Browsing by Subject "agroinfiltration"

Molecular biology has created a new pathway for plant breeding in cut flower industry. It focuses on studying flower gene functions and provides a more direct and effective way of breeding new flower cultivars using genetic transformation. Besides flower color, disease resistance, quality and vase life, modification of flower architecture is an important target for flower breeding. Previous studies have showed that various transcription factors encoded by the corresponding genes are involved regulating flower development and flower architecture. The most studied are MADS domain and TCP domain transcription factors. For targeted breeding, it is crucial to study the functions of the corresponding genes in detail. For both MADS and TCP domain proteins, previous studies have indicated that protein-protein interactions are important for their function. GhCYC1, GhCYC2, GhCYC3 and GhCYC4, isolated from gerbera (Gerbera hybrida), are CYCLOIDEA –like genes affecting inflorescence development. The protein-protein interactions among these four genes have previously been studied by yeast two-hybrid system. The aim of this thesis was to verify the interactions in living plant cells, using both BiFC and split luciferase assays. Protoplast electroporation and agroinfiltration were used to introduce the genes in planta. The results from the two assays were compared in order to find an effective in planta method for detecting protein-protein interactions. The experiment also provided information about DNA transformation efficiency using protoplast electroporation and agroinfiltration. The results of the split luciferase assay showed that GhCYC1+GhCYC4, GhCYC3+GhCYC4 as well as GhCYC4+GhCYC4 interacted quite strongly in plant cells while GhCYC1+GhCYC1, GhCYC2+GhCYC2 as well as GhCYC4+GhCYC2 had almost no interactions. The interactions between GhCYC3+GhCYC4, and GhCYC4+ GhCYC4 were also shown in yeast two-hybrid, but the other results were different. According to the BiFC assay, no signals of interactions were detected from GhCYC2+GhCYC2, while strong signals were observed from GhCYC2+GhCYC3, and weak signals were seen from GhCYC2+GhCYC4. The interactions between GhCYC2+GhCYC3, GhCYC2+GhCYC4 were also observed in yeast two-hybrid, but the other results were unconfirmed. Large standard deviations were observed in the split luciferase assay and thereby reliable conclusions cannot be drawn from it. However, BiFC turned out to be a better method to detect the protein-protein interactions in planta and clear signals from interactions could be observed. Comparison of the transformation methods indicated that agroinfiltration is a better way of introducing DNA into plant cells than protoplast electroporation. For further study, BiFC assay still needs to be repeated to confirm the efficiency of this assay, and factors affecting the transformation efficiency in protoplast electroporation need to be optimized in the future studies.

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.