Androgenic processes in diploid potato (2n=2x=24) were studied in three interspecific hybrids. Five incubation temperature treatments were examined. Temperature shock (35°C) during the first 12 h of anther culture and elevated incubation temperature (30°C 16h/20°C 8h) (hereafter 35°C-30°C/20°C) enhanced androgenic embryo production. Variation among experiment dates was highly significant. Temperature treatment (35°C-30°C/20°C) during anther culture did not influence the subsequent conversion rate of androgenic embryos, thus providing a simple and effective way to enhance androgenic embryo yield. Repeated experiments were conducted to study extended anther culture by replacing anthers into medium following the usual harvest 6 weeks after culture initiation. Embryos continued to be produced after the first harvest. Embryo yield at the first harvest was significantly correlated with that at the second harvest (P <0.01). Significantly more embryos were produced when anthers were put back into the same medium compared to fresh medium in extended anther culture. Although relatively high embryo yield was produced in extended anther culture, high contamination and low regeneration rate eliminate any practical use unless a better regeneration protocol is developed. Randomly amplified polymorphic DNA (RAPD) techniques were applied to analyze the genetic composition of anther-derived plants, whose ploidy level was predetermined by flow cytometry. The RAPD fragments amplified from various anther derived diploid plants (2n=2x=24) were compared with those from anther donor (2n=2x=24) and anther-derived monoploids (2n=1x=12). Anther donor and anther-derived monoploids were distinguished by scoring segregating bands as well as total number of scorable bands that exhibited polymorphism. Thus RAPD has the potential to separate homozygous from heterozygous diploids, since the frequency of RAPDs present in homozygous diploids is expected to be the same as in a group of known monoploids, whereas heterozygous ones will be similar to the anther donor. / Master of Science

Competence for leaf disc regeneration, anther culture, and protoplast culture was tested in the parental, F₁, and F₂ generations of a diploid cultivated primitive potato, S. phureja (2n=2x=24). The parental pair consisted of AM3-8, an anther culture derived homozygous diploid, and NBP2, a heterozygous, field selected line. AM3-8 produced embryos in anther culture, and shoots on cultured leaf discs, but its cells did not divide after protoplast isolation. Cells of NBP2 divided to form calli and shoots in protoplast culture, but the clone did not respond to anther culture or leaf disc regeneration. All the individual plants in the F₁ generation were responsive to both anther culture and protoplast culture; however, there was segregation for the ability to regenerate shoots from leaf discs. The F₂ population, the result of a sib-cross, segregated for all three tissue culture competencies. Segregation data fit a one gene model for anther culture competence with the homozygous dominant genotype expressing the highest response, the heterozygous resulting in a marginal response, and the homozygous recessive resulting in no response. A two-gene model applied to the protoplast culture data, with a dominant allele at both loci required for division to occur after protoplast isolation. Leaf disc regeneration data could only be explained by a two gene model with recessive alleles at each locus required for the highest response, a dominant allele at either of the loci resulting in a marginal response, and dominant alleles at both loci resulting in no response. No significant correlation was found among these traits, implying three separate genetic mechanisms which segregate independently. Several temperature regimes were used in an attempt to enhance caulogenesis following protoplast isolation during the p-callus growth and regeneration phase of a single F₂ clone from this S. phureja population. Each of eight treatments was applied to 120 p-calli in six replications of 20 each. Shoot regeneration was scored at 94, 105, 121, and 131 days after protoplast isolation. P-calli cultured at 30°C days and 20°C nights produced significantly more shoots than those cultured at a constant 25°C. Therefore, the standard 25°C used for p-calli regeneration in potato may not be optimal; elevated temperatures or simply a diurnal temperature fluctuation may enhance morphogenesis. / Master of Science

