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为拓宽白菜育种的基因资源,改良白菜品质,以白菜(Brassica campestris,2n=20,AA)和甘蓝(B.oleracea L.var.capitata,2n=18,CC)的子叶和下胚轴为材料分离、制备原生质体。采用40%聚乙二醇(Polyethylene glycol,PEG)进行原生质体融合。融合细胞在以0.3 mol/L蔗糖、0.3 mol/L葡萄糖为渗透稳定剂,附加1.0 mg/L 2,4-D+0.5 mg/L 6-苄氨基嘌呤(6-BA)+0.1 mg/L 1-萘乙酸(NAA)+1.0 mg/L激动素(Kinetin,Kin)的改良K8p培养基中培养并诱导细胞分裂。小愈伤组织经增殖培养后在MS+0.2 mg/L玉米素(Zeatin,ZEA)+1 mg/L 6-BA+0.5 mg/L Kin+0.4 mg/L NAA的固体分化培养基上诱导出不定芽。30 d后再转入MS基本培养基,获得完整的再生植株。将生根的植株转移到花盆,并对其杂种性质进行形态学、细胞学和分子生物学鉴定。结果表明,经细胞融合分裂出的320个愈伤组织中,获得了35棵再生植株,其再生率达10.94%。形态学观察显示,绝大多数再生植株的叶面积较大,株型和叶型为两种杂交亲本的中间型,部分植株的叶片浓绿、肥厚。染色体计数结果显示,36.4%的再生植株染色体数为2n=38;36.4%的再生植株的染色体数为2n=58 60;27.2%的再生植株的染色体数为2n=70 76,超过两个融合亲本的染色体数的总和。流式细胞仪测定DNA含量显示,再生植株DNA含量变化比较大,其结果与染色体鉴定结果相吻合。随机扩增多态性DNA(Randomamplified polymorphic DNA,RAPD)和基因组原位杂交(Genomic in situ hybridization,GISH)分析结果证明再生植株具有双亲基因组。体细胞杂种花粉育性比较低,杂交、回交后其育性逐渐获得恢复,与白菜回交后代逐渐恢复了育性。通过体细胞杂交和回交、杂交获得了形态变化广泛的个体,为白菜的品种育种提供多样的种质资源。
In order to broaden the genetic resources of cabbage breeding and improve cabbage quality, the cotyledon and hypocotyls of Brassica campestris (2n = 20, AA) and B. oleracea L.var.capitata (2n = 18, CC) Isolated, protoplasts were prepared. Protoplast fusion was performed with 40% polyethylene glycol (PEG). The fusion cells were treated with 0.3 mol / L sucrose and 0.3 mol / L glucose as osmotic stabilizers, with 1.0 mg / L 2,4-D + 0.5 mg / L 6-benzylaminopurine 1-naphthaleneacetic acid (NAA) +1.0 mg / L Kinetin (Kin) in a modified K8p medium and induce cell division. Small callus was induced by solid differentiation medium with MS + 0.2 mg / L zeatin (ZEA) +1 mg / L 6-BA + 0.5 mg / L Kin + 0.4 mg / L NAA Adventitious buds. After 30 days, the medium was transferred to MS medium to obtain a complete regenerated plant. The rooted plants were transferred to pots and their hybrids were characterized by morphology, cytology and molecular biology. The results showed that among 320 callus split by cell fusion, 35 regenerated plants were obtained with the regeneration rate of 10.94%. Morphological observation showed that the vast majority of regenerated plants had larger leaf area, plant type and leaf type were intermediate between the two hybrid parents, some of the plants had thick green leaves and hypertrophy. Chromosome counting results showed that the chromosome number of 36.4% regenerated plants was 2n = 38, that of 36.4% regenerated plants was 2n = 5860, that of 27.2% regenerated plants was 2n = 7076, more than two fusion parents The sum of the chromosome number. The results of flow cytometry showed that the content of DNA in the regenerated plants varied greatly, and the results were consistent with the results of chromosome identification. Random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analysis showed that the regenerated plants had a parental genome. Somatic hybrids pollen fertility is relatively low, after hybridization, back fertility and gradually restore fertility, and cabbage backcross progeny gradually restore fertility. By somatic hybridization and backcrossing, hybrids obtained a wide range of morphological changes in individuals, for the breeding of cabbage variety germplasm resources.