Particle bombardment and the genetic enhancement of crops: myths and realities

Abstract

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.

Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize

“…We constructed individual expression vectors for 5 carotenogenic genes each under the control of a different endosperm-specific promoter to avoid potential gene silencing caused by promoter homology (20) and to ensure the coordinated, restricted expression of all transgenes, a feat that has not been achieved in previous reports of multigene transfer using marker genes to our knowledge (21). Direct DNA transfer with separate vectors usually results in transgene integration at a random single locus, in the form of a multigene array (22,23) containing any number of transgenes from 1 to n, with the distribution within the transgenic population tending to describe a normal curve as would be expected from random sampling (24). Input transgenes once integrated remain linked and do not segregate in subsequent generation as is the norm for direct DNA transfer of multiple transgenes irrespective of whether these are on 1 cointegrate vector or independent plasmids (cotransformation) (19)(20)(21)(22)(23)(24).…”

“…Direct DNA transfer with separate vectors usually results in transgene integration at a random single locus, in the form of a multigene array (22,23) containing any number of transgenes from 1 to n, with the distribution within the transgenic population tending to describe a normal curve as would be expected from random sampling (24). Input transgenes once integrated remain linked and do not segregate in subsequent generation as is the norm for direct DNA transfer of multiple transgenes irrespective of whether these are on 1 cointegrate vector or independent plasmids (cotransformation) (19)(20)(21)(22)(23)(24). Plants carrying 1-5 transgenes in a roughly normal distribution could be identified from colored seed phenotypes arising from the accumulation of specific carotenoids, and these could be correlated with mRNA and metabolite profiles to identify and quantify the specific carotenoid molecules present in the endosperm.…”

Integration and Expression Stability of Transgenes in Hybriding Transmission of Transgenic Rice Plants Produced by Particle Bombardment

“…Many studies have analysesed the progenies of the primary rice transformants, revealing that transgene stability was significantly related to differences in transgene structure and expression levels between transgenic lines, particularly in transgenic plants derived from direct DNA transfer such as particle bombardment (Vain et al, 2002;Altpeter et al, 2005). In transgenic cereals, more than 50% of transgenes can be inactivated over successive generations (Iyer et al, 2000).…”

“…Additionally, they create difficulties in predicting transgene behavior when transgene needs to be transferred by conventional crossing (Vain et al, 2002). Altpeter et al (2005) speculated that particle bombardment might be advantageous over Agrobacterium-mediated transformation in respect of transferring the transgenes into a new genetic background via traditional breeding, because by particle bombardment multiple transgenes are tend to be integrated into the same locus. But there are few direct evidences for this question up to date.…”

Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways

“…Here we have gone beyond the current state of the art in vitamin enhancement by simultaneously increasing the levels of ␤-carotene, ascorbate, and folate in corn endosperm. These 3 vitamins represent 3 entirely different metabolic pathways, and the only way to achieve such a radical change in the nutritional properties of an elite breeding variety of corn so rapidly is to take advantage of multigene engineering via direct DNA transfer (9). Currently, only direct DNA transfer has the potential to facilitate the transfer of multiple genes to plants routinely and reliably, and only direct DNA transfer is versatile enough to achieve the direct transformation of commercially important germplasm.…”