基于甘蓝型油菜小孢子培养和胚状体诱导的植株再生、转化与诱变体系的构建
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摘要
1.本试验研究了不同因素对两个甘蓝型油菜品种浙双758和浙双72的小孢子出胚率和胚状体不定芽再生率的影响。试验结果表明,30℃暗培养2天后转入24℃下培养,具有较高的正常胚产率。在活性炭浓度为0.25 mg/ml时,两品种的小孢子胚产率最高。当蔗糖浓度为130 g/L时,两个品种的小孢子正常胚产率均为最高。在固体培养基上接种两个品种不同胚龄的小孢子胚,发现胚龄对芽再生率和再生外植体芽数有显著的影响。以4周胚龄,即子叶期的胚状体芽再生率最大。研究发现,当激素BA和NAA的配比浓度为0.3 mg/L和2 mg/L时,浙双758和浙双72两品种的芽再生率达到最大。GA浓度对甘蓝型油菜胚再生不定芽有显著的影响。其中以添加0.1 mg/L GA不定芽再生能力最强,浙双758和浙双72两品种芽再生率分别达到82.5%和79.4%。
2.本研究利用秋水仙素直接处理再生植株根系,研究秋水仙素对再生植株加倍效率的影响。结果表明,甘蓝型油菜小孢子再生植株一般以单倍体植株为主,自发二倍体植株数次之。流式细胞仪(FCM)倍性分析表明,不同浓度的秋水仙素(150、300 mg/L)对甘蓝型油菜均有不同程度的加倍效果,处理后的再生植株平均二倍体率达到40%以上。采用合适的加倍剂浓度对于染色体加倍有利,如采用过高的秋水仙素浓度则会对植物细胞产生毒害,如采用过低的浓度又达不到较好的加倍效果。本实验还研究了荧光素二乙酸酯(FDA)气孔保卫细胞荧光染色来鉴定小孢子再生植株的倍性。通过对大量材料进行荧光染色显微观察,发现单倍体植株的气孔细胞显著小于二倍体或对照,同时密度要高于二倍体或对照。
3.本研究在台湾大学叶开温教授实验室构建的双价抗虫基因(sporamin和chitinase)表达载体的基础上,进行农杆菌介导抗性基因转化,添加羧苄青霉素具有抑制农杆菌的生长以降低农杆菌对受体造成深度伤害。结果发现,甘蓝型油菜胚状体对高浓度的羧苄青霉素(>500 mg/L)较为敏感,在细菌被杀死的同时,正常的生长细胞也逐渐褐化,最终死亡。本实验通过设定不同浓度的潮霉素浓度,未经转化的胚状体愈伤组织在含有大于5 mg/L潮霉素的分化培养基中愈伤组织全部褐化死亡,不能再生出芽,而添加相对较低浓度的潮霉素(2.5 mg/L)的筛选分化培养基中,未经转化的胚状体仍有再生苗产生。此外,在油菜转化中,在生根培养基中添加100 mg/L的头孢霉素能有效抑制农杆菌的生长,同时能最大限度的降低抑菌素本身对再生苗生根的影响。将双价抗虫基因导入受体,经PCR检测已初步获得转基因植株。
4.本研究试图通过基因枪介导方式将双价抗虫基因转殖到甘蓝型油菜中,筛选获得了生长正常的转基因植株,期望可以得到高效能的抗虫转基因植株来减少农药的使用量。结果表明,对胚状体进行预培养,其基部膨大,代谢活动较旺盛,可提高胚状体受轰击的面积、子弹的利用率及外源DNA整合机率,从而可适当提高转化效率。此外,研究表明轰击2次比轰击1次抗性胚状体数多,可能是基因枪轰击是随机的,轰击2次比轰击1次概率大。据此建立了甘蓝型油菜小孢子胚状体的转化体系,将双价抗虫基因整合到胚状体中,经初步检测已获得转基因植株。
5.本研究基于油菜小孢子高效胚胎发生技术,利用EMS诱变与ZJ0273选择培养,筛选抗除草剂ZJ0273的再生植株,并对再生植株进行除草剂抗性检验,获得了一些抗性植株。结果表明,EMS浓度越大、诱变时间越长,胚状体受到的毒害作用也越大。本试验选择了半致死剂量诱变后的胚状体进行ZJ0273选择培养,进行植株再生。抗性结果表明,在较高浓度ZJ0273 (100mg/L)培养基中筛选得到的再生植株,具有较高的抗性比例。由没经过选择培养获得再生植株进行除草剂喷施可看出,只有极少量的植株在喷施100 mg/L ZJ0273后能够存活。由此可见,在正常情况下利用除草剂ZJ0273筛选抗性植株的效率非常低,而运用本研究的诱变与选择培养进行除草剂抗性筛选相对较高。
6.为了从基因水平上探索乙酰乳酸合成酶(ALS)和酮醇酸还原异构酶(KARI)对除草剂敏感性表现差别的原因,本研究通过添加不同浓度丙酯草醚(产品代号ZJ0273)于培养基中,结合对不同处理时间胚状体样品的荧光定量PCR检测,探讨了油菜小孢子胚状体分化过程中ALS和KARI基因的表达含量差异及其响应特征。研究表明,添加高浓度的ZJ0273对油菜ALS酶活有一定的抑制作用,且随着处理时间的延长,酶活力降低,对ALS酶的抑制作用增加。而对照和在低浓度处理下,胚状体存活率无显著变化,有正常的分化能力。研究结果发现,ZJ0273对油菜胚状体中的ALS基因和KARI基因的调控因其浓度高低和处理时间不同而异。在高浓度的ZJ0273处理下,ALS基因和KARI基因对其响应呈下调表达模式,其表达水平很低或基本不表达；而在低浓度的ZJ0273处理下,ALS基因和KARI基因的表达水平较高,这表明ALS基因和KARI基因的表达在高浓度除草剂ZJ0273处理下可能受到了抑制。
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.
