I read this book the same weekend that the Packers took on the Rams, and the experience of the latter event, obviously, colored my judgment. Although I abhor anything that smacks of being a handbook (like, \"How to Earn a Merit Badge in Neurosurgery\") because too many volumes in biomedical science already evince a boyscout-like approach, I must confess that parts of this volume are fast, scholarly, and significant, with certain reservations. I like parts of this well-illustrated book because Dr. Sj6strand, without so stating, develops certain subjects on technique in relation to the acquisition of judgment and sophistication. And this is important! So, given that the author (like all of us) is somewhat deficient in some areas, and biased in others, the book is still valuable if the uninitiated reader swallows it in a general fashion, realizing full well that what will be required from the reader is a modulation to fit his vision, propreception, adaptation and response, and the kind of problem he is undertaking. A major deficiency of this book is revealed by comparison of its use of physics and of chemistry to provide understanding and background for the application of high resolution electron microscopy to problems in biology. Since the volume is keyed to high resolution electron microscopy, which is a sophisticated form of structural analysis, but really morphology in a modern guise, the physical and mechanical background of The instrument and its ancillary tools are simply and well presented. The potential use of chemical or cytochemical information as it relates to biological fine structure , however, is quite deficient. I wonder when even sophisticated morphol-ogists will consider fixation a reaction and not a technique; only then will the fundamentals become self-evident and predictable and this sine qua flon will become less mystical. Staining reactions (the most inadequate chapter) ought to be something more than a technique to selectively enhance contrast of morphological elements; it ought to give the structural addresses of some of the chemical residents of cell components. Is it pertinent that auto-radiography gets singled out for more complete coverage than other significant aspects of cytochemistry by a high resolution microscopist, when it has a built-in minimal error of 1,000 A in standard practice? I don't mean to blind-side (in strict football terminology) Dr. Sj6strand's efforts for what is \"routinely used in our laboratory\"; what is done is usually well done. It's just that …

Methods in Enzymology.

We have developed a general method for electrically introducing DNA into plant cells. Gene transfer occurs when a high-voltage electric pulse is applied to a solution containing protoplasts and DNA. Carrot protoplasts were used as a model system to optimize gene-transfer efficiency, which was measured 24-48 hr after electroporation by the amount of chloramphenicol acetyltransferase activity resulting from the expression of the introduced chimeric plasmids. Gene-transfer efficiency increased with the DNA concentration and was affected by the amplitude and duration of the electric pulse as well as by the composition of the electroporation medium. Our optimized gene-transfer conditions were effective when applied to tobacco and maize protoplasts, demonstrating that the method is applicable to both monocot and dicot protoplasts.

https://www.pnas.org/content/pnas/82/17/5824.full.pdf

Expression of genes transferred into monocot and dicot plant cells by electroporation

Diverse and rapidly evolving pathogens cause plant diseases and epidemics that threaten crop yield and food security around the world. Research over the last 25 years has led to an increasingly clear conceptual understanding of the molecular components of the plant immune system. Combined with ever-cheaper DNA-sequencing technology and the rich diversity of germ plasm manipulated for over a century by plant breeders, we now have the means to begin development of durable (long-lasting) disease resistance beyond the limits imposed by conventional breeding and in a manner that will replace costly and unsustainable chemical controls.

http://science.sciencemag.org/content/sci/341/6147/746.full.pdf

Pivoting the Plant Immune System from Dissection to Deployment

Plants have the capacity to synthesize, accumulate and emit volatiles that may act as aroma and flavor molecules due to interactions with human receptors. These low-molecular-weight substances derived from the fatty acid, amino acid and carbohydrate pools constitute a heterogenous group of molecules with saturated and unsaturated, straight-chain, branched-chain and cyclic structures bearing various functional groups (e.g. alcohols, aldehydes, ketones, esters and ethers) and also nitrogen and sulfur. They are commercially important for the food, pharmaceutical, agricultural and chemical industries as flavorants, drugs, pesticides and industrial feedstocks. Due to the low abundance of the volatiles in their plant sources, many of the natural products had been replaced by their synthetic analogues by the end of the last century. However, the foreseeable shortage of the crude oil that is the source for many of the artificial flavors and fragrances has prompted recent interest in understanding the formation of these compounds and engineering their biosynthesis. Although many of the volatile constituents of flavors and aromas have been identified, many of the enzymes and genes involved in their biosynthesis are still not known. However, modification of flavor by genetic engineering is dependent on the knowledge and availability of genes that encode enzymes of key reactions that influence or divert the biosynthetic pathways of plant-derived volatiles. Major progress has resulted from the use of molecular and biochemical techniques, and a large number of genes encoding enzymes of volatile biosynthesis have recently been reported.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2008.03446.x

Biosynthesis of plant-derived flavor compounds.

