In order to identify organizational differences between sterile Polima (pol) and fertile Campestris (cam) mitochondrial genomes that could be linked to cytoplasmic male sterility (CMS), the physical map of the pol mitochondrial genome was constructed and compared to the physical map of the cam mitochondrial genome. The only structural differences between the two genomes are confined to a region encompassed by a 4.5 kb segment, present in pol mtDNA but absent in cam mtDNA. This 4.5 kb CMS-associated pol segment contains a chimeric gene called orf224 that is cotranscribed with atpG and comprises the single mtDNA region expressed differently in fertile, sterile and fertility restored plants which makes it a good candidate for specifying the sterility trait. Sequence analysis of the pol 4.5 kb segment has shown that orf224 was the only significant open reading frame (ORF) within the segment that gives rise to abundant transcripts, strengthening the view that the orf224/atp6 gene region is conferring pol male sterility. The pol 4.5 kb segment is also present and similarly organized in the common Brassica napus nap mtDNA but the sequences flanking the two segments are unrelated. Thus, the 4.5 kb segment appears to have transposed during the evolution of the pol and nap mitochondrial genomes and appears to have been lost in the cam mitochondrial genome. Sequence analysis of the nap segment revealed the presence of an ORF related to but divergent from orf224. This open reading frame (orf222) potentially encodes a protein of 222 amino-acids with 79% homology to the predicted product of orf224. orf222 is co-transcribed with the third exon of the trans-spliced gene, nad5, and another ORF of unknown function. Expression of the orf222 gene region is tightly associated with nap CMS since the levels of orf222 transcripts are significantly reduced upon restoration while the expression of 22 other mitochondrial genes do not consistently correlate with nap CMS. Antibodies were rai

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.

Recalcitrant seeds are those that are shed at high water contents, are actively metabolic throughout development, when they are, and remain, desiccation-sensitive, and may also be chilling sensitive. These properties preclude their conventional storage. Because recalcitrant seeds lose viability rapidly (within a few days to several months depending on the species) the long-term storage of their germplasm is achievable only by cryopreservation [i.e. storage at very low temperatures, generally in or over, liquid nitrogen at -196°C or -150°C, respectively. Generally the seeds are far too large to be cryostored, thus explants - most conveniently, excised zygotic embryonic axes - are used. As the axes of recalcitrant seeds are highly hydrated, specific pre-treatments prior to freezing have to be applied in order to avoid lethal ice crystal formation. During the course of this study, cryopreservation protocols were developed for excised zygotic embryonic axes of two different species (Quercus robur L. and Ekebergia capensis Sparrm.). Surface-sterilisation regimes were tested for axes of both species, with the use of a 1% sodium hypochlorite solution containing a wetting agent, emerging as the best. For both species, the vigour and viability of axes, assessed by in vitro germination performance, was tested after the implementation of four different rates of desiccation (achieved by a laminar-airflow; silica-gel-; flash- and fast flash-drying). The most rapid dehydration rate (fast flash-drying) facilitated the best germination rates (vigour) for both Q. robur and E. capensis axes after 240 and 20 min, when water contents were reduced to 0.37 ±0.04 and 0.39 ±0.06 g g-1 (dmb), respectively. Consequently, fast flash-drying was used in combination with three different freezing rates (slow, intermediate and ultra-rapid cooling). While axis viability was lost after slow or intermediate cooling, good survival was obtained for each species after ultra-rapid cooling. In addition to the optimisation of culture conditions, desiccation and freezing rates, the efficacy of different thawing media (distilled water, mannitol, sucrose, full-strength MS medium supplemented with sucrose and a 1 µM calcium/1 mM magnesium solution) was also assessed. The only thawing medium that ensured normal seedling production was the Ca2+Mg2+ solution, in which electrolyte leakage was significantly curtailed. In addition to vigour and viability assessment the responses of the embryos to the various manipulations were monitored by light microscopy and/or transmission electron microscopy. The results of the various manipulations are discussed in terms of the stresses imposed on the excised axes, by each of the procedures. For axes of Q. robu, the outcome of the presently developed successful procedure and two unsuccessful protocols from the published literature are compared and contrasted. It is concluded that while in vitro germination media need to be assessed on a species basis, use of the mildest effective surface-sterilant, in conjunction with the most rapid means to achieve dehydration and cooling/freezing, are likely to underlie generally successful cryopreservation. Additionally, thawing parameters have emerged as being critically important. / Thesis (M.Sc.)-University of Natal, Durban, 2000.