Being the third most cultivated crop in South Africa, potatoes are of great economic importance. As potatoes originated from cooler areas in the world, they do not easily adapt to South African conditions. The main objective of potato breeding is, therefore, to extend the crop's limited genetic base. Progress in crop improvement is slow due to dominance, segregation and other factors caused by the tetraploid character of cultivated potatoes. A new breeding program for rapid progress has been initiated at the Vegetable and Ornamental Plant Institute, Roodeplaat, South Africa, which comprises the combination of conventional and unconventional breeding techniques. The program is based on the reduction of the ploidy level from the tetraploid to the dihaploid level to facilitate crossings with diploid wild species. Anther culture is the preferred technique for the rapid reduction of the ploidy level and has been successfully applied on different members of the Solanaceae. Cultivated potato, Solanum tuberosum is, however, an important exception. In this study various potato genotypes (tetraploid cultivars, dihaploid breeding lines and a diploid wild species) were used in experiments concerning microtechniques, alternative culture methods and medium manipulation. The main objectives were to evaluate and compare the androgenetic ability of the various genotypes used and to try and identify the factors limiting their in vitro response. Regarding microtechnique, the study focussed on the investigation of the frequency of androgenesis - as a function of plant age - and the determination of defined flower bud lenqths representative of the correct microspore developmental stage for optimal androgenetic response. Combined with an extensive histological study on the microspore development within anthers, from the time of flower selection, after a cold-pretreatment and at various time-intervals during the culture period of 42 days, the following conclusions were reached: In vitro androgenetic response proved optimal when flowers of responsive genotypes were selected during the first seven to 21 days of the flowering period. Both microspore derived embryoid- and callus development were visible within responsive anthers after a culture period of only seven days. The flower bud length required for anthers to be in the optimal stage of microspore development, e.g. the uninucleate stage, varied between the different genotypes but could readily be determined with the DAPI (4,6-diamidino-2- phenylindole) technique. It was also concluded that anthers of the tetraploid cultivar Atzimba should be selected later, between the late-uninucleate and the early-binucleate developmental stages. This suggested a limited selection period for Atzimba anthers, as starch depositioning - which prevent embryogenesis - occurs within anthers during the binucleate stage. Histologically, Atzimba showed limited embryoid development with no embryoid release, while the diploid wild species, S. canasense, proved androgenetically unresponsive. Alternative culture methods were applied to study the effect of different culture phases (liquid, double layered and agar solidified) and anther orientations (lateral, dorsal and ventral) on the androgenetic response of the potato genotypes used. Liquid cultures, based on the so-called shed-pollen technique, enhanced the androgenetic response of the tetraploid cultivar Atzimba. Optimal embryogenesis was obtained for responsive breeding line 87.2002/3 with the utilization of agar solidified media, with maximal response when anthers were cultured in the lateral orientation. No response was observed from S. canasense. The effect of medium manipulation on the androgenetic response of the three genotypes was investigated. The utilization of various combinations of different concentrations of indole-3-acetic acid (1M) and benzyladenine (BA), the alteration of the initial time of incubation of anthers on the initiation media and the use of media without growth regulators compared to that containing gibberellic acid (GA[3]), were investigated. BA had to be present in the initiation media and had a major, though not exclusive, effect on embryogenesis compared to 1M. The optimal BA concentration varied between the two trials. IAA also had an increasing effect on anther response, both in the absence of BA and, especially, in addition with relatively high BA concentrations. In this experiment, only breeding line 87.2002/3 responded. The initial culture of anthers, during the first seven to 21 days of the culture period, on media containing growth regulators proved essential for microspore derived embryoid production in the tetraploid cultivar Atzimba. As these growth regulators are metabolized in the culture media, the regular transfer at shorter, two-weekly intervals to media containing metabolically active substances, proved important. GA[3] had no enhancing-effect on embryogenesis in any of the three tetraploid cultivars. The results obtained in this study suggest that the first 21 days is the critical stage in the anther culture period in terms of the optimal time for flower selection, embryoid induction and the increase in embryogenetic response due to growth regulator influence. It is important to pre-determine the developmental stage when most microspores were in the uninucleate stage of development and to correlate this stage with a specific flower bud length. This would assure maximum response of those genotypes amenable to anther culture. It also implies a more practical and economical starting pOint to anther culture experiments. Following the determination of microspore developmental stage and pollen fertility, flowers should be selected from the donor plants only during the first three weeks of the flowering period. The composition of the nutrient media used for potato anther cultures were sufficient with respect to growth regulators. The growth regulators SA, IAA and the amines glutamine and asparagine had to be present in the initiation media, especially during the first three weeks of the culture period. As microspore development within anyone anther was found to be asynchronous, the regular transfer of anthers to fresh media is recommended to assure proper development of all microspores. The use of a slightly higher IAA concentration could be considered, but care should be taken as too-high concentrations would induce callus production. Microspore derived embryoid production is preferred, as the ploidy level of callus derived plantlets normally varies and somaclonal variation can occur. Liquid media should be considered for anther culture of tetraploid genotypes, while embryoid production can be increased by culturing the anthers of responsive genotypes on agar solidified media on the lateral orientation. Finally, the diploid wild species S. canasense seemed androgenetically unresponsive, or the media and culture conditions used did not satisfy the specific requirements of this genotype. Androgenetic amenability should first be transferred by means of interspecific crossings with a responsive dihaploid genotype, such as the breeding line 87.2002/3. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.

