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Browsing by master's degree program "Erasmus-Mundus Master's Programme in Plant Breeding (EMPLANT)"

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  • Shehata, Reham (2022)
    Cell walls (CWs) are the safeguards of plant cells and have a crucial role in controlling cell integrity and plant-environment interactions. Perception of cell wall damage (CWD) results in activation of signaling pathways leading to activation of plant defiance responses. Previous research utilizing murus1 GDP-L-fucose deficient mutant has shown the role of GDP-L-fucose biosynthesis in controlling the structure and integrity of the CWs. Here, we investigated whether phenotypical consequences provoked by the lack of GDP-L-fucose, observed in mutants lacking MURUS1 can be suppressed by blocking signal transduction pathways involving THESEUS1 (THE1) receptor-like kinase or WALL-ASSOCIATED KINASE 2 (WAK2)- MAP KINASE 6 (MPK6) signaling module involved in CWD responses. For this, mur1 the1 and mur1 mpk6 double mutants were generated and analyzed. We found that the phenotypes of double mutants closely resemble that of mur1 mutants, indicating that the morphological consequences of GDP-L-fucose deficiency develop independently of THE1 and MPK6 signaling. Further, we aimed to investigate whether GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductases GER1 and GER2 controlling the final step of GDP-L-fucose synthesis are functionally redundant. Due to the inability of generating ger1 ger2 double mutants, we have applied the artificial microRNA (amiRNA) strategy to generate ger1 ger2 amiRNA knockdown plants. We found that these knockdown plants exhibit severe developmental growth defects similar to those observed in plants impaired in import of GDP-L-fucose into the Golgi apparatus and these growth defects exceeded those observed in plants lacking MUR1. Therefore, we have concluded that GER1 and GER2 are functionally redundant.
  • LU, Jinghong (2022)
    Inflorescence meristem development in plants generally falls into two types: one is the indeterminate type when the main axis keep growing and new flowers are initiated on its flank; while the other is the determinate type when the main axis terminates as a flower and growth continues sympodially. The Asteraceae plant family harbors a unique type of inflorescence, called the flower head. It combines up to hundreds of individual florets into a single structure, but its infloresecence meristem has a determinate fate and can only produce a certain number of florets before getting consumed. The genetic regulation of such determinacy has been recently brought into attention in Gerbera hybrida, a model system used for studying inflorescence and flower development in Asteraceae. So far, several genetic regulators have been identified regulating the determinacy of inflorescence meristem in Gerbera. This thesis aims to characterize new Gerbera transgenic lines to study the function of the Gerbera homolog of TERMINAL FLOWER 1 (GhTFL1). In Arabidopsis and other species, TFL1 is known to counteract with the flower meristem identity genes and maintain the indeterminacy of the inflorescence meristem. Previous results by overexpressing GhTFL1 under constitutive 35S promoter in Gerbera convert the determinate fate of inflorescence meristem into indeterminate. To better study the GhTFL1 function, an inducible overexpression system pOpON2-pOp6/LhGR-N was introduced, and the transgenic lines have been made for GhTFL1. In this this thesis, in total of 19 candidate lines were screened with GUS staining assay and RT-PCR. Two promising lines (TR4 and TR7) with strongest responses in GUS staining and high expression of the target gene were identified. Further treatment of dexamethasone was conducted in these two lines to the growing rosette; however, no clear phenotypes was observed in these lines. The treatment results suggested that further optimization should be made, in particular, the timing of treatment shall be the determining factor for a successful treatment.
  • Bubolz, Jéssica (2022)
    Late blight, caused by Phytophthora infestans (Mont.) de Bary, is considered the most devastating disease in potato (Solanum tuberosum L.) production worldwide. Control methods involve mostly the use of fungicides, which are costly and are under political pressure for reduction in Europe. Potatoes from the major potato cultivar in Sweden, King Edward, previously stacked with three resistance (R) genes (RB, Rpi-blb2 and Rpi-vnt1.1) were tested in a local Swedish field, with spontaneous P. infestans infection over three seasons to evaluate the effectiveness and stability of the resistance on leaves. In addition, testing of resistance was done in both in leaves and tubers. Field results demonstrated that the 3R stacked into the cultivar King Edward, showed practically full resistance to infections of P. infestans, with no difference to fungicide use. Moreover, the resistance was effective in both leaves and tubers. The results reveal the 3R potatoes offer a functional field resistance, that could, alone, reduce the total use of fungicides in agriculture by several percent in Sweden, in an event of modifications in the EU legislation.
