Zinc oxide nanoparticles are highly variable materials capable of producing positive and negative responses to their effects on plants. Negative effects are often associated with their excessive concentration, which can cause oxidative stress in plants.Conversely, suitable dosages lead to positive manifestations of growth and improvement in the properties studied. ZnO nanoparticles provide plants with a more acceptable form of zinc that is well accessible and easier to use. However, information about their pos-sible toxic effect in combination with another stress factor is lack. The aim of this work was to compare and evaluate the effects of zinc in the form of nanoparticles (ZnO NPs) with the effects of zinc bound in chelate bonding (Zn-EDTA), in combination with UV-C stress factor in plants Solanum lycoperscicum L. Results of this pilot experiment show that both ZnO and Zn-EDTA nanoparticles of 10.95 mg Zn2+ /l in combination with UV-C radiation have a negative effect on vitality and induce oxidative stress in treated plants. The decisive effect on the toxic effect was based on the concentration used and the form of zinc applied. Differences were expres-sed in expression levels of genes affected by oxidative stress. The results obtained from the expression analysis of the genes made it possible to observe the individual transcripts used to respond to oxidative stress and also to provide information on the possible sy-nergistic effect of UV-C radiation and zinc in both studied forms (Zn-EDTA and ZnO NPs).

N-acylethanolamines (NAEs) are fatty acid ethanolamides that occur in a wide range of eukaryotes and their composition is specific to species, developmental stage and tissue type. In plants, NAEs negatively regulate growth and mediate stress responses. The function of NAEs is terminated by a highly conserved enzyme fatty acid amide hydrolase (FAAH). In plants, FAAH homologue has been characterized only for model plant Arabidopsis. Under normal growth conditions, AtFAAH overexpressor seedlings showed enhanced growth suggesting that FAAH might act as a modulator of endogenous NAE levels and regulate growth. Because of the significant role NAEs were shown to play in model plant Arabidopsis, it is pertinent to elucidate this conserved metabolic pathway in crop species such as tomato (Solanum lycopersicum) that may lead to improved productivity. We hypothesized that there is a functional FAAH in tomato that hydrolyzes NAEs. With AtFAAH as a template and using BLASTX, we identified two putative FAAH sequences in tomato. CLUSTALW alignment showed conserved amidase signature sequence and the catalytic triad. Molecular visualization system (PyMOL) revealed that the protein structures of putative SlFAAH1and 2 were similar in domain structure to AtFAAH, with minor differences in spatial arrangement. For further biochemical characterization, full-length coding sequence of SlFAAH1 and SlFAAH2 were isolated and cloned into a heterologous expression system. The expressed protein will be characterized for its hydrolytic activity against radiolabelled NAE substrates. This research is expected to lead to characterization of NAE pathway in a crop plant. Long-term implications of this study include development of molecular and biochemical tools necessary to improve tolerance to abiotic stress and increase crop productivity.

No description available.

