Utilization of engineered resistance to viruses in crops of the developing world, with emphasis on sub-Saharan Africa.

Viral diseases in crop plants constitute a major obstacle to food security in the developing world. Subsistence crops, including cassava, sweetpotato, potato, banana, papaya, common bean, rice and maize are often infected with RNA and/or DNA viruses that cannot be controlled with pesticides. Hence, healthy planting materials and virus-resistant cultivars are essential for high yields of good quality. However, resistance genes are not available for all viral diseases of crop plants. Therefore, virus resistance engineered in plants using modern biotechnology methods is an important addition to the crop production toolbox.

Protocol: Optimised methodology for isolation of nuclei from leaves of species in the Solanaceae and Rosaceae families.

In this study, a protocol is described for rapid preparation of an enriched, reasonably pure fraction of nuclear proteins from the leaves of tobacco (Nicotiana tabacum), potato (Solanum tuberosum) and apple (Malus domestica). The protocol gives reproducible results and can be carried out quickly in 2 hours. Tissue extracts clarified with filtration were treated with non-ionic detergent (Triton X-100) to lyse membranes of contaminating organelles. Nuclei were collected from a 60% Percoll layer of density gradient following low-speed centrifugation. Western blot analysis using antibodies to marker proteins of organelles indicated that the nuclear protein fractions were highly enriched and free or nearly free of proteins from the endoplasmic reticulum and chloroplasts.

Advances in plant gene-targeted and functional markers: a review.

Public genomic databases have provided new directions for molecular marker development and initiated a shift in the types of PCR-based techniques commonly used in plant science. Alongside commonly used arbitrarily amplified DNA markers, other methods have been developed. Targeted fingerprinting marker techniques are based on the well-established practices of arbitrarily amplified DNA methods, but employ novel methodological innovations such as the incorporation of gene or promoter elements in the primers. These markers provide good reproducibility and increased resolution by the concurrent incidence of dominant and co-dominant bands. Despite their promising features, these semi-random markers suffer from possible problems of collision and non-homology analogous to those found with randomly generated fingerprints. Transposable elements, present in abundance in plant genomes, may also be used to generate fingerprints. These markers provide increased genomic coverage by utilizing specific targeted sites and produce bands that mostly seem to be homologous. The biggest drawback with most of these techniques is that prior genomic information about retrotransposons is needed for primer design, prohibiting universal applications. Another class of recently developed methods exploits length polymorphism present in arrays of multi-copy gene families such as cytochrome P450 and ß-tubulin genes to provide cross-species amplification and transferability. A specific class of marker makes use of common features of plant resistance genes to generate bands linked to a given phenotype, or to reveal genetic diversity. Conserved DNA-based strategies have limited genome coverage and may fail to reveal genetic diversity, while resistance genes may be under specific evolutionary selection. Markers may also be generated from functional and/or transcribed regions of the genome using different gene-targeting approaches coupled with the use of RNA information. Such techniques have the potential to generate phenotypically linked functional markers, especially when fingerprints are generated from the transcribed or expressed region of the genome. It is to be expected that these recently developed techniques will generate larger datasets, but their shortcomings should also be acknowledged and carefully investigated.