sites ranged in age from 3 to 28 years after human disturbance, mean 14.6 years ± 6.3 (SD). Capture sites had been abandoned after clearing (65%), converted to goat pasture (26%), burned (2%), or were young second growth following unknown disturbance (7%). Canopies in 104 capture plots were lower (mean 1.8 m) than canopies in random plots (mean 2.7 m) and plots of late-successional tall coppice (mean 6.3 m). At seven sites mean foliage density in capture plots was consistently greatest at 0.5 to 1.0 m height, but the sites were heterogeneous for other foliage-height classes <3 m and for time since disturbance, canopy height, stem density, and five ground-cover traits. Plots did not differ by the sex of the captured bird except for a difference (P = 0.05) in foliage density at heights <3 m. Kirtlands Warblers frequently consumed fruit (69% of 499 observations), especially from Lantana involucrata, Erithalis fruticosa, and Chiococca alba. Foliage of these plants was more abundant in capture plots than random plots. Because the warblers consume fruit extensively and fruit is more abundant in early successional habitat, this species, like other nearctic—neotropical migrants that breed in early successional habitats, is absent from mature forests on the wintering grounds.

The distribution of tree species in steepheads of the Apalachicola River Bluffs, Florida'

KWIT, C. (Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803), M. W. SCHWARTZ (Center for Population Biology, University of California, Davis CA 95616), W. J. PLATT (Department of Biological Sciences, Louisiana State University, Baton Rouge, LA 70803), and J. P. GEAGHAN (Department of Experimental Statistics, Louisiana State University, Baton Rouge, LA 70803). The distribution of tree species in steepheads of the Apalachicola River Bluffs, Florida. J. Torrey Bot. Soc. 125:309-318. 1998.-Steephead ravine forests of the Apalachicola River Bluffs region of northern Florida contain tree species that are widespread in the Southeastern Coastal Plain and species that are highly endemic, such as the Florida yew, Taxusfloridana. We sampled woody vegetation in three ravine systems within the Apalachicola Bluffs and Ravines Preserve (Liberty Co., Florida). We used transects randomly located along north-facing and south-facing slopes, as well as transects along north-facing slopes that contained adult individuals of T. floridana. Slope position (lower slope, midslope, and upper slope) greatly affected important species composition and densities. In contrast, aspect of steepheads had little influence on the densities of most species. American beech (Fagus grandifolia), southern magnolia (Magnolia grandiflora), and a number of evergreen understory species were more abundant along north-facing slopes containing T. floridana than along random north-facing slopes. North-facing slopes containing T. floridana, with their characteristic mesic midslope areas, represent a unique subset of north-facing slopes along steepheads of the Apalachicola River Bluffs. The discontinuous distribution of such north-facing slopes within ravine systems potentially results in a discontinuous distribution of T. floridana.

Stand structure and dynamics of sand pine differ between the Florida panhandle and peninsula

Size and age structures of stand populations of numerous tree species exhibit uneven or reverse J-distributions that can persist after non-catastrophic disturbance, especially windstorms. Among disjunct populations of conspecific trees, alternative distributions are also possible and may be attributed to more localized variation in disturbance. Regional differences in structure and demography among disjunct populations of sand pine (Pinus clausa (Chapm. ex Engelm.) Vasey ex Sarg.) in the Florida panhandle and peninsula may result from variation in hurricane regimes associated with each of these populations. We measured size, age, and growth rates of trees from panhandle and peninsula populations and then compiled size and age class distributions. We also characterized hurricanes in both regions over the past century. Size and age structures of panhandle populations were unevenly distributed and exhibited continuous recruitment; peninsula populations were evenly sized and aged and exhibited only periodic recruitment. Since hurricane regimes were similar between regions, historical fire regimes may have been responsible for regional differences in structure of sand pine populations. We hypothesize that fires were locally nonexistent in coastal panhandle populations, while periodic high intensity fires occurred in peninsula populations over the past century. Such differences in local fire regimes could have resulted in the absence of hurricane effects in the peninsula. Increased intensity of hurricanes in the panhandle and current fire suppression patterns in the peninsula may shift characteristics of sand pine stands in both regions.

Native Grasses as a Management Alternative on Vegetated Closure Caps

Capped waste sites often are vegetated with commercial turf grasses to increase evapotranspiration and prevent erosion and possible exposure of the barrier. Fertilizer, frequent watering, and mowing may be required to establish the turf grass and prevent invasion by trees and shrubs. Oldfield vegetation of grasses and forbs is a possible sustainable alternative to turf grass communities. To determine if oldfield vegetation can establish on caps, we (1) compared establishment of a dominant oldfield grass and a commercial turf grass under different combinations of new closure cap management: spring or summer planting and presence or absence of amendments to alleviate drought (watering, mulch) or increase soil fertility (fertilizer, lime, a nitrogen-fixing legume); (2) surveyed existing caps to determine if oldfield species establish naturally; and (3) performed a greenhouse experiment to compare growth of two native grasses under low and amended (added water, soil nutrients) conditions. Both the commercial grass and oldfield species established under new cap conditions; fertilizer, water, and mulch improved vegetation establishment in spring or summer, but legumes decreased grass cover. In the greenhouse, both native grasses grew best with amendments; however, substantial stem and root length were obtained with no fertilizer and only once-weekly watering. Existing vegetated caps supported planted grasses and naturally established oldfield species. Overall, the results indicate native grasses can establish on new caps and oldfields can serve as a management model; further work is needed to determine the management strategy to maintain herbaceous vegetation and slow woody species invasion.