Thesis(M.Sc.( Agriculture, Horticulture)) -- University of Limpopo, 2019 / Worldwide, tomato (Solanum lycopersicum L.) is one of the most important crops grown for nutritional value and health benefits, and are highly susceptible to root-knot (Meloidogyne species) nematodes. Following the withdrawal of synthetic chemical nematicides, Nemarioc-AL and Nemafric-BL phytonematicides have been researched and developed as alternatives to synthetic chemical nematicides. However, Nemarioc-AL and Nemafric-BL phytonematicides contains allelochemicals namely, cucurbitacin A (C32H46O9) and cucurbitacin B (C32H46O8) as their active ingredients. Therefore, the objective of this study was to determine whether increasing concentration of Nemarioc AL and Nemafric-BL phytonematicides would result in cucurbitacin residues in tomato plant, to generate mean concentration stimulation point (MCSP) values, overall sensitivity (∑k) and selected foliar mineral elements of tomato plant. Two parallel trials of Nemarioc AL and Nemafric-BL phytonematicides were conducted under field conditions, with each validated the next season. Each trial had seven treatments, namely, 0, 2, 4, 8, 16, 32 and 64% of Nemarioc-AL or Nemafric-BL phytonematicide concentrations, arranged in a randomised complete block design (RCBD), with five replications. In each trial, the seasonal interaction on variables was not significant and therefore data were pooled across the two seasons (n = 70). In both phytonematicides, the cucurbitacin residues were not detected in soil and tomato fruit. Plant variables and selected foliar nutrient elements were subjected to the Curve-fitting Allelochemical Response Data (CARD) model to generate biological indices which allowed for the calculation of MCSP of phytonematicides on tomato and their ∑k values of tomato to Nemarioc-AL and Nemafric BL phytonematicides. In Nemarioc-AL phytonematicide experiment, MCSP for tomato plant variables was at 1.13%, with the ∑k of 60 units, while the MCSP for selected tomato nutrient elements in leaf tissues was at 2.49%, with the ∑k of 21 units. Plant height, chlorophyll content, stem diameter, number of fruit, dry fruit mass, dry shoot mass and dry root mass each with increasing concentration of Nemarioc-AL phytonematicide exhibited positive quadratic relations with a model explained by 95, 82, 96, 89, 83, 83 and 92%, respectively. Similarly, K, Na and Zn each with increasing Nemarioc-AL phytonematicide concentration exhibited positive quadratic relations with a model explaining a strong relationship by 91, 96 and 89%. In Nemafric-BL phytonematicide experiment, MSCP for tomato plant variables was at 1.75%, with the ∑k of 45 units, whereas MCSP for selected tomato nutrient elements in leaf tissues was at 3.72% with the ∑k of 33 units. Plant height, chlorophyll content, stem diameter, number of fruit, dry fruit mass, dry shoot mass and dry root mass and increasing Nemafric-BL phytonematicide concentration exhibited positive quadratic relations with the model explaining a strong relationship by 92, 83, 97, 96, 87, 94 and 96%. Likewise, Na and Zn each with increasing Nemafric-BL phytonematicide concentration exhibited positive quadratic relations with a model explaining their relationship by 93 and 83%, respectively. In contrast, K with increasing Nemafric-BL phytonematicide concentration exhibited negative quadratic relations with a model explaining the relationship by 96%. In conclusion, tomato plant variables and selected foliar nutrient elements over increasing concentration of phytonematicides exhibited DDG patterns, characterised by three phases, namely, stimulation, neutral and inhibition. The developed non-phytotoxic concentration would be suitable for successful tomato production under field conditions.

Recent advances in plant genetic engineering offer substantial benefits to farmers throughout the world. Genetic research has identified many exogenous genes that could considerably decrease production costs through transgene-mediated resistance to insect, viral, fungal and bacterial pathogens. Potato can be produced from true potato seed (TPS) through a sexual polyploidization step, known as 4x-2x hybridization. Little is known regarding the stability of transgenes through sexual polyploidization in potato, although studies have associated ploidy elevation with transgene silencing in plants such as Arabidopsis thaliana. In the present study, potato was transformed with two different transgenes, cry3Aa and PVYo cp, and transgene expression was analyzed through 4x-2x hybridization. Transgene introgression did not affect fertility or agronomic performance (tuber set, average tuber weight, total tuber yield) of the resulting 4x-2x hybrids; however, reduced seed germination was observed for several transgenic lines in an in vitro study. Ploidy elevation did not affect a highly expressed single copy cry3Aa transgene, simplex or duplex, transmitted through pollen to 4x-2x hybrids. By contrast, multiple copies of cry3Aa triggered significant transgene silencing in diploids and silencing was further pronounced upon pollen transmission to 4x-2x hybrids. Crosses between two, single insert plants demonstrated additional evidence that multiple cry3Aa transgenes resulted in reduced expression, as well as provided evidence for maternal effects on expression of the cry3Aa transgene. Finally, Cry3Aa expression levels of progeny derived from low expressing, multiple copy 4x-2x hybrids indicated that reduction of transgene number in progeny, through meiotic segregation, could increase Cry3Aa expression. The results suggest that 4x-2x hybridization using single copy, male parents can result in high expressing, transgenic 4x-2x hybrids while segregating for a low frequency of non-transgenic hybrids that create a "refuge" to inhibit development of resistance to transgenes in pest populations. / Ph. D.

