| Based on tissue culture methods combined with colchicines-induced rice polyploidy, Nipponbare-2X was used as the research material, after callus induction and subculture, chromosome doubled, differentiation, rooting process, the seedlings produced, then transplanted to the field. Making use of root tip chromosome technology to observe the doubled plants, the number of chromosome is2n=48, from which it can be inferred the Nipponbare-4X was successfully established. Analyze the shape and density of stomata, the results indicated that the second leaf from the bottom of the diploid and tetraploid plants showed very significant differences in stomata density, length, width and stomata chloroplast number. Tetraploid leaf stomata density reduced by8.3%than the diploid, and the difference was very significant. Tetraploid stomata length, width and stomata chloroplast number respectively increased by25.51%,16.52%and121.64%than the diploid, the stomata chloroplast number showed the most obvious differences. Tetraploid pollen dyeing rate was37%, decreased by49%compared with86%of the diploid, the difference is very significant. The analysis of morphological observation and agronomic traits of tetraploid and diploid showed that the former plant type was tall, thick stem, leaves number and tiller number were reduced, larger anther and grain, had awns, reduced seed set rateBased on the PMES HN2026-4X which was obtained the early time in the research group and Nipponbare-4x that has been gained in this study. Callus of HN2026-2X, HN2026-4X, Nipponbare-2X and Nipponbare-4X were transformed through Agrobacterium-mediated transformation method. Transgenic plants were identified in molecular and cellular levels. The distribution of microfilaments and microtubules were observed in different tissues and cell levels. All these are advantageous to see the dynamic changes of microfilaments and microtubules in pollen developmental stage and nutritional organizations after rice polyploidy.The results are as follows:1. pBA-YFP-TUA6plasmid verification and conversionThe pBA-YFP-TUA6vector was correct through PCR validation and restriction enzyme verification. YFP and TUA6were transformed into the calluses of HN2026-2X, HN2026-4X and Nipponbare-2X, Nipponbare-4X by Agro bacterium mediated method. Then the transgenic plants of HN2026-2X and HN2026-4X were obtained after screening and differentiation. After co-cultured for two days, YFP fluorescence detection was carried out under a fluorescence microscope to have a preliminary identification. The obtained transgenic plants were detected by PCR techniques. And the roots and pollen cells were observed by fluorescence microscopy for further identification. The results show that rice plants which have the fluorescent labeling microtubules were obtained. It provides the material for the study of a-tubulin dynamic changes in the pollen meiosis.2. pUbi-sGFP-ABD2-sGFP plasmid establishment and transformationThe constitutive promoter Ubiquitin was cloned from the vector p1301and then constructed the pUbi-sGFP-ABD2-sGFP vector. HN2026-2X callus was used as a receptor and did genetic transformation research through the Agro bacterium-mediated of green fluorescent protein gene (sGFP). Embryogenic callus after infection by co-cultured under a fluorescence microscope were detected sGFP fluorescence, to make the initial identification. sGFP fluorescence of roots and pollen cells of transgenic plants was identify using PCR techniques and fluorescence microscopy. The results show that we get transgenic plants with strong fluorescence microfilament. Provide a guarantee for the visualization of pollen development and pollen tube growth in the dynamic changes of actin filament. |
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