白菜与甘蓝之间体细胞杂交种获得与遗传特性鉴定

Production and characterization of a somatic hybrid of Chinese cabbage and cabbage

摘要:

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 (Random amplified 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。

Abstract:

In order to broaden Chinese cabbage gene pool, we conducted interspecific somatic hybridization between Chinese cabbage (Brassica campestris, 2n=20, AA) and Cabbage (B. oleracea, 2n=18, CC). Protoplasts were isolated from 10-day-old cotyledons and hypocotyls of young seedlings, and fused by 40% polyethylene glycol (PEG). Fused cells were cultured in modified K8p liquid medium supplemented with some plant growth regulators. Fusion products were characterized by their morphological, cytological and molecular biological traits. The results showed that, a total of 35 regenerated green plants were obtained from 320 calli, the plant regeneration frequency was 10.94%, and eleven of which were survived in greenhouse. All regenerants were true hybrids as confirmed by randomly amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH) analysis. Ploidy levels of hybrid plants were determined by chromosome counting and flow cytometry. The sum of the chromosome number (2n = 38) from the two fusion patents were found in 36.4% of regeneratns; another 36.4% had chromosomes range to 58-60; 27.2% had more chromosomes ranges to 70-76. All regenerated plants produced normal flowers. We investigated the pollen fertility and seed set after self-pollination and backcrossing with the parental species. For hybrids with chromosomes more than 38 it was possible to obtain some seeds when they after self-pollination. Within the group of hybrids with 38 chromosomes, seed set were very variable, only 0.11 seeds per pod by self-pollination, 0.23-0.76 by open-pollination, 0.02-0.04 by backcrossing with Chinese cabbage. Progeny lines obtained by self-pollination had larger leaves and leaf shapes intermediate of the parental species. Pollen fertility was gradually recovered in the first and second progenies. The backcrossing progeny lines, as a whole, exhibited morphologies were similar to Chinese cabbage. Morphological variations were observed among the somatic hybrids and their progenies.