The anther culture response of diploid Solanum chacoense (chc) - S. phureja (phu) hybrids and the regeneration potential of anther-derived monoploids was evaluated. In vivo evaluation of interspecific hybrids was also performed. Three hybrids were anther cultured to observe the effects of reduced nitrogen source on androgenesis. Anthers were distributed to five reduced nitrogen sources. The N concentration was 30 mM. No tested reduced nitrogen source proved superior to the control. Genotype significantly affected embryo production. Eleven monoploid genotypes were included in a leaf disc regeneration procedure utilizing three separate transfers to fresh medium differing by growth regulator composition; six genotypes responded. Silver thiosulfate (STS) at either of two steps in the process proved detrimental to diploid recovery. Hybrids between phu and chc involving six phu clones and eight chc clones or accessions, all resistant to Colorado potato beetle (CPB), were used. No inter-family differences for germination, fruit/pollination, or seed/fruit were observed. Substantial mortality, ascribed to the phenomenon of “hybrid breakdown”, occurred in three families by month four of the study. Field plantings revealed adequate CPB resistance, while Ambush (147 g/ha) application increased total tuber weight per plant and average tuber weight. Hybrids produced less total tuber weight than S. tuberosum (tbr), while chc genotypes produced the smallest average tuber size. Interspecific hybrids produced tuber sizes intermediate between chc and phu. Tbr tubers were the largest. Chc families, regardless of selection for leptine glycolakaloids, suffered the least CPB damage and phu parental clones and hybrid families suffered the most. / Master of Science

Investigations of potato (Solanum tuberosum) have been hampered by its complicated genetics and high genetic load. This dissertation applies genome reduction techniques to investigate a broad swath of genomic and physiological phenomena. It begins with the presentation and evaluation of a protocol to characterize endoreduplication within potato tubers, demonstrating substantial variation between tissue types and among wild species which may facilitate research into the genesis and growth of these starchy underground stems. Next, we transitioned to explore the distribution and consequences of a specific class of transposable element, Miniature Inverted Transposable Elements (MITEs), showing that they comprise approximately 5% of the potato genome, occur more frequently in genes with stress-related functions, and may be associated with changes, especially decreases, in gene expression. We then combined homology and sparsity based approaches to predict recent MITE activity, identifying five families as especially active. Finally, we expose the gene underlying the potato flower color locus, a homolog of AN2, while showing the effects it exerts on the flavonoid biosynthesis and fruit ripening pathways. This region was shown to be particularly dynamic, replete with MITEs and structural variants which we hypothesize to be the ultimate cause of differences in AN2 expression within the germplasm we examined. While the separate topics of this dissertation are quite disparate, each addresses an important topic in potato genetics, the in-depth study of which is only possible through the utilization of genomic reduction approaches to acquire homozygous genotypes for study and currently available genomic resources. / Ph. D. / Despite their humble appearance and routine consumption, potatoes have a complex genetic structure and a life cycle capable of both sexual reproduction through flowers, fruit and seed, and asexual reproduction through the tubers which also comprise the edible product. From an agronomic perspective, one of the most important qualities of a potato tuber is size, a feature influenced by genetics and environment. Cell-to-cell variation for the amount of DNA per cell, one component that influences tuber size, is known to occur, yet our ability to measure DNA content in starchy tuber cells has been obscured by debris generated through routine preparation techniques. We present and evaluate a new method for measuring the DNA content of potato tuber cells, which provides reliable results across a range of different potato varieties and species. ‘Jumping genes’ also known as transposons, first reported in maize but now known to occur in most advanced plant and animal species, have been found to comprise ~5% of the recently sequenced potato genome. We show that a particular class of transposons is more likely to occur adjacent or actually in certain types of genes, such as those which confer resistance to disease, where they may have meaningful effects on how those genes operate. We then proceed to predict the current activity of the various families of these jumping genes to understand how they continue to alter the genetic landscape of potato. Finally we identify a particular gene which dictates flower color in potato (purple vs. white). We demonstrate that several transposons occur in some forms of the flower color gene. Originally we hypothesized that transposons were associated with the turning off of the purple flower color form; however, on closer examination, we could express the white flower form in transgenic plants that were originally white-flowered and convert them to have purple flowers, demonstrating that even the white flower form was functional. While the separate topics of this dissertation are quite disparate, each addresses an important topic in potato genetics, the in-depth study of which is only possible through the availability of the special strains of potatoes with reduced chromosome number and the publication of the potato genome.

Tomato (Solanum lycopersicum) is a member of the Solanaceae family. In this research project tomato, more specifically the M82 cultivar was chosen as a model plant for Agrobacterium-mediated gene transfer by cotyledon inoculation. Our objective was to transform tomato with a T-DNA construct bearing a transposon from maize that can be used for mutagenesis when it transposes or moves around the genome of the tomato. The vector used is a two-component in-cis Ac-Ds system which needs a single transformation event. It was proved that it worked in Arabidopsis and rice according to Trijatmiko (2005). The construct consists of the BAR gene conferring resistance to herbicide Basta, hygromycin (HYG) gene conferring resistance to the antibiotic hygromycin and the green fluorescent protein (GFP) gene, which are driven by specific plant promoters. The selectable marker genes such as HYG and BAR were used to select the rare transformation events by making the transformed tomato tissue resistant to the toxic chemicals (antibiotic and herbicide) compared to the untransformed tissue in which growth was inhibited. The results described consist of developing a transformation protocol which enabled the production of transgenic tomato lines by the help of the antibiotic augmetin (amoxicillin/clavulanic acid). The transgenic lines were tested through polymerase chain reaction (PCR) and herbicide bioassays. / Master of Science