  • Laugel, Henri (2022)
    Faba bean (Vicia faba L.) is an annual herbaceous cool-season food legume widely cultivated worldwide, especially for its high seed protein content. However, its major limitation in being used as food and feed, is the presence of antinutritional factors in its seeds, especially vicine and convicine (VC), two related compounds, which may be harmful to livestock and G6PD-deficient humans. To remove VC, the most sustainable method is breeding for low-VC faba bean cultivars. To improve the efficiency and speed of breeding programs, breeders use marker-assisted selection (MAS). The identification of genes responsible for VC content allows the development of reliable DNA markers and a better understanding of the molecular basis of this trait. The major-effect QTL controlling VC content named “VC1”, was identified in faba bean chromosome 1, and a few minor-effect QTLs were detected in previous studies. Hence, a total of 165 RILs from the cross Mélodie/2 (low-VC) x ILB 938/2 (high-VC) were genotyped and evaluated for VC content. Composite interval mapping was run on R/qtl software with accurate phenotypic data associated with a high-density SNP-based genetic map. Results revealed two minor-effect QTLs in addition to VC1. One was on chromosome 4 and had about 15% effect on convicine content. The other was on chromosome 5 and had 15% effect on vicine and total VC content. This research also reports candidate genes for the newly detected minor-effect QTLs through comparative genomics with the Medicago truncatula genome. Hypotheses were proposed on the role of these candidate genes on the VC biosynthetic pathway or transportation into the embryo beans for further testing.
  • Omran, Mohammad (2022)
    Strawberry breeding in Finland began 60 years ago and has largely relied on traditional breeding methods. The geography of Finland, as well as local consumer preferences for darker-coloured strawberries, have been the main focus of selection in all implemented Finnish breeding programs. With several successful and popular cultivars have been released, the current focus is on increasing yield and disease resistance while maintaining the excellent fruit quality of breeders' selections. A panel of 175 garden strawberry accessions was created and trialled at the Natural Resources Institute Finland (Luke) as part of a Nordic-Baltic pre-breeding collaboration between the institute and the Norwegian breeding company Graminor AS. A medium-density, genome-wide scan for 50K SNPs has been performed on each individual of the panel. This panel represents the genetic diversity present and typical for cultivated strawberry in the Nordic-Baltic region, and it is expected to provide Nordic breeders with markers tailored to their plant material diversity, supporting their future selection decisions and accelerating their breeding cycles. In this study, symptom severities of two strawberry leaf diseases (leaf spot caused by Mycosphaerella fragariae and leaf scorch caused by Diplocarpon earlianum) and seven fruit quality traits (berry weight, skin colour, flesh colour, evenness of flesh colour, basket appearance, predominant berry shape and tip type) were scored in the panel. GAPIT and statgenGWAS statistical packages in R were used to run five different GWAS models: Significant SNP-trait associations were found using single-locus (GLM and MLM) and multi-locus (FarmCPU and BLINK) association mapping analyses. The study had shed a light on the importance of considering several statistical models and parameters for a maximum benefit of association mapping studies. Among significant SNP-trait associations for fruit weight, fruit skin colour and fruit flesh colour, four were partially characterized by inspecting their allelic effects. On chromosome Fvb6-1, appearance of consensus, significant signals from the flesh colour trait of secondary-position berries is discussed. Nine significant SNP-marker associations were detected for berry weight.
  • Bour, Charlotte (2022)
    Bilberry is a soft fruit, growing wild in the forests of Europe, and especially Nordic and East European countries. Its demand is growing on the market, thanks to its heath beneficial components like anthocyanins, present in both skin and flesh of the berry. It however has a very short shelf-life. The goal of this study was to identify pectinase genes, with a focus on two subfamilies, polygalacturonase (PG) and pectate lyase (PL). The main interest was to study those that are expressed during bilberry’s ripening, to get a better understanding of this process. Bioinformatics were used to identify the annotated genes from the bilberry genome, and point out candidates, from which transcripts are found during ripening, with BLAST searches within a transcriptome of ripening bilberry fruit. The expression of the PL candidates was then studied with qPCR analysis. The study identified 70 PG-coding genes and 25 PL-coding genes, of which 35 and 12, respectively, were found in the ripening berry. The expression of five PL genes was increased during ripening, suggesting a role in the softening of the fruit. Two of those had a notably higher relative increase, making them prime candidates for further study.
