Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots

Abstract

Organization of transgenes in rice transformed through direct DNA transfer strongly suggests a twophase integration mechanism. In the ''preintegration'' phase, transforming plasmid molecules (either intact or partial) are spliced together. This gives rise to rearranged transgenic sequences, which upon integration do not contain any interspersed plant genomic sequences. Subsequently, integration of transgenic DNA into the host genome is initiated. Our experiments suggest that the original site of integration acts as a hot spot, facilitating subsequent integration of successive transgenic molecules at the same locus. The resulting transgenic locus may have plant DNA separating the transgenic sequences. Our data indicate that transformation through direct DNA transfer, specifically particle bombardment, generally results in a single transgenic locus as a result of this two-phase integration mechanism. Transgenic plants generated through such processes may, therefore, be more amenable to breeding programs as the single transgenic locus will be easier to characterize genetically. Results from direct DNA transfer experiments suggest that in the absence of protein factors involved in exogenous DNA transfer through Agrobacterium, the qualitative and͞or quantitative efficiency of transformation events is not compromised. Our results cast doubt on the role of Agrobacterium vir genes in the integration process.Efficient genetic manipulation of major crops, including cereals, has been made possible by transfer of foreign DNA into the host genome through direct DNA transfer procedures such as particle bombardment (1-3). Recently, Agrobacterium-mediated transformation of rice (4), maize (5), and barley (6) has been reported.Much research has been undertaken to understand the mechanism of Agrobacterium-mediated transfer and integration of foreign DNA into plant genome (7). However, little is known about equivalent mechanisms for foreign DNA integration into plants generated through bombardment or other direct DNA transfer methods. Understanding the mechanism of integration of transgenes through direct DNA transfer is important in elucidating, at the plant cell level, events that characterize the process of DNA integration, without the bacterial gene products involved in Agrobacterium-mediated transformation. This understanding will be crucial to achieve the targeted gene delivery. As most transgenic plants (certainly all cereals) in field trials and on the market have been generated through particle bombardment, it assumes more importance.Transgenic plants generated either through Agrobacteriummediated transformation or direct DNA transfer share a common feature, that of transgenic loci frequently consisting of multiple copies of the transgenes. This varies from species to species, and in general, it is accepted that transgenic solanaceous plants generated through Agrobacterium-mediated transformation contain lower transgene copy numbers and simpler integration patterns (8). In species such as rice, maize, and barley (...

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Direct gene transfer in potato: A comparison of particle bombardment of leaf explants and PEG-mediated transformation of protoplasts

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