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.

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.