The graminaceous monocots, including the economically important cereals, seem to be refractory to infection by Agrobacterium tumefaciens, a natural gene transfer system that has been successfully exploited for transferring foreign genes into higher plants. Therefore, direct transfer techniques that are potentially applicable to all plant species have been developed using a few dicot and monocot species as model systems. One of these techniques, electroporation, uses electrical pulses of high field strength to permeabilize cell membranes reversibly so as to facilitate the transfer of DNA into cells. Electroporation-mediated gene transfer has resulted in stably transformed animal cells and transient gene expression in monocot and dicot plant cells. Here we report that electroporation-mediated DNA transfer of a chimaeric gene encoding neomycin phosphotransferase results in stably transformed maize cells that are resistant to kanamycin.

Stable transformation of maize after gene transfer by electroporation

Genetic engineering requires a procedure for introducing DNA into host cells, followed by integration into the host genome and gene expression. Although several procedures for DNA-medi-ated gene transfer in mammalian cells, yeast and bacteria have been reported, no such methods are yet available for plant cells. The major obstacle to DNA uptake in plant cells is the cell wall, but this can be circumvented by using plant protoplasts, cells freed of their cell walls by enzymatic digestion. However, none of the reports on the uptake of DNA into plant protoplasts1,2 has produced conclusive evidence for the integration of DNA into the host genome, that is, that stable transformation occurs. The tumour-inducing bacterium Agrobacterium tumefaciens, which causes crown gall disease, is a natural system for the introduction of foreign DNA into plants. This bacterium introduces part of its tumour-inducing (Ti) plasmid, called T-DNA, into plant cells, where it becomes integrated into the nuclear DNA of the host3,4 and is transcribed into mRNA5,6. T-DNA encodes tumour-specific enzymes responsible for the formation of amino acid derivatives such as octopine or nopaline7, which the bacterium can use as a sole source of carbon and nitrogen. The transformed cells have also acquired the ability to grow in the absence of phytohormones (autotrophy). An in vitro system for infection of Nicotiana tabacum protoplasts by A. tumefaciens has already been reported8. Transformants are selected by their ability to divide and grow in tissue culture without the addition of plant phytohormones to the synthetic culture medium. Here, we report a reproducible method for the stable transformation of tobacco protoplasts with Ti-plasmid DNA, using a similar selection procedure.

In vitro transformation of plant protoplasts with Ti-plasmid DNA

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The testing and release of genetically modified organisms (GMOs)—in particular GM plants—is tightly regulated internationally to prevent any negative effects on the environment or human health. However, these regulations are based on transgenic organisms and do not discriminate between transgenic plants and cisgenic plants, although we believe that they are fundamentally different (see sidebarNow, cisgenic plants fall under regulations designed for transgenic organisms, possibly because there have not yet been any applications for the approval of the deliberate release of cisgenic plants into the environment.

Definitions of key terms in relation to plants

Cisgenesis is the genetic modification of a recipient plant with a natural gene from a crossable—sexually compatible—plant. Such a gene includes its introns and is flanked by its native promoter and terminator in the normalsense orientation.Cisgenic plants can harbour one or more cisgenes, but they do not contain any transgenes.

Transgenesis is the genetic modification of a recipient plant with one or more genes from any non‐plant organism, or from a donor plant that is sexually incompatible with the recipient plant. This includes gene sequences of any origin in the anti‐sense orientation, any artificial combination of a coding sequence and a regulatory sequence, such as a promoter from another gene, or a synthetic gene.

Traditional breeding encompasses all plant breeding methods that do not fall under current GMO regulations.As the European legal framework defines GMOs and specifies various breeding techniques that are excluded from the GMO regulations,we use this framework as a starting point, particularly the European Directive 2001/18/EC on the deliberate release of GMOs into the environment (European Parliament, 2001). Excluded from this GMO Directive are longstanding cross breeding, in vitro fertilization, polyploidy induction,mutagenesis and fusion of protoplasts from sexually compatible plants (European Parliament, 2001).

Although transgenesis and cisgenesis both use the same genetic …

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Cisgenic plants are similar to traditionally bred plants: International regulations for genetically modified organisms should be altered to exempt cisgenesis

Public concerns about the issue of the environmental safety of genetically modified plants have led to a demand for technologies allowing the production of transgenic plants without selectable (antibiotic resistance) markers. We describe the development of an effective transformation system for generating such marker-free transgenic plants, without the need for repeated transformation or sexual crossing. This system combines an inducible site-specific recombinase for the precise elimination of undesired, introduced DNA sequences with a bifunctional selectable marker gene used for the initial positive selection of transgenic tissue and subsequent negative selection for fully marker-free plants. The described system can be generally applied to existing transformation protocols, and was tested in strawberry using a model vector in which site-specific recombination leads to a functional combination of a cauliflower mosaic virus 35S promoter and a GUS encoding sequence, thereby enabling the histochemical monitoring of recombination events. Fully marker-free transgenic strawberry plants were obtained following two different selection/regeneration strategies.

