Construction Of Plant Chromosomal BAC Library And Characterization Analysis Of Endopolyploidy In Plants Using Flow Cytometric Technique

Flow cytometric technique (FCM), a new measurement method developed at 1960s -1970s with the combination of technologies of laser, electronics, chemistry etc., can allowed rapid measurement of cells with multi-parameter and high-throughout analysis and precise isolation of cell population. This technique has been widely used for diagnosis and research of diseases in medical sciences at the last decades. Flow cytometric technique has been adopted into cell biology of plants since 1980s and shown a great potential in estimation of DNA content, detection of plant cell cycle, monitor of large-scale cell suspension culture and chromosome sorting etc. However, the studies and utilization of FCM in plants was still drop behind the world in China. Therefore, this thesis was dedicated to establish the key techniques on the utilization of FCM in plants. The construction procedure of plant chromosomal BAC library was established through synchronization the root cells, flow sorting and identification, preparation and purification of high quality chromosomal DNA and vector, ligation with vector, as transformation and screening of BAC clones as well on wheat and double-telomere wheat lines. The endopolyploidy in cabbage and its related species was characterized through flow detection analysis of nuclear DNA, their characters among different species, progeny and tissues at different stages were observed. Those results and methods obtained in this study could be widely used in plants research in China and to provide a foundation for promoting plant researches in China to the advanced levels.Synchronization of cell division of root tip cell is the first step for flow isolation of chromosome. There are a few methods for the synchronization of cell division, but those methods are not applicable to wheat due to the different duration of cell division at cell mitosis of different genotype. Therefore the method to synchronize the cell division needs to be established. The results showed that the metaphase index of meristem can be increased dramatically through synchronization of wheat root tip treated with HU treatment, followed by recovery and triflurolin treatment. The maintain of the highest proportion of G1-phase nuclei or the lowest proportion of S-phase in cell population of wheat root tip cells was considered as a standard for evaluating effect of HU treatment. The best time of HU treatment was optimized at nine hours. The recovery treatment is to promote DNA synthesis in cells restrained by HU. Whether a highest proportion of S phase cells can be obtained was the evaluation standard of this treatment. In our research, a 2-4h or 6-7h of recovery treatment would result in the cell transferring into rapid synthesis of DNA and showed an obvious S phase peak between G1 peak and G2 peak on the flow karyotype histogram. After the root tip was incubated in Hongland solution with 100μM triflurolin for 3.5-6h, the most of root tip cells would stay at metaphase and the chromosomes peaks also can be observed on the flow karyotype histogram. The proportion of G2/M phase nuclei was used to assess the effect of trifluorlin treatment and a high proportion of G2/M cell was recommended. There was no significant difference on resolution of flow karytype histogram between ice-water incubation and without ice-water treatment. Perhaps, ice-water incubation make more cell go into M phase, but it may be not necessary for some plants.Rapid isolation of high quantity target chromosome is the base to construction of chromosomal BAC library. The most important steps involved are the preparation, analysis, sorting and identification of high quality chromosome suspension solution. Results showed that synchronized root tips were fixed with 2% paraformaldehyde for 20 min will prevent the cell division and improve suspension of chromosomes. Homogenization was benefit for release of chromosomes from root tip cell. Homogenization at 9600rpm for 12s will release most of chromosomes in cell and maintain intact structure of chromosome. The chromosome suspension should possess a clear background and chromosomes dissociating in solution as a single particle. The suspensed chromosome was stained by DAPI at final concentration of 2μg/ml. A less than 2% of CV was acceptable for chromosome sorting. Chromosome sorted at less than 20 pieces/s of flow speed would improve purity of sorted chromosomes. The sorted chromosomes can be identified by C-PRINS, FISH and PCR. The C-PRINS was the most potential method. PCR method is simple and rapid. The three methods can be used together. The construction of chromosomal BAC library is different with the normal method of BAC construction. Based on our results on wheat chromosomal BAC construction, the procedure could be optimized as followed. 80, 000 pieces of sorted chromosome were imbedded in gel to form a unit. Most of DNA was in the range of 100-400kb in size after digested by BamHI at 10 units for 15 min. The concentration of HMW DNA dialyzed by a dialysis membrane was about 4ng/μl. After extraction, purification, digestion and de-phosphorus, the vectors were ligated with HMW DNA on a ratio of 1:4 for 12-14h. The ligation products were transformed into competent cell at a ratio of 1:20. 1,500 single clones would be obtained from 200μl of recovery solution. 30 clones were picked and cultured, their DNA were extracted and digested with NotI. PFGE result showed that the average insert fragment was more than 100kb. The decomposition of chromosome was a main problem in construction of BAC library. DNA discompose would be avoided or decreased by adjustments of component and pH of chromosome extraction buffer or pre-electrophoresis for 6-8h before digestion.Identification of inter-species hybrids was a necessary step for creating a new germplasm. Two samples were selected from offspring of hybridization and backcross of a cross between cabbage and Chinese cabbage. There was no significant difference in DNA content among them and Chinese cabbage. Thus, these two samples were speculated as interspecies hybrids of cabbage and Chinese cabbage. The identification method is simple and rapid comparing with convenient methods. It is suitable for identification in a big population.Endopolyploidy was existed widely in seed plants. The endopolyploidy among cabbage and its related species were detected through flow cytometric technique. The results showed that the endopolyploidization was specific for species/genus, tissue organ, developmental stage and progress. The old–mature transportation tissues seem have a higher endopolyploidization level than young un-transportation organ have. The endopolyploidization level can be boosted at the first stage of organ development. When the organ developed to mature, the endopolyploidization level would not increase. The endopolyploidization was controlled by nucleolus genes. The cytoplasm of acceptor parent was no impact on endopolyploidization level of BC2 plants. The petiole possessed a higher endopolyploidization level than laminae do.