No description available.

The fungus Ophiocordyceps camponoti-floridani (Ophcf) infects Camponotus floridanus carpenter ants and manipulates them to climb to a high tree branch, bite down on foliage and die. Post ant death, the fungus grows out of the ant and spreads spores for reproduction. I investigated the function of an Ophcf gene product highly activated during the behavioral manipulation of these “zombie ants”; an enterotoxin. I have created an expression vector and heterologously expressed this enterotoxin in Cordyceps bassiana (Cbass), a related fungus that does not normally manipulate behavior. This process includes gene amplification, Golden Gate vector cloning in E. coli, A. tumefaciens-mediated transformation to Cbass, and RT-qPCR to verify heterologous gene expression. This was followed by carpenter ant infections with transgenic Cbass (EntB), wildtype Cbass (infected control), and sham (non-infected control) infected ants. Subsequent behavioral observations using tracking system MARGO (Werkhoven et al., 2019) have detected changes in activity levels of ants infected with both transgenic and WT Cbass compared to sham infected ants. This supports previously qualitative descriptions of increased activity caused during infection with WT Cbass (Trinh, 2020). There is a slight but insignificantly higher activity response from EntB compared to WT infected ants over the course of the trial that may be indicative of Ophcf induced changes that are different from general sickness behavior. Additional replicates are necessary to discern if these findings are statistically robust. Future studies should administer this enterotoxin expressing Cbass to observe inter-social behaviors of Carpenter ants. If the enterotoxin is sufficient to elicit one of the side effects of typical Ophcf infection, this would justify further characterization of the proteins and their functions in altering C. floridanus behavior. This characterization could yield information applicable to other parasite-host relationships as well.

No description available.

The genetic manipulation of perennial woody tree species presents a range of additional challenges compared to that of annual weedy crop species. These include long generation times and reproductive cycle, the heterogeneity of plants under investigation and, when investigating xylogenesis, a number of physical and biochemical limitations to microscopic and molecular experimentation. Efforts have been made to understand molecular aspects of xylogenesis and have involved functional gene testing using transgenic approaches. These methods involve the production of plantlets from a variety of plant tissues using in vitro full plant regeneration techniques. Although these systems are effective, the time taken from transformation event, to plant establishment and growth, then finally to secondary wood production can take up to several years and requires high labor and technical inputs. (For complete abstract open document)

Philosophiae Doctor - PhD / Starch is a major carbohydrate reserve in many plants, providing energy during heterotrophic growth and it is contained in large amounts in staple foods such as potatoes, wheat, maize, rice, sorghum and cassava. Apart from being a major product for use in the food industry, starch is also attracting interest from the biofuels industry as a source of bioethanol. This study reports on the development of genetic tools aimed at increasing starch production in sorghum (Sorghum bicolor L Moench), a crop of key agronomic importance worldwide by exploiting a new discovery of a transcription factor gene that regulates starch accumulation in Arabidopsis thaliana namely LEAFY COTYLEDON I (LECl). Ectopic over expression of this gene in arabidopsis has previously been shown to induce a massive hyper accumulation of starch in vegetative tissues. Therefore, we set out to investigate the function of its orthologous gene counterpart in sorghum with the aim of manipulating starch yield directly. Deduced protein sequence analyses showed that the putative sorghum LEAFY COTYLEDON I gene (SbLEC1) cloned in this study shares an overall high amino acid sequence identity (70 %) with the arabidopsis LEC 1, while the functional central B domain shows an even higher percentage sequence identity (91 %) with the same region of arabidopsis LEC 1. The putative SbLEC1 protein shares 14 out of the 16, signature ammo acids characteristic of the Central B Domain with arabidopsis. Furthermore, the putative SbLEC1 protein was also shown to share a significantly high sequence identity (> 80 %) with other well-characterized LEC1 protein sequences from organisms such as maize, rice, rapeseed as well as other organisms documented in the NCB I database. Similarly, much of the sequence similarity lies within the functional central B domain compared to any other region. Gene expression profiling using semi-quantitative PCR showed that SbLEC1 transcripts accumulated in developing seeds as well as in embryogenic calli tissue and no SbLEC 1 transcripts were detectable in leaf, root or sheath tissue. In order to confirm that the identified transcription factor is a functional ortholog, the full cDNA encoding putative SbLEC 1 transcription factor was identified, isolated and cloned from the sweet sorghum MN 1812 genotype. Plant transformation gene constructs based on the pCAMBIA1305.2 binary vector harbouring the transcription factor gene under the control of different promoter sequences were then assembled and immobilized into Agrobacterium tumefaciens strain LBA4404 in preparation for sorghum and arabidopsis transformation. Transient GUS expression studies showed that the five SbLEC1 gene constructs developed in this study were successfully transformed into arabidopsis (Ws ecotype) and sorghum (variety MN1812) callus and cell suspension cultures. The transformed tissues thus represent essential tools that are useful to evaluate the effect of over expressing the putative SbLEC1 protein. Transient GUS expression assays also further revealed differences in efficiency among promoters in driving transgene expression. Transient GUS activity was highest for the maize ubiquitin promoter (MUbi1), followed by the sorghum LEC1 promoter (SLECP), the arabidopsis LEC1 promoter (ALECP) and lastly the maize alcohol dehydrogenase promoter (MAdh1). The ability of the putative SbLEC 1 gene to complement the arabidopsis lecI mutation was also investigated and our findings were not conclusive as they only revealed partial complementation. A detailed comparison of SbLECI full cDNA sequences isolated and cloned from twenty-eight different F2 population plants from different sorghum varieties revealed the existence of sequence variation within the SbLEC 1 gene, which appeared to be allelic. The allelic variation was further shown to affect the amino acid composition of the putative SbLEC 1 protein. Heterologous protein expression studies of the SbLECI gene using an E. coli system showed that the predicted 29.16 kDa putative SbLEC 1 protein could be expressed in vitro both as an development of an efficient tissue culture protocol is a prerequisite for plant genetic engineering, this study also reports on the evaluation of thirteen sorghum genotypes from different genetic backgrounds for their in vitro culture response. A tissue culture protocol for three previously unexplored sorghum genotypes namely Agricol white, AS4 and MNI812 was established. The effect of plant genotype, explant and medium composition on in vitro culture response was highly significant (95 % Cl) in this study. Taken together, the findings in our study demonstrate efforts to draw a baseline foundation for the development of molecular technologies that can be used to increase starch production in sweet sorghum as a water efficient and sustainable feedstock for biofuel production.