Inheritance of androgenetic competence was studied in 10 diploid potato species hybrids and 16 backcross progeny. Ten hybrid families including three reciprocals were generated between competent clones of S. phureja and incompetent clones of S. berthaultii, S. microdontum and S. phureja. The F₁ hybrid families segregated for androgenetic competence with some highly competent and some incompetent genotypes in all families. The expression of androgenetic competence was modified by parents lacking competence. The cytoplasm of species lacking competence exerted a greater influence on the expression of androgenesis in intraspecific than in interspecific hybrids. The segregation data of 16 backcross progeny between a highly competent hybrid and its incompetent parent suggested that competence may be under control of a single dominant gene. Androgenetic competence can be transferred among sexually compatible potato species. The transfer of desirable traits to a monoploid background can be expected using an- drogenetically competent selections in hybrid combination with germplasm expressing the desired attributes. In an attempt to determine genetic changes of regenerated potato plants following anther and callus cultures, 20 callilines of two S. phureja clones were examined. Four of 20 callilines selected for fertility and diploidy were morphologically indistinguishable among themselves and from the parental clone that had not undergone a tissue culture cycle. Even though morphologically indistinct from the parental clone, all four callilines exhibited higher seed set as pollen parents in 4x-2x crosses and two of the four exhibited higher recombination frequency between the centromere and the y gene. The estimated increase in map distance of the y locus ranged from 3.4 to 10.0 units. Progeny analysis revealed no significant morphological differences among 4x-2x hybrid families under field conditions, and only a single difference among 2x-2x hybrid families under screenhouse conditions. Hence, variation induced in tissue culture may have occurred without detectable morphological change. Assuming no adverse tissue culture effects, breakage of undesirable linkage groups may be an advantage of caulogenesis before backcrossing. / Ph. D.

Monoploid genotypes (2n = x = 12), derived by anther culture of a diplandrous genotype of Solanum phureja, a South-American diploid potato species, were examined for their utility in germplasm development. Nine monoploid genotypes and the diploid anther-donor plant were grown in photoperiod chambers at The Southeastern Plant Environment Laboratories (SEPEL) at North Carolina State University to examine the effect of photoperiod on tuber yield and to determine the variability for critical photoperiod for tuberization. Significant differences were found among the monoploid genotypes for total tuber weight and tuber number. Longer photoperiod treatments both decreased and delayed tuberization. Axillary tuber formation from single-node cuttings was used to estimate the onset of tuber induction and demonstrated variability among monoploid genotypes for critical photoperiod for tuberization. Leaf-disc culture of 24 monoploid genotypes yielded calli which regenerated plants from three genotypes. SDS-polyacrylamide gel electrophoresis of leaf extracts demonstrated variability among diploid and tetraploid calliclones of one monoploid genotype for total protein banding pattern. Absence of stainable pollen and lack of seed set after crosses to diploid species and tetraploid cultivars illustrated infertility among doubled (2n = 2x = 24) and twice doubled (2n = 4x = 48) monoploid-derived lines. Flow-cytometric analysis of pollen obtained from the diploid anther-donor genotype grown under three photoperiods at SEPEL yielded two populations of pollen based on propidium iodide staining of DNA. These populations corresponded to pollen separation based on size parameters alone, introducing the potential for flow sorting of pollen to increase seed set in 4x-2x crosses to tetraploid cultivars. Protoplast isolation from in vitro material and extraction of leaf nuclei both in vitro and in vivo were performed on the anther-donor plant, one of its anther-derived monoploids, and a diploid and tetraploid plant derived from callus culture of the monoploid genotype. Flow-cytometric analysis of propidium-iodide Stained cells and nuclei showed a greater ploidy stability for plant material grown in vitro and a limit to endopolyploidization imposed by initial ploidy level. Flow-cytometric analysis of protoplast-derived nuclei from nine monoploid genotypes derived from anther culture of a single diploid genotype exhibited Significant differences for 4C DNA content, but not for 1C DNA content, indicating that ploidy stability, rather than monoploid status per se, is influenced by genotype. / Ph. D.

