Developmental progression and differentiation of distinct cell types depend on the regulation of gene expression in space and time. Tools that allow spatial and temporal control of gene expression are crucial for the accurate elucidation of gene function. Most systems to manipulate gene expression allow control of only one factor, space or time, and currently available systems that control both temporal and spatial expression of genes have their limitations. We have developed a versatile two-component system that overcomes these limitations, providing reliable, conditional gene activation in restricted tissues or cell types. This system allows conditional tissue-specific ectopic gene expression and provides a tool for conditional cell type- or tissue-specific complementation of mutants. The chimeric transcription factor XVE, in conjunction with Gateway recombination cloning technology, was used to generate a tractable system that can efficiently and faithfully activate target genes in a variety of cell types. Six promoters/enhancers, each with different tissue specificities (including vascular tissue, trichomes, root, and reproductive cell types), were used in activation constructs to generate different expression patterns of XVE. Conditional transactivation of reporter genes was achieved in a predictable, tissue-specific pattern of expression, following the insertion of the activator or the responder T-DNA in a wide variety of positions in the genome. Expression patterns were faithfully replicated in independent transgenic plant lines. Results demonstrate that we can also induce mutant phenotypes using conditional ectopic gene expression. One of these mutant phenotypes could not have been identified using noninducible ectopic gene expression approaches.

The plant life cycle alternates between two distinct multi-cellular generations, the reduced gametophytes and the dominant sporophyte. Little is known about how generation-specific cell fate, differentiation, and development are controlled by the core regulators of the cell cycle. In Arabidopsis, RETINOBLASTOMA RELATED (RBR), an evolutionarily ancient cell cycle regulator, controls cell proliferation, differentiation, and regulation of a subset of Polycomb Repressive Complex 2 (PRC2) genes and METHYLTRANSFERASE 1 (MET1) in the male and female gametophytes, as well as cell fate establishment in the male gametophyte. Here we demonstrate that RBR is also essential for cell fate determination in the female gametophyte, as revealed by loss of cell-specific marker expression in all the gametophytic cells that lack RBR. Maintenance of genome integrity also requires RBR, because diploid plants heterozygous for rbr (rbr/RBR) produce an abnormal portion of triploid offspring, likely due to gametic genome duplication. While the sporophyte of the diploid mutant plants phenocopied wild type due to the haplosufficiency of RBR, genetic analysis of tetraploid plants triplex for rbr (rbr/rbr/rbr/RBR) revealed that RBR has a dosage-dependent pleiotropic effect on sporophytic development, trichome differentiation, and regulation of PRC2 subunit genes CURLY LEAF (CLF) and VERNALIZATION 2 (VRN2), and MET1 in leaves. There were, however, no obvious cell cycle and cell proliferation defects in these plant tissues, suggesting that a single functional RBR copy in tetraploids is capable of maintaining normal cell division but is not sufficient for distinct differentiation and developmental processes. Conversely, in leaves of mutants in sporophytic PRC2 subunits, trichome differentiation was also affected and expression of RBR and MET1 was reduced, providing evidence for a RBR-PRC2-MET1 regulatory feedback loop involved in sporophyte development. Together, dosage-sensitive RBR function and its genetic interaction with PRC2 genes and MET1 must have been recruited during plant evolution to control distinct generation-specific cell fate, differentiation, and development.

SummaryPotato is the third most important global food crop and the most widely grown noncereal crop. As a species highly amenable to cell culture, it has a long history of biotechnology applications for crop improvement. This review begins with a historical perspective on potato improvement using biotechnology encompassing pathogen elimination, wide hybridization, ploidy manipulation and applications of cell culture. We describe the past developments and new approaches for gene transfer to potato. Transformation is highly effective for adding single genes to existing elite potato clones with no, or minimal, disturbances to their genetic background and represents the only effective way to produce isogenic lines of specific genotypes/cultivars. This is virtually impossible via traditional breeding as, due to the high heterozygosity in the tetraploid potato genome, the genetic integrity of potato clones is lost upon sexual reproduction as a result of allele segregation. These genetic attributes have also provided challenges for the development of genetic maps and applications of molecular markers and genomics in potato breeding. Various molecular approaches used to characterize loci, (candidate) genes and alleles in potato, and associating phenotype with genotype are also described. The recent determination of the potato genome sequence has presented new opportunities for genomewide assays to provide tools for gene discovery and enabling the development of robustly unique marker haplotypes spanning QTL regions. The latter will be useful in introgression breeding and whole-genome approaches such as genomic selection to improve the efficiency of selecting elite clones and enhancing genetic gain over time.

The intragenic vector system involves identifying functional equivalents of vector components from the genome of a specific crop species (or related species to which it can be hybridised) and using these DNA sequences to assemble vectors for transformation of that plant species. This system offers an attractive alternative to current genetic engineering strategies where vectors are based on DNA sequences that usually originate from bacteria. The construction of intragenic vectors enables the well-defined genetic improvement of plants with all transferred DNA originating from within the gene pool already available to plant breeders. In this manner genes can be introgressed into elite cultivars in a single step without linkage drag and without the incorporation of foreign DNA. The resulting plants are non-transgenic, although they are derived using the tools of molecular biology and plant transformation. The use of intragenic vectors for the transfer of genes from within the gene pools of crops may help to alleviate some of the major public concerns over the deployment of GM crops in agriculture, notably the ethical issue associated with the transfer of DNA across wide taxonomic boundaries. This paper reviews the progress toward the development and use of intragenic vectors and the implications of their use for the genetic improvement of crops.

Analysis of female meiosis (megasporogenesis) and embryo sac development (megagametogenesis) in angiosperms is technically challenging because the cells are enclosed within the nucellus and ovule tissues of the female flower. This is in contrast to male sporogenesis and gametogenesis where development can readily be observed through the easily dissectable developing anthers. Observation of embryo sac development is a particular problem in crassinucellate ovules such as those of maize. To overcome the problems in observing reproductive development, we developed a simple Feulgen staining procedure optimized for use with confocal microscopy to observe reproductive progression in the crassinucellate ovules of maize. The procedure greatly facilitates the observation of nuclei and cell structures of all stages of megasporogenesis and embryo sac development. The high resolution obtained using the technique enabled us to readily visualize chromosomes from individual cells within ovule tissue samples of maize. A propidium iodide staining technique was also used and compared with the Feulgen-based technique. Static cytometry of relative DNA content of individual nuclei was possible using Imaris software on both Feulgen and propidium iodide-stained samples. The techniques also proved successful for the observation of Arabidopsis and Hieracium aurantiacum female gametophyte and seed development, demonstrating the general applicability of the techniques. Using both staining methods, we analysed the maize meiotic mutant elongate1, which produces functional diploid instead of haploid embryo sacs. The precise defect in meiosis from which diploid embryo sacs arise in elongate1 has not previously been reported. We used confocal microscopy followed by static cytometry using Imaris software to show that the defect by which diploid embryo sacs arise in the maize mutant elongate1 is the absence of meiosis II with one of the dyad cells directly initiating megagametogenesis.