Theodore M. Klein

DELIVERY OF SUBSTANCES INTO CELLS AND TISSUES USING A PARTICLE BOMBARDMENT PROCESS

ABSTRACT Biologists commonly wish to introduce a wide range of substances into living cells. Such substances include biological stains, proteins (antibodies or enzymes), and genetic material (either RNA or DNA). The cell membrane and, in the case of plant ceils, the cell wall pose formidable barriers which exclude many macromolecules. The concept of particle bombardment has been put forward as a universal mechanism for transporting substances into any living cell. An acceleration device has been designed and constructed which can accelerate small tungsten particles (1 to 4 urn in diameter) to velocities of about 1,000 to 2,000 ft/sec. We have found that these particles can penetrate cell walls and membranes and enter cells in a nonlethal manner. Thousands of cells can be penetrated simultaneously, in situ, as they occur in tissues. Particle bombardment has been shown to be effective in delivering foreign substances into a variety of plant species, including onion, tobacco, corn, and rice. This new method ...

Transient expression of foreign genes in rice, wheat and soybean cells following particle bombardment

The development of an efficient transformation system is a prerequisite for the molecular analysis of gene expression in plants. In crop plants, this development has been hindered by difficulties encountered both in whole plant regeneration from protoplasts and in the general insusceptibility of monocots to Agrobacterium-mediated transformation. We have circumvented these difficulties by transferring foreign genes directly into the intact cells (with cell walls) of three important crop plants including rice, wheat and soybean by a particle bombardment device. Oryza sativa and Triticum monococcum cells were bombarded with accelerated tungsten particles coated with plasmids containing a β-glucuronidase gene as the reporter. Blue transformed cells were detected in an in situ enzyme assay. The number of blue cells was next used as a convenient criterion to study several factors affecting gene transfer efficiency. After optimal conditions were defined, gene transfer into intact cells of O. sativa, T. monococcum and Glycine max was successfully carried out with chloramphenicol acetyltransferase (CAT) gene as the reporter.

Effect of simulated acid precipitation on nitrogen mineralization and nitrification in forest soils

After exposure of samples of three forest soils (pH 3.4 to 3.9) from the Adirondacks region of New York to 60, 230, or 400 cm of simulated rain of pH 3.5 or 5.6 in 4, 14, or 24 weeks, respectively, the soil samples were separated into the 0 to 2 and 2 to 5 cm organic layers and further incubated. The rates of N mineralization in Woods soil exposed to the simulated precipitation were less for rain at pH 3.5 than at pH 5.6, but the inhibition decreased with increasing exposure of the 0 to 2 cm layer. In Panther soil, the rates of mineralization were usually not affected by the acidity of the simulated rain. In the upper layer of Sagamore soil, mineralization was not influenced by pH of the simulated rain, but the transformation was faster in the bottom layer of soil after prolonged exposure to simulated rain at pH 3.5 than at pH 5.6. The rate of nitrate formation in Panther and Woods soil amended with ammonium was inhibited by the more acid rain. Studies with 15NH4 indicated that ammonium was oxidized to nitrate even though ammonium levels did not decline or declined only slightly after prolonged exposure of Panther or Woods soil to rain at pH 3.5. The growth of orchardgrass in Panther and Woods soil was inhibited by the more acid simulated rain.

Simultaneous inhibition of carbon and nitrogen mineralization in a forest soil by simulated acid precipitation.

One method to simulate the long-term exposure of soil to acid rain involves the addition of single doses of concentrated acid. The inhibition of carbon mineralization accompanied by a stimulation of nitrogen mineralization may result from this severe, unnatural treatment. The present study was designed to determine whether the inhibition of carbon mineralization and the accompanying enhanced nitrogen mineralization would occur when soils are treated with more dilute acid for long periods of time, as takes place in nature.

Transformation of pollen by particle bombardment

The development of pollen as a vector for direct gene transfer would be a significant advance in our ability to introduce genes into plants. Such methodology should be of general utility for many plant species, and in particular for the major monocotyledonous crop plants such as maize, wheat and barley that are recalcitrant to protoplast regeneration and that are not amenable to Agrobacterium based transformation techniques. A further advantage would be the avoidance of tissue culture steps that are time consuming and known to result in undesirable somaclonal variation. The potential of pollen as a vector for direct gene transfer has long been realized. For more than 10 years numerous investigators have attempted pollen-mediated transformation, several of which have claimed success [5, 8, 19, 23]. However the ultimate proof that transformation has taken place, that is, the demonstration of integration of foreign DNA into the nuclear genome at the molecular level and the genetic transmission of this DNA, is still lacking. This chapter presents a summary of research that has been directed towards pollen-mediated gene transfer, a detailed protocol for the delivery of DNA into pollen using particle bombardment and a discussion of factors that may be important for the successful application of this technique to obtain stably transformed plants.

