published_or_final_version / Botany / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

In this study the gene expression pattern of a novel proteinase inhibitor II (PI-II) gene, PiA, in potato (Solanum tuberosum ), was analyzed using RT-PCR. The PiA gene was found to have enhanced expression in expanding tubers of a greenhouse-grown diploid clone (DC), different stages of 'Russet Burbank' tubers and expanding microtubers of in vitro-grown 'Russet Burbank' and 'Bintje'. PiA mRNA was more abundant in the tuber vascular ring and perimedullary zone areas than in other tuber tissues of DC. The PiA gene was also expressed in flower buds/flowers, shoot apices, leaf blades, stems and roots of 'Russet Burbank'. The PiA mRNA levels did not show significant change after IAA treatment in detached leaf blades and tubers of 'Russet Burbank'. Results suggest that the PiA gene is constitutively expressed and could be involved in tuber developmental processes. To the best of our knowledge, PiA is the first reported PI-II gene expressed in most tissues/organs of potato plants.

No description available.

True potato (Solanum tuberosum) seed (TPS) is used for preservation of variable genetic lines of wild and cultivated potatoes (Hawkes et al., 2000) and for propagation of food crops in some developing countries. TPS has advantages over seed potato tubers in storage and transportation and favors lower virus infection levels in fields. However, TPS has thermodormancy and will not readily germinate at 25°C and above (D'Antonio and McHale, 1988; Pallais, 1995a, b; Alvarado et al., 2000). TPS can be extremely unreliable when planted directly in fields due to poor emergence related to diseases and soil crusting. Germination tests were conducted with two lots of TPS derived from cvs. EB-8109 and All Blue, respectively, to study dormancy mechanisms. Seeds were germinated under four temperature regimes (10°C, 15°C, 20°C and 25°C). The two lots showed distinctly different germination characteristics. EB-8109 seeds showed only thermodormancy whereas All Blue seeds showed very deep dormancy. A carotenoid synthesis inhibitor, fluridone, which blocks abscisic acid (ABA) synthesis, effectively broke thermodormancy in EB-8109 TPS but did not break primary dormancy in All Blue seeds. Additional treatments, including pre-chilling and hormonal regimes, also failed to break All Blue deep dormancy. When the micropylar region of the endosperm (endosperm cap) was removed from seeds of both seed lots, radicle elongation was observed, suggesting that mechanical resistance from the endosperm cap restrains radicle protrusion, and that weakening of the endosperm cap is requisite for TPS germination. Endo-β-mannanase expression was measured to help characterize mechanism underlying the weakening of endosperm cap tissues. This enzyme is thought to permit radicle protrusion by degrading cell walls thereby weakening the tissues of the endosperm cap (Groot et al., 1988). The coding region of germination-specific mannanase was isolated from the potato genome by use of polymerase chain reaction (PCR) with primers specifically designed for the tomato germination-specific mannanase gene (LeMAN2, Nonogaki et al., 2000). The cDNA of the TPS mannanase was identical to that of LeMAN2. The expression of mannanase mRNA was detected in the endosperm cap of germinating TPS after 72 h of imbibition at 15°C, while no expression was detected at 25°C (thermodormant condition). Fluridone induced mannanase expression in the micropylar region of the endospem at 25°C. Thus, there was a correlation between induction of mannanase and dormancy breakage. A major increase in TPS post-germinative endo-β-mannanase activity was detected by use of gel diffusion assay. Two isoforms of mannanases were detected in the protein extracts of germinated TPS by activity staining of native polyacrylamide gel electrophoresis. The post-germinative mannanase was detected in the whole endosperm of germinated TPS by using tissue printing with the LeMAN1 (Bewley et al., 1997) RNA probe. These results suggest that, as with tomato, TPS also expresses post-germinative mannanase activity. The promoter region of a new tomato mannanase was isolated during this research. This promoter was shown to be involved in anther-specific expression of mannanase. / Graduation date: 2003

No description available.

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.

This research was carried out to identify salt tolerant potato genotypes in vitro among 131 tetraploid potato cultivars (Solanum tuberosum), 9 diploid simple hybrid clones (4 clones of S. chacoense $ times$ S. tuberosum, 4 clones of S. phureja/S. stenotomum $ times$ S. tuberosum, and 1 clone of S. tuberosum $ times$ S. tuberosum), 1 primitive cultivated diploid S. phureja/S. stenotomum accession, 12 tetraploid complex hybrids, and 13 diploid S. chacoense accessions. Four levels of NaCl (0, 40, 80, and 120 mM) were used. The cultivars, and the simple and complex hybrids were tested for salt tolerance at the vegetative stage in the nodal cutting bioassay. The thirteen S. chacoense accessions were tested for salt tolerance at the germination and early seedling growth stage, in a seedling bioassay. Eleven of these S. chacoense accessions were further tested at the vegetative stage, in the nodal cutting bioassay. There was a progressive decline in the morphological parameters measured, with increased salt levels, in the nodal cutting bioassay. The parameters were used collectively in ranking the different genotypes, averaged over three NaCl levels (40, 80, and 120 mM). Twenty potato cultivars, two clones of the simple hybrid S. chacoense $ times$ S. tuberosum, and one complex hybrid were all considered salt tolerant at the vegetative stage. Ranking of seven S. chacoense accessions was similar between early seedling growth and later vegetative stage. Two of these accessions were promising as sources of salt tolerance.

No description available.

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.