The ultimate goal of the· breeding scheme, of which the present study is a part, is to introduce exotic germplasm into existing cultivars of Solanum tuberosum through· 4x X 2x crosses using the South American diploid potato species Solanum phureja as the pollen parent. The first phase of this program includes the 'reconstruction' of a highly heterozygous diploid, pollen parent by .first : reducing the chromosome number of the S. phureja clones to the monoploid level and subsequently fusing genomes of two unrelated monoploid plants either by somatic hybridization or by cross-pollination between fertile doubled monoploids. Within this framework, the objectives of this research were to analyze variation among anther-derived plants of Solanum phureja regarding their: 1) ploidy level and morphology, 2) net photosynthesis and its biochemical components, and 3) nuclear genomic structure, particularly with regard ·to the amplification of rRNA genes as influenced by the anther-culture process. Based on the analysis of several morphological characters of the anther-derived plants by canonical discriminant function, four characters (anther length, number of chloroplasts/pair of guard cells, leaf width, corolla width) were selected for most effective assignment of plants to their ploidy groups by clustering procedures. Clustering of the anther-derived plants proved to be an efficient means of separating monoploids from higher ploidy levels. To assess the impact of the process of anther-culture on the physiology of the resulting plants and to evaluate the possibility of selecting anther-derived genotypes for further breeding efforts, monoploid, diploid and tetraploid anther-derived plants were studied regarding their net photosynthesis and its component characteristics. Leaf area, net photosynthesis and chlorophyll content increased with increasing ploidy' Among .the. monoploids I. Rubisco activity and concentration displayed a. significant genotypic effect; whereas in the diploid group variation among genotypes was significant for total protein content and maximum specific activity of ribulose bisphosphate carboxylase. Among the tetraploid genotypes, significant differenc.es were found with respect to net photosynthesis and specific leaf weight. Two exceptional genotypes were identified: a monoploid with an increase of 28% fcfr maximum activity of ribulose bisphosphate carboxylase and a tetraploid with an increase of 30% for net photosynthesis over the anther-donor plant. To evaluate DNA variation among the anther-derived plants, the nuclear genomes of anther-derived monoploid and diploid plant were studied by DNA reassociation kinetics. It was found that the nuclear genome of the monoploid has undergone differential replication resulting in an increase of sequences consisting of highly· repetitive DNA. Free solution RNA-DNA hybridization showed that the monoploid DNA contained 30% more rDNA sequences than the diploid. Southern blot analysis using rRNA as the probe revealed variation for copy number of certain restriction fragments and for restriction enzyme cleavage sites. / Ph. D.

The sequence of 3553 nucleotides corresponding to the 3'-terminal region of potato virus S (PVS) has been determined from cloned cDNA. The sequence obtained contains six open reading frames with the potential to encode proteins of Mr 10,734, Mr 32,515, Mr 7,222, Mr 11,802, Mr 25,092 and at least Mr 41,052. The amino acid sequence of the 33K ORF has been confirmed to be that of the viral coat protein gene. The nucleotide sequence of this ORF was obtained from expression plasmids which were isolated by binding with a specific monoclonal antibody to PVS, and the expression of coat protein fusion products was verified by Western blots of bacterial cell lysates. The deduced amino acid sequence of a 70 amino acid portion from the central region of the PVS coat protein was 59% identical to the analogous region of potato virus X. In addition, the 7K, 12K and 25K ORF's displayed significant sequence homology with similar sized ORF's from a number of potexviruses. The partial 41K ORF was homologous with the C-terminal portion of the viral replicase proteins of potato virus X and white clover mosaic virus. While the biological functions of the 12K and 25K non-structural proteins coded for by PVS and members of the potexvirus group remain unknown, the 12K protein displays a hydropathicity profile consistent with a membrane associated protein and the 25K protein contains a conserved sequence motif found in a number of nucleoside triphosphate binding proteins. Members of the carlavirus group are distinguished from the potexviruses by the presence of a small [11K (PVS, potato virus M) - 16K (lily symptomless virus)] 3' terminal ORF which appears to contain a sequence motif similar to the 'zinc-finger' domain found in many nucleic acid binding proteins. The coat protein gene from potato virus S (PVS) was introduced into Nicotiana debneyii tobacco as well as a commercial potato cultivar, 'Russet Burbank', by leaf disc transformation using Agrobacterium tumefaciens. Transgenic plants expressing the viral coat protein were highly resistant to subsequent infection following mechanical inoculation with the Andean or ME strains of PVS as indicated by a lack of accumulation of virus in the upper leaves. The coat protein mediated protection afforded by these transgenic plants was sufficient to prevent the accumulation of virus in the tissues of non-transformed 'Russet Burbank' shoots which had been grafted onto transgenic plants inoculated with PVS, and in reciprocal grafts, transgenic shoots accumulated less than 2% (6 weeks after grafting) of the concentration of PVS found in non-transformed shoots similarly grafted onto plants systemically infected with PVS. These transgenic plants also displayed a measure of resistance to inoculation with a related carlavirus from potato, potato virus M. In agreement with previous reports for plants expressing PVX coat protein, plants expressing PVS coat protein were also protected from inoculation with PVS RNA. These results provide further evidence that coat protein mediated protection for these two groups of viruses, which share similar genome organizations, may involve inhibition of some early event in infection, other than, or in addition to, virus uncoating. Specific monoclonal antibodies were prepared against a C-terminal derived 18 kDa portion of the 25K protein of PVS expressed as an in-frame chimeric fusion protein with the glutathione S-transferase gene. The in vivo expression of this non-structural protein in virus infected tissue, as well as tissue from transgenic tobacco (var Xanthi-nc) engineered to contain the entire 25K gene, was verified by Western immunoblot labelling. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Potato (Solanum tuberosum L.) is the world's fourth largest food crop and large financial losses are incurred each year from wound and bruise related injuries. However, little is known about the coordinate induction of genes that may be associated with, or mark major wound-healing and periderm maturation events. Also, one of the key defense mechanisms for potato tubers is the robust barrier provided by the phellem (skin) of the native periderm. Many biological processes are involved in the formation of this stout tissue. However, little is known about induction of genes that may be associated with this process. The objectives of this research were to molecularly assess the processes of wound periderm development and maturation, and native periderm maturation in potato tubers. In this study, these processes were determined in coordination with expression profiles of selected genes. The cell cycle, cell wall protein, and pectin methyl esterase genes were determined from two diverse potato genotypes and two harvests NDTX4271-5R (ND) and Russet Burbank (RB) tubers; 2008 and 2009 harvests. Cell cycle genes encoding epidermal growth factor binding protein (StEBP), cyclin-dependent kinase B (StCDKB), and cyclin-dependent kinase regulatory subunit (StCKS1At) expression profiles were coordinated with related phellogen formation and the induction and cessation of phellem cell formation. Genes encoding the structural cell wall proteins extensin (StExt1) and extensin-like (StExtlk) expression profiles suggested involvement with closing layer formation and subsequent phellem cell layer formations. The coordinate induction and expression profile of StTLRP, a gene encoding a cell wall strengthening "tyrosine- and lysine-rich protein," suggested a role in the formation of the closing layer followed by phellem cell generation and lastly cell wall thickening in nonmeristematic phellogen cells. StPME and StPrePME expression increased during periderm development, implicating involvement in modifications for closing layer and phellem cell formation. Collectively, these results indicate that the genes monitored were involved in and their expression profiles markedly coordinated with periderm formation and the on-set of periderm maturation; results were more influenced by harvest than genotype. Importantly, StTLRP was the only gene examined that may be involved in phellogen cell wall strengthening or thickening after cessation of cell division.