Sensitivity of a real-time PCR method for the detection of transgenes in a mixture of transgenic and non-transgenic seeds of papaya ( Carica papaya L.)

BackgroundGenetically engineered (GE) ringspot virus-resistant papaya cultivars ‘Rainbow’ and ‘SunUp’ have been grown in Hawai’i for over 10 years. In Hawai’i, the introduction of GE papayas into regions where non-GE cultivars are grown and where feral non-GE papayas exist have been accompanied with concerns associated with transgene flow. Of particular concern is the possibility of transgenic seeds being found in non-GE papaya fruits via cross-pollination. Development of high-throughput methods to reliably detect the adventitious presence of such transgenic material would benefit both the scientific and regulatory communities.ResultsWe assessed the accuracy of using conventional qualitative polymerase chain reaction (PCR) as well as real-time PCR-based assays to quantify the presence of transgenic DNA from bulk samples of non-GE papaya seeds. In this study, an optimized method of extracting high quality DNA from dry seeds of papaya was standardized. A reliable, sensitive real-time PCR method for detecting and quantifying viral coat protein (cp) transgenes in bulk seed samples utilizing the endogenous papain gene is presented. Quantification range was from 0.01 to 100 ng/μl of GE-papaya DNA template with a detection limit as low as 0.01% (10 pg). To test this system, we simulated transgene flow using known quantities of GE and non-GE DNA and determined that 0.038% (38 pg) GE papaya DNA could be detected using real-time PCR. We also validated this system by extracting DNA from known ratios of GE seeds to non-GE seeds of papaya followed by real-time PCR detection and observed a reliable detection limit of 0.4%.ConclusionsThis method for the quick and sensitive detection of transgenes in bulked papaya seed lots using conventional as well as real-time PCR-based methods will benefit numerous stakeholders. In particular, this method could be utilized to screen selected fruits from maternal non-GE papaya trees in Hawai’i for the presence of transgenic seed at typical regulatory threshold levels. Incorporation of subtle differences in primers and probes for variations in cp worldwide should allow this method to be utilized elsewhere when and if deregulation of transgenic papaya occurs.

Pollen-mediated gene flow from transgenic to non-transgenic switchgrass ( Panicum virgatum L.) in the field

BackgroundSwitchgrass is C4 perennial grass species that is being developed as a cellulosic bioenergy feedstock. It is wind-pollinated and considered to be an obligate outcrosser. Genetic engineering has been used to alter cell walls for more facile bioprocessing and biofuel yield. Gene flow from transgenic cultivars would likely be of regulatory concern. In this study we investigated pollen-mediated gene flow from transgenic to nontransgenic switchgrass in a 3-year field experiment performed in Oliver Springs, Tennessee, U.S.A. using a modified Nelder wheel design. The planted area (0.6 ha) contained sexually compatible pollen source and pollen receptor switchgrass plants. One hundred clonal switchgrass ‘Alamo’ plants transgenic for an orange-fluorescent protein (OFP) and hygromycin resistance were used as the pollen source; whole plants, including pollen, were orange-fluorescent. To assess pollen movement, pollen traps were placed at 10 m intervals from the pollen-source plot in the four cardinal directions extending to 20 m, 30 m, 30 m, and 100 m to the north, south, west, and east, respectively. To assess pollination rates, nontransgenic ‘Alamo 2’ switchgrass clones were planted in pairs adjacent to pollen traps.ResultsIn the eastward direction there was a 98% decrease in OFP pollen grains from 10 to 100 m from the pollen-source plot (Poisson regression, F1,8 = 288.38, P < 0.0001). At the end of the second and third year, 1,820 F1 seeds were collected from pollen recipient-plots of which 962 (52.9%) germinated and analyzed for their transgenic status. Transgenic progeny production detected in each pollen-recipient plot decreased with increased distance from the edge of the transgenic plot (Poisson regression, F1,15 = 12.98, P < 0.003). The frequency of transgenic progeny detected in the eastward plots (the direction of the prevailing wind) ranged from 79.2% at 10 m to 9.3% at 100 m.ConclusionsIn these experiments we found transgenic pollen movement and hybridization rates to be inversely associated with distance. However, these data suggest pollen-mediated gene flow is likely to occur up to, at least, 100 m. This study gives baseline data useful to determine isolation distances and other management practices should transgenic switchgrass be grown commercially in relevant environments.