Anther-derived monoploids of Solanum phureja were utilized to investigate factors essential to an efficient method of regenerating doubled monoploids (DMs). The presence of silver thiosulfate (STS) in the MS basal medium did not affect the frequency of cells with 2x nuclei but increased the proportion of cells with 1x nuclei and decreased the proportion with 4x nuclei. Results indicated that STS lower the occurrence of endopolyploid nuclei. The production of DMs was not affected by the presence of STS in MS basal medium on which the source of leaf explants was maintained. The incubation of leaf cultures in the dark or light during the callus induction phase did not influence the subsequent regenerative ability of the monoploids. However, there was a significant genotype by incubation condition interaction. Overnight incubation on MS medium with benzyladenine (BA) pulse prior to transfer to regeneration medium did not affect regeneration. Field evaluation showed various responses of DMs in terms of growth and yield compared to their anther donors or corresponding F₁ progeny. Female fertility was observed in a majority of the DMs verifying their applicability as parental genotypes in practical breeding. Efforts to generate potato hybrids based on selection of genetically diverse parents using RAPD markers and to develop high yielding diploid potato germplasm that may be instrumental in new cultivar development were addressed. Genetic diversity among in vitro plantlets of S. phureja monoploids (which represent DM maternal genotypes) and diploid heterozygous pollinators (ID lines) was estimated using RAPD markers. Field evaluation revealed significant differences among fourteen F₁ hybrids of S. phureja DM x ID with respect to total tuber number, total tuber yield, average tuber weight and vigor. Using simple matching or Jaccard coefficients, the largest parental genetic distance was associated with the highest total tuber yield among the progenies of DM parents. Based on our results, RAPDs have the potential to facilitate the identification of diverse parents to maximize the expression of heterosis in S. phureja hybrids. SSR markers were used to analyze the genetic composition of anther-derived potato plants. Anther-derived monoploids exhibited a single allele as expected. Both homozygous and heterozygous diploids were identified. SSRs were also utilized to study allelic segregation in an F₁ population. Results of this experiment revealed Mendelian inheritance of SSR alleles. / Ph. D.

Potato cyst nematodes (PCN), Globodera rostochiensis and G. pallida, are economically important pests of potato (Solanum tuberosum) crops in potato growing regions worldwide. Integrated management is under threat, with effective nematicides increasingly being withdrawn on environmental and health grounds. Alternative strategies are urgently needed and trap cropping could be one of them. The non-tuber-bearing Solanum sisymbriifolium is regarded as an effective trap crop for PCN with strong hatching ability and immunity to PCN infection and has been used in the UK and The Netherlands. However, its mode of action is unknown. In order to shed light on the mode of action so that a novel control strategy could be identified, the interactions between G. pallida and S. sisymbriifolium were investigated using in vitro bioassays. In choice assays, G. pallida J2s were equally attracted to the roots of S. sisymbriifolium and to those of S. tuberosum. However, potato root diffusate (PRD), which is routinely used to induce PCN hatch, failed to attract G. pallida J2s in chemotaxis bioassays, indicating hatching factors (HFs) and soluble compounds present in PRD are not involved in attraction of G. pallida J2s to potato roots. The J2s invaded the roots of S. sisymbriifolium in large numbers but failed to develop further. To facilitate continuous observation of nematode development, a novel in vitro method was devised with the use of Pluronic F-127, which requires no sterilisation, and the life cycle of G. pallida was successfully observed in S. tuberosum roots. Quantitative real-time polymerase chain reaction analyses of defence related genes of S. tuberosum and S. sisymbriifolium infected with G. pallida revealed up-regulation of the chitinase gene (ChtC 2.1) at 3 days post inoculation in S. sisymbriifolium but not in S. tuberosum. Electrospray ionisation-mass spectrometry analyses of root exudate extracts of the two Solanum species and subsequent bioassay-guided fractionation showed that the HF of S. sisymbriifolium differs from that of S. tuberosum. Previously, attention had been solely paid to the hatching ability of the root exudate of S. sisymbriifolium, but this study revealed for the first time that the aerial part extract possesses a significant hatching ability.