Text in English / Potato is an important source of energy to most micronutrient malnourished affected population in South Africa. Improvements through bio-fortification can therefore enhance access to essential micronutrients. The study was aimed at determining the level of variability of iron and zinc concentration among 20 potato genotypes as a preliminary step for future breeding program. The materials were evaluated using inductively coupled plasma optical emission spectrometry. Statistical analysis indicated significant (P<0.001) variation of Fe and Zn among the genotypes. The average concentration ranges from 34.67 to 76.67 mg kg-1 and 12.88 to 66.1 mg kg-1 for iron and zinc respectively. The best performing genotypes were cultivar Mnandi, Hertha, Buffelspoort and breeding lines-N105-1, 00-S100-33 and 03-627-50. Iron concentration was positively correlated with Zinc concentration. The study showed that enough variability of Fe and Zn concentration exist among the evaluated genotypes, which can be exploited for use in potato bio-fortification breeding programme. / Agriculture, Animal Health and Human Ecology / M.Sc. (Agriculture)

Potato is an important source of energy to most micronutrient malnourished affected population in South Africa. Improvements through bio-fortification can therefore enhance access to essential micronutrients. The study was aimed at determining the level of variability of iron and zinc concentration among 20 potato genotypes as a preliminary step for future breeding program. The materials were evaluated using inductively coupled plasma optical emission spectrometry. Statistical analysis indicated significant (P<0.001) variation of Fe and Zn among the genotypes. The average concentration ranges from 34.67 to 76.67 mg kg-1 and 12.88 to 66.1 mg kg-1 for iron and zinc respectively. The best performing genotypes were cultivar Mnandi, Hertha, Buffelspoort and breeding lines-N105-1, 00-S100-33 and 03-627-50. Iron concentration was positively correlated with Zinc concentration. The study showed that enough variability of Fe and Zn concentration exist among the evaluated genotypes, which can be exploited for use in potato bio-fortification breeding programme. / Agriculture, Animal Health and Human Ecology / M.Sc. (Agriculture)