Effect of the quantity and duration of application of simulated acid precipitation on nitrogen mineralization and nitrification in a forest soil

A study was conducted of the influence of the rate of application of simulated acid rain on N mineralization and nitrification in a forest soil. The rates were varied by applying different quantities of simulated rain for varying periods of time. The soil was exposed in the laboratory to simulated rain at pH 3.5, 4.1, or 5.6 at rates equivalent to 1.5, 2.3, 4.6, 7.1 or 15 times the average rate of precipitation in the field, and then mineralization of soil N or oxidation of added ammonium was determined. The rates of N mineralization were inhibited by precipitation at pH 3.5 or 4.1 when applied for 27 to 234 day at rates 1.5 times greater than that which occurs in nature. Nitrogen mineralization was not affected by simulated rain at pH 3.5 or 4.1 in soils exposed for 156 day at 2.3 times the natural rate of precipitation, for 27 or 81 day at 4.6 times the natural rate, for 54 day at 7.1 times the natural rate, or for 234 day at 15 times the natural rate. On the other hand, mineralization was fastest in soil exposed to pH 3.5 rain for 234 day at 4.6 times the natural rate of precipitation and for 81 day at 15 times the natural rate. Nitrate formation in soil amended with ammonium was inhibited by rain of pH 3.5 regardless of the intensity of rain or the duration of exposure. For a constant rate of rain application, the inhibition of nitrate formation in ammonium-amended soil generally increased with longer periods exposure. The data show that the use of different rates of additions of artificial rain or different periods of exposure to the simulated precipitation will lead to different conclusions on the influence of acid rain on N mineralization in soil.

National Academies report has broad support

VOLUME 35 NUMBER 4 APRIL 2017 NATURE BIOTECHNOLOGY Giddings and Henry Miller2 published in your December issue. After reading their Correspondence, we wish to share our assessment of the NAS report with your readers. We represent a subset of Forum participants. Although our views have not been formally endorsed by all of our respective scientific societies, we represent a wealth of diverse scientific expertise and experience. As a whole, our professional assessment is that the NAS report offers an extensive and authoritative review of peer-reviewed scientific literature on a wide range of topics related to the agronomic performance of GE crops, the social, economic, political, health, safety, and regulatory context that guides the trajectory of GE technological innovation, and the costs and benefits of these technologies. We broadly agree with key conclusions of the NAS report that: • “...no differences have been found that implicate a higher risk to human health safety from these GE foods than from their non-GE counterparts” (p. 19); • GE crops “have generally had favorable economic outcomes for producers who have adopted these crops, but there is high heterogeneity in outcomes” (p. 20); • the ability of GE crops “to benefit intended stakeholders will depend on the social and economic contexts in which the technology is developed and diffused” (p. 22); and finally, • the scientific evidence suggests that “it is the product, not the process, that should be regulated” (p. 26). The NAS report notes that most of the extant peer-reviewed scientific research is focused on resistance to herbicides (mainly glyphosate) and resistance to insect pests (via Bacillus-thuringiensis-derived Cry proteins). We concur with the committee’s conclusion that GE crops have been adopted on millions of hectares without the emergence of scientific evidence of serious health and environmental problems that were expected by early critics of the technology. At the same time, we applaud the report for not overstating what is known about potential shortand long-term health, environmental, and socioeconomic implications of emerging GE traits. Giddings and Miller criticize the qualified language of the report because they were hoping for the NAS to “overtly back GE crops.” But in our view, the more nuanced phrasing in the NAS report represents a balanced and objective reading of the peer-reviewed evidence. The NAS committee reported that, on a national scale, rates of yield increases in maize, cotton, and soybean were the same before the advent of GE crops as afterward, concluding Science and Policy, Jaharis Family Center for Biomedical and Nutrition Sciences, Boston, Massachusetts, USA. 11Purdue University, Dept. of Food Science, West Lafayette, Indiana, USA. 12University of California, Los Angeles, Institute of the Environment and Sustainability, Los Angeles, California, USA. 13Johns Hopkins University School of Advanced International Studies, Washington, DC, USA. 14Oregon State University, Dept. of Crop and Soil Science, Corvallis, Oregon, USA. 15CIMMYT (Centro Internacional de Mejoramiento de Maíz y Trigo), Texcoco CP, Edo. de México, Mexico. 16University of Richmond, Dept. of Sociology and Anthropology, University of Richmond, Virginia, USA. 17University of Virginia, Dept. of Engineering and Society, Charlottesville, Charlottesville, Virginia, USA. 18Texas A&M University, Dept. of Soil and Crop Sciences, College Station, Texas, USA. 19University of Tennessee, Dept. of Plant Sciences, Knoxville, Tennessee, USA. 20Produce Marketing Association (PMA), Newark, Delaware, USA. e-mail: fred_gould@ncsu.edu