Establishment Of Plant Regeneration, Transformation And Mutagenesis System Based On Microspore Embryogenesis Technology In Brassica Napus L.

1. Composition of nutrient media, inflorescence length and in vitro growth duration of donor plant, as well as heat shock, activated charcoal and sucrose concentrations could have remarkable effects on embryogenesis. Role of these factors towards embryogenesis and shoot regeneration capacity of two oilseed rape (Brassica napus L.) genotypes viz. Zheshuang758 and Zheshuang72 was evaluated in the present study. The highest normal embryo yield was obtained in both cultivars if the NLN induction medium was supplemented with 0.25 mg/ml activated charcoal and 130 g/L sucrose, which was heat shocked in darkness at 30℃for 2 days. The maximum shoot regeneration frequency from microspore-derived (MD) embryos of both cultivars was obtained from the embryos aged 4 weeks. Furthermore, the maximum shoot regeneration frequency of both cultivars was achieved from MS differentiation medium supplemented with 0.3 mg/L BA,2 mg/L NAA and 0.1 mg/L GA3. The plants obtained from both cultivars doubled by colchicine treatment (150 mg/L for 30 h,300 mg/L for 15 h) were successfully transplanted to soil, where plant survival rate and doubling rate were more than 95% and 75%, respectively.2. In another experiment, two colchicine concentrations (150 and 300 mg/L) and two treatment durations (15 and 30 h) were used to treat the plant roots for doubling chromosome. The ploidy level of MD regenerants was determined by flow-cytometer (FCM). Results showed that haploid occupied 50% of total MD regenerated plants. on the other hand, doubled haploid (DH) shared 30% in both rapeseed cultivars before treated by colchicine. However, it was observed that higher doubling frequency was achieved after colchicine treatment. The doubling frequency was approximately 76.8 % in cv. Zheshuang 758 and 82.5% in cv. Zheshuang 72, respectively, when the colchicine concentration was 150 mg/L with the treatment duration of 30 h. Fluorescein diacetate (FDA) was also used to detect the plant ploidy level. The results have shown that the guard cell of haploid is significantly smaller than that of diploid or control, simultaneously the density was higher than that of diploid or control..3. Oilseed rape transgenic system was established through optimizing the factors affecting the Agrobacterium tumefactions. Two varieties viz. Zheshuang 758 and Zheshuang 72 were selected in this experiment, using the anti-insect T-DNA vector constructed by Prof. Ye's laboratory in Taiwan University. MD embryos were used as the explants. The explant was infected with Agrobacterium tumefactions (GV3101) containing plasmids pCAMBIA and constructed with the sporamin and chitinase genes, and co-cultured for 2 d. Explants were screened on the medium containing carb and Hyg, and the transformed shoots were achieved. The result of PCR with the antibiotics resistant cultivar (B. napus) showed that the sporamin and chitinase genes in the T-DNA had been integrated into the genome. The field experiment of transgenic plant is now under way, and further research is required in this aspects.4. Another oilseed rape transgenic system was attempted through the gene gun. Two rapeseed varieties (Zheshuang 758 and Zheshuang 72) were selected in this experiment. The MD embryos were used as the explants. This research focused on the optimization of a protocol to allow the foreign sporamin and chitinase genes to enter the MD embryos in an efficient manner. In this experiment, we tested different options, based on microprojectile bombardment. The best results were obtained through co-transformation by microspore bombardment with DNA-coated microprojectile particles, followed by 6 mg/L Hyg selection. This method provides an efficient mean to integrate extraneous DNA into rapeseed microspores. The primary result of PCR with the antibiotics resistant cultivar (B. napus) showed that the sporamin and chitinase genes in the T-DNA had been integrated into the genome.5. Using two B. napus cultivars (Zheshuang 758 and Zheshuang 72) as donor plants for microspore culture, the experiment was further conducted to select ZJ0273 resistant embryos through EMS mutagenesis and ZJ0273 cultural media with microspore-derived embryos, respectively. The genotypes (Zheshuang 758 and Zheshuang 72) were used to select regenerated plants which showed ZJ0273 resistance for ZJ0273 resistance selection. The embryos at cotyledonary stage were selected to grow on ZJ0273-contained 1/2 MS medium for different duration. The green embryos were transferred into the normal 1/2 MS medium for further root regeneration. Some regenerated plants showed tolerance to ZJ0273 after tested by spraying 100 mg/L ZJ0273 herbicide, indicating that the present embryo selection method of ZJ0273 resistance is effective.6. This experiment describes the response of acetolactate synthase (ALS) and ketol-acid reductoisomerase (KARI) enzymes and ALS and KARI genes of B. napus to different concentrations of ZJ0273 at cotyledonary embryo stage to define the mechanism of this novel herbicide. Oilseed rape variety Zheshuang 758, which has good response to microspore culture, was subjected to various herbicide treatments (0, 1,10 mg/L) at the cotyledonary embryo stage. Differential expressions of two genes (ALS and KARI) encoding the enzymes ALS and KARI in leaves were detected by using real-time fluorescence quantitative PCR (FQ-PCR). The effects of low herbicide ZJ0273 concentration (1 mg/L) on the expression of ALS and KARI genes were not significant, while it was significant with the increase of treatment concentrations and durations. Down regulation was observed for ALS and KARI genes under high ZJ0273 concentration (10 mg/L). Both ALS and KARI enzymes play a key role in the synthesis of branched-chain amino acids in B. napus, and correlated with each other closely during this process.