  • Ndah, Renata (2022)
    Aluminum (Al3+) toxicity is a major limiting factor in acidic soils when pH<5.5 and faba bean experiences yield decreases in these conditions. The multidrug and toxic compound extrusion (MATE) family plays a vital role in Al3+ tolerance across species. This study searched for an ortholog of MtMATE66, a gene involved in Al3+ resistance in barrel medic, in faba bean and quantified the ortholog’s relative expression in 4 faba bean genotypes (GPID_0022, GPID_0153, GPID_0178 and GPID_0191). pBLAST of MtMATE66 in the faba bean unpublished genome identified the ortholog jg20333.t1, with 88.6 % identity, e-value 0.0 and bit score 880. InterPro Scan and NCBI CDD conserved domain queries classified jg20333.t1 as a MATE in the DinF subfamily. MEME Suite identified the 50-amino acid citrate exuding motif characteristic to MATEs exuding citrate in Al3+ tolerance while a multiple sequence alignment and phylogenetic analysis clustered jg20333.t1 with Al3+ -tolerant citrate exuding MATEs. The SWISS Model 3D structure and DeepTMHMM predicted an α-helical, twelve-transmembrane protein topology. The plasma membrane was predicted as the subcellular localisation of jg20333.t1 by ProtComp, WoLFSPORT and YLoc, however SignalP identified no signal peptides. The molecular weight 54.57kDa, theoretical isoelectric point 8.60 and grand average of hydropathicity 0.67 of jg20333.t1 were calculated by ProtParam. EMBOSS Needle and GSDS aligned 13 exons. Four biological replicates of faba bean plants were set up for 3 different treatments: acidic of pH 3.81 (Ac), acidic of pH 3.81 plus Al3+ (Al) and neutral of pH 6.01 (N) in a greenhouse and root tip samples were collected 45 days after transplanting in peat media for RNA extraction. The relative expression of jg20333.t1 was determined by RT-qPCR of jg20333.t1 as target gene, Vfactin as reference gene and N as internal calibrator. The Cq values generated were analysed using the 2-ΔΔCq method and showed high relative fold change in both Ac and Al. The upregulation in Al confirmed the implication of jg20333.t1 in faba bean tolerance to Al3+. The upregulation in Ac suggests upstream regulation by STOP transcription factor. The four genotypes had no significant difference in fold change. Based on these results, it is concluded that jg20333.t1 is a faba bean MATE gene, VfMATE, implicated in Al3+ tolerance by citrate exudation.
  • Jyakhwa, Sarkal (2022)
    Turnip mosaic virus (TuMV) is an economically important plant RNA virus causing huge damage to wide range of arable and vegetable crops. A study was conducted in Nicotiana benthamiana to know if a TuMV mutant carrying a mutation in a thoroughly conserved WD-domain interacting motif and WG motif in HCPro protein can be mechanically transmitted to a healthy plant or not. HCPRoWD is a mutation in “AELPR” motif where glutamic acid and arginine are replaced by alanine. This mutated virus is here referred as TuMVWD. Similarly, in TuMVAG the tryptophan residue in the WG pair is changed to alanine and this mutated HCPro is called as TuMVAG. Four treatments, TuMVWT (positive control), Mock (negative control), TuMVWD and TuMVAG were made. Three plants per treatment were agroinfiltrated and five plants per treatment were used for mechanical inoculation experiment. Green fluorescent protein (GFP), a quantitative reporter of gene expression, was measured followed by qPCR for quantification of vRNA (viral RNA) accumulation. In agroinfiltrated plants, newly emerged leaves showed strong fluorescence in TuMVWT and TuMVAG by 14 dpi (days post inoculation), but TuMVWD showed poor GFP as compared to TuMVWT. During mechanical inoculation experiment, none of the treatments developed GFP in systemic leaves by six dpi but by 14 dpi GFP accumulation in the upper leaves of TuMVWT and TuMVAG was increased. TuMVWD was not used for 2nd mechanical experiment as it did not cause systemic infection during 1st mechanical inoculation experiment even by 14 dpi. Results from vRNA accumulation showed that mechanical transmission of virus was reduced with TuMVAG and not possible with TuMVWD. However, mutations had negative effect on vRNA accumulation.
