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Genetic engineering is important to enhance crop characteristics and certain traits. Genetically engineered crop cultivation brings environmental and ecological concerns with the potential of unwanted transgene escape and introgression. Transgene escape has been considered as a major environmental and regulatory concern. This concern could be alleviated by appropriate biocontainment strategies. Therefore, it is important to develop efficient and reliable biocontainment strategies. Removing transgenes from pollen has been known to be the most environmentally friendly biocontainment strategy. A transgene excision vector containing a codon optimized serine resolvase CinH recombinase (CinH) and its recognition sites RS2 were constructed and transformed into tobacco (Nicotiana tabacum cv. Xanthi). In this system, the pollen-specific LAT52 promoter from tomato was employed to control the expression of CinH recombinase. Loss of expression of a green fluorescent protein (GFP) gene under the control of the LAT59 promoter from tomato was used as an indicator of transgene excision. Efficiency of transgene excision from pollen was determined by flow cytometry (FCM)-based pollen screening. While a transgenic event in the absence of CinH recombinase contained about 70% of GFP-synthesizing pollen, three single-copy transgene events contained less than 1% of GFP-synthesizing pollen based on 30,000 pollen grains analyzed per event. This suggests that CinH-RS2 recombination system could be effectively utilized for transgene biocontainment. A novel approach for selective male sterility in pollen was developed and evaluated as a biocontainment strategy. Overexpression of the EcoRI restriction endonuclease caused pollen ablation and/or infertility in tobacco, but exhibited normal phenotypes when compared to non-transgenic tobacco. Three EcoRI contained 0% GFP positive pollen, while GFP control plants contained 64% GFP positive pollen based on 9,000 pollen grains analyzed by flow cytometry-based transgenic pollen screening method. However, seven EcoRI events appeared to have 100% efficiency on selective male sterility based on the test-crosses. The results suggested that this selective male sterility could be used as a highly efficient and reliable biocontainment strategy for genetically engineered crop cultivation.

Genetic variation exists in <i>Raphanus</i> that could be of use to <i>Brassica</i> breeders. Of particular interest is the Ogura system of cytoplasmic male sterility (CMS) which has been worked on extensively in a <i>Brassica napus</i> background. Problems have been experienced in <i>B. napus</i>restorer lines due to the inheritance of a large segment of <i>Raphanus</i> chromosome containing the fertility restoring locus. This restorer introgression is located on the <i>Brassica</i> C genome making it only of use for <i>B. napus</i> and not for <i>B. rapa</i> or <i>B. juncea</i>. This thesis describes the development of the materials necessary for the introgression into the <i>Brassica</i> A genome of a defined segment of <i>Raphanus</i> chromosome containing a restorer locus. Defined genetic stocks of <i>Raphanus</i> were developed that contained specific loci controlling restoration of Ogura CMS. This material was used to develop populations segregating for specific restorer loci. Extensive RFLP maps of three <i>Raphanus</i> populations were developed and aligned, resulting in a robust consensus map of the entire <i>Raphanus</i> genome. Three restorer loci were accurately mapped on three separate linkage groups. The segment of <i>Raphanus</i> that is implicated in the restoration of Ogura CMS in a <i>B. napus</i> restorer line developed by INRA was identified and it did not correspond to any of the regions containing the three mapped restorer loci, suggesting the presence of more restorer loci in <i>Raphanus</i>. Comparative mapping between the <i>Raphanus</i> genome map and previously generated <i>Brassica</i> A genome RFLP maps demonstrated large regions of collinearity between segments of chromosomes of the two species. Preliminary examination of the two genome maps suggest they contain essentially the same overall genetic content but with large segments of the genomes rearranged with respect to each other. Likely sites of <i>Raphanus</i> restorer introgression into the <i>Brassica</i> A genome were predicted. Trigenomic tetraploids were developed in which pairing and recombination between homoeologous segments of <i>Raphanus</i> and <i>Brassica</i> A chromosomes should result. Progeny of these individuals will allow an assessment of the pattern and extent of recombination that occurs between the chromosomes of the <i>Raphanus</i> and <i>Brassica</i> A genomes and should lead to the development of 'B. napus' lines carrying Ogura CMS restorer alleles from <i>Raphanus</i>.