Effective production of marker‐free transgenic strawberry plants using inducible site‐specific recombination and a bifunctional selectable marker gene

1. Andow, D.A. & Hilbeck, A. BioScience 54, 637–649 (2004). 2. EuropaBio. Safety Assessment of GM crops. Document 1.1 Substantial Equivalence—Maize (EuropaBio, Brussels, 2003). <http://www.projectgroepbiotechnologie.nl/download/SubstantialEquivalence-Maize. pdf> (accessed 20 May 2006). 3. Bradford, K.J., Van Deynze, A., Gutterson, N., Parrott, W. & Strauss, S.H. Nat. Biotechnol. 23, 439–444 (2005). 4. Environmental Protection Agency. Guidelines for Ecological Risk Assessment. EPA 630/R-95-002F (Environmental Protection Agency, Washington, DC, USA, 1998). 5. Hill, R.A. & Sendashonga, C. Environ. Biosafety Res. 2, 81–88 (2003). 6. European Food Safety Agency. Eur. Food Safety Agency J. 99, 1–94 (2004). 7. Rose, R.I. IOBC/wprs Bull. 29 (5), 145–152 (2006). 8. Prasifka, J.R., Hellmich, R.L., Dively, G.P. & Lewis, L.C. Environ. Entomol. 34, 1181–1192 (2005). 9. Croft, B.A. Arthropod Biological Control Agents and Pesticides (John Wiley & Sons, New York, USA, 1990). 10. Birch et al. in Environmental Risk Assessment of Transgenic Organisms: A Case Study of Bt Maize in Kenya (eds. Hilbeck, A. & Andow, D.A.) 117–185 (CAB International, Wallingford, UK, 2004). 11. Vogt, H. et al. in Guidelines to Evaluate Side-effects of Plant Protection Products to Non-Target Arthropods (ed. Candolfi, M.P. et al.) 27–44 (IOBC/WPRS, Gent, 2000). 12. Dutton, A., Klein, H., Romeis, J. & Bigler, F. Ecol. Entomol. 27, 441–447 (2002). 13. Obrist, L., Dutton, A., Romeis, J. & Bigler, F. BioControl 51, 31–48 (2006). 14. <http://www.bauernverband.ch/de/markt_preise_statistik/pflanzen/se_2004_0217.pdf> (accessed 20 May 2006). 15. <http://www.biosuisse.ch/de/produkte/ackerkulturen/aktuellesvommarkt2.php> (accessed 20 May 2006).

/pdf/do-cisgenic-plants-warrant-less-stringent-oversight-3hit38s0qh.pdf

Do cisgenic plants warrant less stringent oversight

Background Apple allergy is dominated by IgE antibodies against Mal d 1 in areas where birch pollen is endemic. Apples with significantly decreased levels of Mal d 1 would allow most patients in these areas to eat apples without allergic reactions. Objective The aim of this study was to inhibit the expression of Mal d 1 in apple plants by RNA interference. Methods In vitro –grown apple plantlets were transformed with a construct coding for an intron-spliced hairpin RNA containing a Mal d 1–specific inverted repeat sequence separated by a Mal d 1–specific intron sequence. The presence of the construct in transformants was checked by PCR. Expression of Mal d 1 in leaves was monitored by prick-to-prick skin testing in 3 patients allergic to apples and by immunoblotting with a Mal d 1–reactive mAb and with IgE antibodies against Mal d 1. Results After transformation, plantlets were selected on the basis of having a normal phenotype and growth rate. With PCR, in 6 of 9 selected plantlets, the presence of the gene-silencing construct was demonstrated. By skin prick test it was shown that a wild-type plantlet had significantly ( P r of 17 kDa. This band was virtually absent in the transformants that carried the gene-silencing construct. With human IgE antibodies, the same observations were made. Conclusions Mal d 1 expression was successfully reduced by RNA interference. This translated into significantly reduced in vivo allergenicity. These observations support the feasibility of the production by gene silencing of apples hypoallergenic for Mal d 1.

Frans A. Krens

Papers

In vitro transformation of plant protoplasts with Ti-plasmid DNA

Cisgenic plants are similar to traditionally bred plants: International regulations for genetically modified organisms should be altered to exempt cisgenesis

Effective production of marker‐free transgenic strawberry plants using inducible site‐specific recombination and a bifunctional selectable marker gene

Do cisgenic plants warrant less stringent oversight

Silencing the major apple allergen Mal d 1 by using the RNA interference approach.