  • Reda, Shaimaa Roshdy Abdullah (2022)
    Abstract Nuclear bodies (NB) have been studied for their importance of being one of the sites for gene regulation activities. RADICAL INDUCED CELL DEATH1 (RCD1) has proven to be a potential nuclear protein in A.thaliana that localizes to NB. It can interact with different transcription regulators responsible for many physiological functions. One of which is light signaling. Hence, it shares mutual functions with some phytochrome photoreceptors (PHYs), e.g., PHYB. It also contains intrinsically disordered regions (IDRs) that stabilize RCD1 protein upon phosphorylation. This study aims to examine the colocalization events of RCD1 that coexpressed with PHYB in full-length RCD1 complementation line and domain deletion lines, given the fact that both RCD1 and PHYB have previously shown mutual interaction with some of PHYTOCHROME INTERACTING FACTORS (PIFs). To achieve this aim, the colocalization of Venus-tagged RCD1 and Green fluorescent protein (GFP)-tagged PHYB using confocal Microscopy was performed. Another objective is to study the phosphorylation effect of one of the IDRs between WWE and PARP-like domain -IDR2- on RCD1 NB localization. Two phosphomutants -non-phosphorylatable and phosphomimetic- constructs were transiently and stably expressed in the rcd1-4 background. Furthermore, they were screened using Confocal Microscopy. We were able to see the colocalization events in 2 domain deletion lines, RCD1-∆WWE-3xVenus and RCD1 ∆PARP 3xVenus. On the other hand, we could not see any colocalization in the RCD1 ∆RST 3xVenus, which indicates the importance of the RST domain in the colocalization. In addition, phosphorylation was found to affect the abundance of RCD1 protein in both transiently and stably expressed lines. Our study showed non-phosphorylatable forms of IDR2 having a higher abundance of RCD1 NB than the control line RCD1-3xVenus, whereas phosphomimetic IDR2 showed no signal. Collectively, Our experiments showed the effect of phosphorylation on RCD1 NB localization and the importance of the RST domain in the colocalization of RCD1 with PHYB.
  • Bhadhury, Suzan Kumer (2022)
    Plant vascular cambium development involves dynamic changes in transcriptional profiles, which are mediated by key transcription factors. Understanding secondary growth through complex molecular processes of vascular cambium development has remarkable significance in maximizing carbon storage in trees and is crucial for the genetic improvement of trees for wood production. Here, we studied the functions of NO TRANSMITTING TRACT (NTT)/WIP DOMAIN PROTEIN2 (WIP2) zinc-finger transcription factor in regulating vascular cambium activity and xylem formation using Arabidopsis roots and tree stems as model organisms. Reporter lines carrying red fluorescent protein driven by the promoter of NTT/WIP2 showed cambium specific expression in Arabidopsis roots under confocal microscope. Inducible overexpression of NTT/WIP2 after cambium activation reduced vascular diameter, primary phloem pole distance and secondary vessel number in Arabidopsis roots. In loss of function ntt mutants, significant reductions of vascular diameter, phloem pole distance and number of secondary vessels were also evident. Our data suggests that, both increased expression and loss of function of NTT/WIP2 gene significantly reduce secondary growth in Arabidopsis roots. Simultaneous to our Arabidopsis experiments we also tested the possibility of increasing wood formation amounts through manipulation of PttNTT (putative Populus ortholog of Arabidopsis NTT/WIP2 gene) expression in hybrid aspen trees. But increased expression of NTT/WIP2 in Poplar significantly reduced plant height, stem girth and internode numbers. Altogether, we hypothesize that, appropriate spatiotemporal expression pattern and levels of NTT/WIP2 is required for cambial activity to proceed properly in plants. These findings represent a new foundation toward a more comprehensive understanding of the molecular mechanisms of NTT/WIP2 gene in regulation of cambium development and secondary growth in plants.