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)

Sweet potato (Ipomoea batatas Lam.) is ranked as the seventh most important food crop in the world and its large biomass and nutrient production give it a unique role in famine relief. However, multiple virus infection is the main disease limiting factor in sweet potato production worldwide. The main objective of this research project was to develop a transgenic sweet potato cultivar with broad virus resistance in South Africa (SA). A review of current literature assembled background information pertaining to the origin, distribution and importance of the sweet potato crop; viruses and complexes infecting sweet potato; and the strategies used in sweet potato virus detection and control. A survey to determine the occurrence and distribution of viruses infecting sweet potato (Ipomoea batatas Lam.) was conducted in major sweet potato-growing areas in KwaZulu-Natal (KZN). A total of 84 symptomatic vine samples were collected and graft inoculated onto universal indicator plants, Ipomoea setosa Ker. and Ipomoea nil Lam. Six weeks post inoculation, typical sweet potato virus-like symptoms of chlorotic flecking, severe leaf deformation, stunting, chlorotic mosaic, and distinct interveinal chlorotic patterns were observed on indicator plants. Under the transmission electron microscope (TEM), negatively stained preparations of crude leaf sap and ultra-thin sections from symptomatic grafted I.setosa plants revealed the presence of elongated flexuous particles and pinwheel type inclusions bodies‟ that are characteristic to the cytopathology of Potyviruses. Symptomatic leaf samples from graft-inoculated I. setosa and I. nil were assayed for Sweet potato feathery mottle virus (SPFMV), Sweet potato mild mottle virus (SPMMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato chlorotic fleck virus (SPCFV), Sweet potato virus G (SPVG), Sweet potato mild speckling virus (SPMSV), Sweet potato caulimo-like virus (SPCaLV), Sweet potato latent virus (SPLV), Cucumber mosaic virus (CMV), and Sweet potato C-6 virus (C-6) using the nitrocellulose membrane enzyme-linked immunosorbent assay (NCM-ELISA). The majority of leaf samples (52%) tested positive for virus disease and showed the occurrence of SPFMV, SPMMV, SPCSV, SPCFV, SPVG, SPMSV, and SPCaLV. Of these 7 viruses, the most frequently detected were SPFMV (39%), SPVG (30%), followed by SPCSV (13%) and SPMMV (12%). SPCaLV and SPCFV at 10% and SPMSV at 7% were found exclusively in samples collected from one area. SPFMV, SPVG, SPCSV, and SPMMV were identified as the most prevalent viruses infecting sweet potato in KZN. The genetic variability of the three major viruses infecting sweet potato (Ipomoea batatas Lam.) in KZN was determined in this study. A total of 16 virus isolates originating from three different locations (Umbumbulu, Umfume and Umphambanyomi River) in KZN were analyzed. These comprised of 10 isolates of Sweet potato feathery mottle virus (SPFMV), five isolates of Sweet potato virus G (SPVG) and one isolate of Sweet potato chlorotic stunt virus (SPCSV). The phylogenetic relationships of the SPFMV, SPVG and SPCSV isolates from KZN relative to isolates occurring in SA and different parts of the world were assessed. The division of SPFMV into four genetic groups (strains) according to the phylogenetic analysis of coat protein encoding sequences revealed mixed infections of the O (ordinary) and C (common) strains in sweet potato crops from KZN. All SPFMV isolates showed close lineage with isolates from South America, East Asia and Africa. The SPVG isolates showed high relatedness to each other and close lineage with other isolates, especially those from China and Egypt. Analysis of the partial sequence of the Heat shock protein 70 homologue (Hsp70h) gene indicated that the SPCSV isolate from KZN belongs to the West African (WA) strain group of SPCSV and showed close relatedness to an isolate from Argentina. The knowledge of specific viral diversity is essential in developing effective control measures against sweet potato viruses in KZN. Multiple virus infections of Sweet potato feathery mottle virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus G (SPVG) and Sweet potato mild mottle virus (SPMMV) cause a devastating synergistic disease complex of sweet potato (Ipomoea batatas Lam.) in KZN. In order to address the problem of the multiplicity and synergism of sweet potato viruses in KZN, this study aimed to develop transgenic sweet potato cv. Blesbok with broad virus resistance. An efficient and reproducible plant regeneration protocol for sweet potato (Ipomoea batatas Lam.) cultivar Blesbok was also developed in this study. The effect of different hormone combinations and type of explants on shoot regeneration was evaluated in order to optimize the regeneration protocol. Coat protein (CP) gene segments of SPFMV, SPCSV, SPVG and SPMMV were fused to a silencer DNA, the middle half of the nucleocapsid (N) gene of Tomato spotted wilt virus (TSWV) and used as a chimeric transgene in a sense orientation to induce gene silencing in the transgenic sweet potato. Transformation of apical tips of sweet potato cv. Blesbok was achieved by using Agrobacterium tumefaciens strain LBA4404 harboring a modified binary vector pGA482G carrying the plant expressible neomycin phosphotransferase ll gene (nptll), the bacterial gentamycin-(3)-N-acetyl-transferase gene and the expression cassette. A total of 24 putative transgenic plants were produced from the transformed apical tips via de novo organogenesis and regeneration into plants under 50mg/L kanamycin and 200 mg/L carbenicillin selection. Polymerase chain reaction (PCR) and Southern blot analyses showed that six of the 24 putative transgenic plants were transgenic with two insertion loci and that all plants were derived from the same transgenic event. The six transgenic sweet potato plants were challenged by graft inoculation with SPFMV, SPCSV, SPVG and SPMMV- infected Ipomoea setosa Ker. Although virus presence was detected using NCM-ELISA, all transgenic plants displayed delayed and milder symptoms, of chlorosis and mottle of lower leaves when compared to the untransformed control plants. These results warrant further investigation under field conditions. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

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)