Currently we can do genetic manipulation by means of direct and indirect methods. Indirect methods use strategy of gram-negative soil bacteria Agrobacterium tumefaciens, which introduces part of its own genes carried by the section of plasmid Ti called T-DNA into the genome of infected plants. It is difficult to reach the transformation of monocotyledonous plants using this method. Application of direct methods of transformation offers the possibility for transformation of monocotyledonous plants. One of the most effective methods of direct transformation is biolistic transformation. This diploma thesis dealt with the comparison of efficacy of biolistic and A. tumefaciens mediated transformation of tomato (Solanum lycopersicum L.).

Douglas-fir (Pseudotsuga menziesii) is an economically important softwood that is clonally propagated for reforestation purposes by somatic embryogenesis. The molecular basis of embryogenesis in conifers is largely unknown and this prevents progress in somatic embryogenesis protocols. In angiosperms, the LEAFY COTYLEDON1 (LEC1) gene, encoding the HAP3 subunit of the eukaryotic CCAAT box-binding factor, is important in embryo formation, and necessary for somatic embryogenesis. A candidate gene strategy was employed to isolate the Douglas-fir LEC1 homologue, PmLEC1, via the polymerase chain reaction (PCR) with degenerate primers based on the Arabidopsis conserved domain, and the full-length cDNA sequence was obtained by rapid amplification of cDNA ends-PCR (RACE-PCR). The putative protein sequence shared high sequence identity with Arabidopsis LEC1. Northern analysis and quantitative real-time PCR indicate that this is an embryo-specific gene, expressed with the highest abundance during early embryogenesis. Antibodies were raised against a synthetic 18-amino acid PmLEC1 peptide, and in contrast to mRNA expression, Western blotting shows that PmLEC1 protein expression persists until the seedling stage. To gain insight into modulation of PmLEC1 expression and its inducibility in mature tissues, stress and hormone treatments were performed on mature seed and the promoter sequence was isolated by genome walking. Sorbitol, mannitol and 2,4-epibrassinolide were found to significantly up-regulate PmLEC1 expression. The PmLEC1 promoter contains a 5’ UTR intron with numerous enhancer elements, and factors that bind to these elements mediate responses to auxin, UV light and developmental cues, osmotic stress, biotic stress, and tissue culture. Some of the regulatory elements are binding sites for seed-specific transcription factors that are well known from angiosperms, providing new evidence that AGL15, ABI3 and VP1 proteins have a direct role on LEC1 expression. In investigating the embryogenic capacity of PmLEC1, ectopic expression of PmLEC1 in the embryo lethal Arabidopsis lec1-1 null mutant complemented the mutation and permitted the production of viable, desiccation tolerant seeds. In addition, transgenic seedlings produced embryo-like structures from vegetative organs and expressed seed-specific genes. In wild type plants, ectopic expression of PmLEC1 resulted in a bushy phenotype but expression of seed-specific genes was not observed. Taken together, these results show that PmLEC1 is an embryo-specific gene with an essential role throughout embryogenesis, and PmLEC1 expression may be induced in mature seeds by stress and hormone treatments. Because mature seeds show only trace amounts of PmLEC1 transcripts and Douglas-fir somatic embryogenesis can only be induced from immature embryos, this information provides useful insight into initiation of embryogenesis from vegetative tissues. The identification of binding sites for transcription factors known from angiosperms in the promoter region of PmLEC1 has revealed the identity of several genes which are expected to play pivotal roles in conifer embryogenesis.