Previous studies have shown that the mitochondrial orf224/ atp6 gene region is correlated with the Polima (pol) cytoplasmic male sterility (CMS) of Brassica napus, and that the effects of nuclear fertility restoration on orf224/ atp6 transcripts co-segregate with the pol restorer gene Rfp in genetic crosses. Results presented in this thesis indicate that the pol CMS restorer gene Rfp acts in an organ-specific manner to promote the processing of primary, dicistronic orf224/atp6 transcripts into new restorer-specific, monocistronic transcripts. The single 1.1 kb atp6 transcript of nap cytoplasm and some orfB transcripts of nap and pol cytoplasm B. napus mitochondria are shown to arise by removal of sequences from the 5' end of a longer precursor. Specific endonucleolytic cleavage of a precursor RNA, followed by non-specific 3' to 5' exonuclease action, may thus represent a common mechanism for tailoring transcripts in plant mitochondria. Northern analysis of a segregating F2 population indicates that the recessive rfp allele at the restorer gene locus, or a very tightly linked gene, acts as a dominant gene designated Mmt (modifier of mitochondrial transcripts). Mmt controls the presence of additional, smaller transcripts of the nad4 and ccl1-like (ccl1-l) genes. These results suggest that Rfp/Mmt is a novel nuclear genetic locus that affects the expression of multiple mitochondrial gene regions, with different alleles or haplotypes exerting specific effects on different mitochondrial genes. One of these genes, ccl1-l, is split in the B. napus mitochondrial genome into two widely separated and independently transcribed parts, contrasting with the situation in other plants where this gene is present as an uninterrupted ORF. Although transcripts of both parts are edited, no "trans-spliced" transcripts spanning both parts of the split ORF, that could be translated into a full sized protein product, were detected. Moreover, an antiserum directed against the product of t

Cytoplasmic male sterility (CMS) is a widespread, maternally inherited trait that results in an inability of plants to produce functional pollen. The Ogura CMS system originated in radish, but has since been transferred to, and confers male sterility on, plants in the related genus Brassica . A gene which restores male fertility is needed for the Ogura CMS system to be exploited commercially for hybrid seed production in oilseed species such as Brassica napus. The restorer gene Rfo is a dominant radish nuclear gene that restores the male fertility to plants with Ogura cytoplasm. This gene has been transferred into Brassica napus through intergeneric crosses; however the introgressed segment of radish DNA contains an unknown number of genes, some of which confer undesirable traits, such as an elevated content of seed glucosinolates, antinutritive compounds that render the seed meal unusable as animal feed. A fine scale linkage map of the region in radish containing Rfo was constructed, and a map-based cloning approach relying on synteny between radish and Arabidopsis was used to clone Rfo. A radish gene encoding a 687 amino acid protein with a predicted mitochondrial targeting presequence was found to confer male fertility upon transformation into Ogura CMS B. napus . This gene, codes for a pentatricopeptide repeat (PPR)-containing protein with multiple, in this case 16, PPR domains. Two similar genes that do not appear to function as Rfo flank this gene. A transcript representing a non-functional allele (rfo) was detected in sterile radish plants. Comparison of the Rfo region with the syntenic Arabidopsis region indicates that a PPR gene is not present at the Rfo-equivalent site in Arabidopsis , although a smaller and related PPR gene is found about 40 kb from this site.

Cytoplasrnic male sterility (CMS) is a maternally inherited defect in the production of pollen, the male gamete of the flower. This sterility can be suppressed by nuclear Restorer of Fertility (Rf) genes that normally downregulate the expression of the CMS-associated novel mitochondrial gene. In Brassica napus, nap CMS and pol CMS are associated with related chimeric mitochondrial genes orf222 and orf224, respectively. CMS in both nap and pol is associated with a polar loss of locule development, loss of synchronous locule development and clumping of sporogenous tissue away from the tapetal cell layer, as well as secondary effects on petal and bud formation. In nap CMS, early accumulation of orf222 transcripts in the locule regions of developing anthers is associated with sterility, while the absence of orf222 transcripts from the locules is associated with fertility restoration. Accumulation of novel antisense transcripts of atp6 in a cell specific manner which matches that of sense transcripts of orf222 and atp6 in nap CMS anthers may be indicative of a post-transcriptional regulatory mechanism associated with CMS in flower buds. / Restoration of fertility in Brassica napus nap and pol CMS is associated with nuclearly encoded genes Rfn and Rfp, respectively. These restorers are very closely linked to one another, and may be allelic. Further efforts to isolate Rfp have narrowed the genomic region to approximately 105 kb of a syntenic region in Arabidopsis thaliana. Cosmid clones isolated from a library of Brassica rapa genomic DNA introgressed with Rfp have been successfully sorted into contigs through the application of the amplified fragment length polymorphism technique. The region to which Rfp is mapped is syntenic to a region of Arabidopsis DNA that is a duplication of a second location at the 23 megabase region of chromosome 1 of that genome. This region contains pentatricopeptide (PPR) motif-encoding genes that are highly related to other restorers of fertility of other species. By inference, Rfp from Brassica napus may encode PPR motifs. The PPR genes related to these previously characterized restorers of fertility are often found alongside the restorer genes existing as mini-clusters of several PPR-encoding genes. This is likely caused by selective pressure acting on PPR-encoding genes that resulted in diversification and multiplication of these genes. In addition, the PPR genes of this duplicated region are not syntenically located, whereas the non-PPR-encoding genes maintain their syntenic locations. The same is true for orthologous comparisons between Arabidopsis and other plant species. PPR genes are therefore malleable and capable of alteration in response to changing environmental pressures, such as the evolution of sterility inducing genes.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning). Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.