DNA transfer by particle bombardment makes use of physical processes to achieve the transformation of crop plants. There is no dependence on bacteria, so the limitations inherent in organisms such as Agrobacterium tumefaciens do not apply. The absence of biological constraints, at least until DNA has entered the plant cell, means that particle bombardment is a versatile and effective transformation method, not limited by cell type, species or genotype. There are no intrinsic vector requirements so transgenes of any size and arrangement can be introduced, and multiple gene cotransformation is straightforward. The perceived disadvantages of particle bombardment compared to Agrobacterium-mediated transformation, i.e. the tendency to generate large transgene arrays containing rearranged and broken transgene copies, are not borne out by the recent detailed structural analysis of transgene loci produced by each of the methods. There is also little evidence for major differences in the levels of transgene instability and silencing when these transformation methods are compared in agriculturally important cereals and legumes, and other non-model systems. Indeed, a major advantage of particle bombardment is that the delivered DNA can be manipulated to influence the quality and structure of the resultant transgene loci. This has been demonstrated in recently reported strategies that favor the recovery of transgenic plants containing intact, singlecopy integration events, and demonstrating high-level transgene expression. At the current time, particle bombardment is the most efficient way to achieve plastid transformation in plants and is the only method so far used to achieve mitochondrial transformation. In this review, we discuss recent data highlighting the positive impact of particle bombardment on the genetic transformation of plants, focusing on the fate of exogenous DNA, its organization and its expression in the plant cell. We also discuss some of the most important applications of this technology including the deployment of transgenic plants under field conditions.

Summary One approach to understanding the Reactive Oxygen Species (ROS)‐scavenging systems in plant stress tolerance is to manipulate the levels of antioxidant enzyme activities. In this study, we expressed in the chloroplast three such enzymes: dehydroascorbate reductase (DHAR), glutathione‐S‐transferase (GST) and glutathione reductase (GR). Homoplasmic chloroplast transformants containing either DHAR or GST, or a combination of DHAR:GR and GST:GR were generated and confirmed by molecular analysis. They exhibited the predicted changes in enzyme activities, and levels or redox state of ascorbate and glutathione. Progeny of these plants were then subjected to environmental stresses including methyl viologen (MV)‐induced oxidative stress, salt, cold and heavy metal stresses. Overexpression of these different enzymes enhanced salt and cold tolerance. The simultaneous expression of DHAR:GR and GST:GR conferred MV tolerance while expression of either transgene on its own didn’t. This study provides evidence that increasing part of the antioxidant pathway within the chloroplast enhances the plant’s ability to tolerate abiotic stress.

This report describes the integration and expression of foreign genes into the plastid genome of a commercial cultivar of potato. Plastid transformation of potato was achieved using two tobacco specific plastid transformation vectors, pZS197 (Prrn/aadA/ psbA3 0 ) and pMSK18 (trc/gfp/Prrn/aadA/ psbA3 0 ). Selection was for spectinomycin resistance after biolistic delivery of plasmid DNA into leaf cells of Solanum tuberosum cv. Desiree. Ten transplastomic lines were obtained from 179 bombarded samples with vector pZS197 and four transplastomic lines selected out of 103 bombarded samples with vector pMSK18. Southern blot and PCR analyses confirmed homoplasmy in the primary regenerants, and incorporation of the aadA and gfp genes into the potato plastid genome by two homologous recombination events via the flanking plastid DNA sequences. Fluorometric measurements confirmed GFP expression in leaves and tubers of pMSK18 lines. No transformants were obtained with a third tobacco vector, pNtcZ7 (Prrn/gfp/ psbA3 0 /trc/aadA/rrnB-ter) in which the selectable marker gene is driven by a bacterial (trc) promoter, which does permit selection of plastid transformants in tobacco, and allows low level expression of the reporter gene, gfp, in potato. #

Antarctic hair grass (Deschampsia antarctica E. Desv.), the only grass indigenous to Antarctica, has well-developed freezing tolerance, strongly induced by cold acclimation. Here, we show that in response to low temperatures, D. antarctica expresses potent recrystallization inhibition (RI) activity that, inhibits the growth of small ice crystals into potentially damaging large ones, is proteinaceous and localized to the apoplasm. A gene family from D. antarctica encoding putative homologs of an ice recrystallization inhibition protein (IRIP) has been isolated and characterized. IRIPs are apoplastically targeted proteins with two potential ice-binding motifs: 1-9 leucine-rich repeats (LRRs) and c. 16 'IRIP' repeats. IRIP genes appear to be confined to the grass subfamily Pooideae and their products, exhibit sequence similarity to phytosulphokine receptors and are predicted to adopt conformations with two ice-binding surfaces. D. antarctica IRIP (DaIRIP) transcript levels are greatly enhanced in leaf tissue following cold acclimation. Transgenic Arabidopsis thaliana expressing a DaIRIP has novel RI activity, and purified DaIRIP, when added back to extracts of leaves from non-acclimated D. antarctica, can reconstitute the activity found in acclimated plants. We propose that IRIP-mediated RI activity may contribute to the cryotolerance of D. antarctica, and thus to its unique ability to have colonized Antarctica.

Most plastid transformation studies with tobacco, and all reports for other species (except tomato [G.D. Nugent, M. ten Have, A. van der Gulik, P.J. Dix, B.A. Uijtewaal, A.P. Mordhorst, Plastid transformants of tomato selected using mutations affecting ribosome structure. Plant Cell Rep. 24 (2005) 341-349]), have used biolistics for plastid transformation. However, nuclear transformation via biolistics has not been reported for any vegetable Brassica species so we used protoplast culture and PEG-mediated DNA uptake, to examine both nuclear and plastid transformation of cauliflower, an important vegetable Brassica. A vector containing genes for hygromycin resistance and b-glucuronidase activity (pGUS-HYG) was used for nuclear transformation, while plastid transformation utilised a vector (pZB1) containing accD-rbcL plastome targeting regions cloned from Brassica napus (oil seed rape), and the selectable marker gene aadA, conferring resistance to spectinomycin. Protoplasts were embedded in agarose and selected on media containing hygromycin or spectinomycin. From five experiments, a single plastid transformant of the commercial cultivar Thalassa was obtained, whereas nuclear transformants were obtained at an absolute transformation frequency up to 1.3 Â 10 À5. No spontaneous spectinomycin resistant mutants were observed in any plastid transformation experiments. PCR and Southern blot analysis confirmed the transgenic status of plants regenerated from the protoplast-derived calli. #