  • Zhanybekova, Zhanargul (2022)
    Abstract Background. Faba bean (Vicia faba L.,) is a protein-rich grain crop and a good source of many nutrients. However, the productivity and composition of this crop can be limited by several biotic and abiotic stresses. Acid soil leads to a reduction in water uptake, and influences on major biological and chemical processes. However, some plants can manage stress. Mostly, low pH reduces plant vegetative organs and a has a negative effect on physiological processes such as gas exchange, photosynthesis, chlorophyll concentration, and transpiration. The aim of this study was to find homolog genes in close spp. and o test selected gene response as a factor in resilience to rootzone acidity in Vicia faba L. Methods. Four faba bean genotypes were selected for our experiment based on germplasm screening in aquaponic conditions from a previous study. According to obtained results of morpho-physiological measures from a previous experiment, IG11204 and Bourdon showed tolerance to acid only. DULUC is tolerant to acid and Al treatments. In contrast, Nora38 demonstrated susceptibility to both stresses. Selected genotypes were grown under the same experimental conditions in order to proceed with samples for further analysis (total RNA extraction, cDNA synthesis, RT-PCR, gel-electrophoresis, validation of selected gene by RT-qPCR). Results. Selected genes have been identified as hub genes in A. thaliana (alternative oxidase 1A, glutamate dehydrogenase 1, zinc fingers superfamily protein), which demonstrated an increase in expression levels under acidic pH stress. We have found homolog genes in the Hedin cultivar. Selected homolog gene were used to check its expression in four genotypes under Al3+ and acidic stresses using RT-qPCR. Finally, up-regulation of the GDH1 was detected by acid and also occurred by Al3+ treatments. Discussion. Our analysis of gene expression revealed that GDH1 was expressed differently between control and treated groups. However, its activity was detected for both stresses. The expression of GDH1 transcripts in this study, also concurs with findings where GDH1 in A. thaliana were expressed under Al3+ stress. Furthermore, it was confirmed that they are novel Al3+ resistance genes, which are regulated by the transcription factor STOP1 (sensitive-to-proton-rhizotoxicity1). GDHs are involved in stress tolerance, and pH-regulated metabolic pathways – GABA shunt. It participates in the regulation of cytosolic pH, carbon fluxes into the TCA cycle, nitrogen metabolism, deterrence of insects, protection against oxidative stress, osmoregulation and signaling in plant cells under different stress conditions. The GABA shunt is enhanced by stresses, which cause cytosol acidification. Genes expressed in conditions of low pH partly overlap with those expressed in plants exposed to Al3+ stress. Generally, acidic soil is associated with the co-occurrence of Al3+ and H+ rhizotoxicities.
  • Pandey, Kailash (2022)
    A well-adjusted regulation of vascular development is crucial for plant growth and development. CLASS III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIP III) genes play a decisive role in cambial stem-cell maintenance, xylem cell fate determination, and ensuring proper patterning of vasculature. LITTLE ZIPPER (ZPR) microproteins are involved in the post-translational regulation of HD-ZIP III via a negative feedback mechanism. The protein-protein docking has predicted that HD-ZIP III and ZPR interact at the b-ZIP domain, which is an evolutionarily conserved region. The expression and mutant phenotype of ZPR in root vasculature haven’t been previously described. In this study, we developed the reporter lines and checked the expression in the Arabidopsis root. ZPR1 and ZPR3 showed a distinct and continuous expression from root tip to elongation and differentiation zone. In the cross-section, the expression was detected in cambium and xylem cells. Unlike ZPR1, ZPR3 showed a stronger expression towards the phloem side. Although ZPR2 didn’t show any endogenous expression, expression was detected in the primary root tip and lateral root initiation site upon xylemin treatment. Exogenous auxin application increased the expression of ZPR3, but it required a longer response time. The expression results suggest that ZPR follows the HD-ZIP III expression pattern, however, each ZPR shows some variation from the others. In the mutant analysis, zpr1 showed increased cellular proliferation in the vasculature, but zpr2 had no difference compared to the wild type. The results suggest that ZPR and HD-ZIP III have their function in a similar region in the root vasculature. However, further empirical evidence is necessary to elucidate the interaction of HD-ZIP III and ZPR, and its role in the regulation of root vascular development.