2.本研究利用秋水仙素直接处理再生植株根系,研究秋水仙素对再生植株加倍效率的影响。结果表明,甘蓝型油菜小孢子再生植株一般以单倍体植株为主,自发二倍体植株数次之。流式细胞仪(FCM)倍性分析表明,不同浓度的秋水仙素(150、300 mg/L)对甘蓝型油菜均有不同程度的加倍效果,处理后的再生植株平均二倍体率达到40%以上。采用合适的加倍剂浓度对于染色体加倍有利,如采用过高的秋水仙素浓度则会对植物细胞产生毒害,如采用过低的浓度又达不到较好的加倍效果。本实验还研究了荧光素二乙酸酯(FDA)气孔保卫细胞荧光染色来鉴定小孢子再生植株的倍性。通过对大量材料进行荧光染色显微观察,发现单倍体植株的气孔细胞显著小于二倍体或对照,同时密度要高于二倍体或对照。
3.本研究在台湾大学叶开温教授实验室构建的双价抗虫基因(sporamin和chitinase)表达载体的基础上,进行农杆菌介导抗性基因转化,添加羧苄青霉素具有抑制农杆菌的生长以降低农杆菌对受体造成深度伤害。结果发现,甘蓝型油菜胚状体对高浓度的羧苄青霉素(>500 mg/L)较为敏感,在细菌被杀死的同时,正常的生长细胞也逐渐褐化,最终死亡。本实验通过设定不同浓度的潮霉素浓度,未经转化的胚状体愈伤组织在含有大于5 mg/L潮霉素的分化培养基中愈伤组织全部褐化死亡,不能再生出芽,而添加相对较低浓度的潮霉素(2.5 mg/L)的筛选分化培养基中,未经转化的胚状体仍有再生苗产生。此外,在油菜转化中,在生根培养基中添加100 mg/L的头孢霉素能有效抑制农杆菌的生长,同时能最大限度的降低抑菌素本身对再生苗生根的影响。将双价抗虫基因导入受体,经PCR检测已初步获得转基因植株。
4.本研究试图通过基因枪介导方式将双价抗虫基因转殖到甘蓝型油菜中,筛选获得了生长正常的转基因植株,期望可以得到高效能的抗虫转基因植株来减少农药的使用量。结果表明,对胚状体进行预培养,其基部膨大,代谢活动较旺盛,可提高胚状体受轰击的面积、子弹的利用率及外源DNA整合机率,从而可适当提高转化效率。此外,研究表明轰击2次比轰击1次抗性胚状体数多,可能是基因枪轰击是随机的,轰击2次比轰击1次概率大。据此建立了甘蓝型油菜小孢子胚状体的转化体系,将双价抗虫基因整合到胚状体中,经初步检测已获得转基因植株。
5.本研究基于油菜小孢子高效胚胎发生技术,利用EMS诱变与ZJ0273选择培养,筛选抗除草剂ZJ0273的再生植株,并对再生植株进行除草剂抗性检验,获得了一些抗性植株。结果表明,EMS浓度越大、诱变时间越长,胚状体受到的毒害作用也越大。本试验选择了半致死剂量诱变后的胚状体进行ZJ0273选择培养,进行植株再生。抗性结果表明,在较高浓度ZJ0273 (100mg/L)培养基中筛选得到的再生植株,具有较高的抗性比例。由没经过选择培养获得再生植株进行除草剂喷施可看出,只有极少量的植株在喷施100 mg/L ZJ0273后能够存活。由此可见,在正常情况下利用除草剂ZJ0273筛选抗性植株的效率非常低,而运用本研究的诱变与选择培养进行除草剂抗性筛选相对较高。
6.为了从基因水平上探索乙酰乳酸合成酶(ALS)和酮醇酸还原异构酶(KARI)对除草剂敏感性表现差别的原因,本研究通过添加不同浓度丙酯草醚(产品代号ZJ0273)于培养基中,结合对不同处理时间胚状体样品的荧光定量PCR检测,探讨了油菜小孢子胚状体分化过程中ALS和KARI基因的表达含量差异及其响应特征。研究表明,添加高浓度的ZJ0273对油菜ALS酶活有一定的抑制作用,且随着处理时间的延长,酶活力降低,对ALS酶的抑制作用增加。而对照和在低浓度处理下,胚状体存活率无显著变化,有正常的分化能力。研究结果发现,ZJ0273对油菜胚状体中的ALS基因和KARI基因的调控因其浓度高低和处理时间不同而异。在高浓度的ZJ0273处理下,ALS基因和KARI基因对其响应呈下调表达模式,其表达水平很低或基本不表达；而在低浓度的ZJ0273处理下,ALS基因和KARI基因的表达水平较高,这表明ALS基因和KARI基因的表达在高浓度除草剂ZJ0273处理下可能受到了抑制。
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.
引文
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