Lian Yuji
College of Life Science, Linyi University, Linyi 276005, Shandong, China.
Sheng Wu Gong Cheng Xue Bao. 2012 Sep;28(9):1080-92.
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.
为了拓宽大白菜基因库，我们开展了大白菜（芸薹，2n = 20，AA）与甘蓝（B. oleracea，2n = 18，CC）的种间体细胞杂交。从10日龄幼苗的子叶和下胚轴中分离原生质体，并用40%聚乙二醇（PEG）进行融合。融合细胞在添加了一些植物生长调节剂的改良K8p液体培养基中培养。通过形态学、细胞学和分子生物学特征对融合产物进行鉴定。结果表明，从320个愈伤组织中共获得35株再生绿色植株，植株再生频率为10.94%，其中11株在温室中存活。经随机扩增多态性DNA（RAPD）和基因组原位杂交（GISH）分析证实，所有再生植株均为真杂种。通过染色体计数和流式细胞术确定杂种植物的倍性水平。在36.4%的再生植株中发现了来自两个融合亲本的染色体数总和（2n = 38）；另外36.4%的植株染色体数范围为58 - 60；27.2%的植株染色体数更多，范围为70 - 76。所有再生植株均能正常开花。我们研究了自交和与亲本回交后的花粉育性和结实率。对于染色体数超过38的杂种，自交后有可能获得一些种子。在染色体数为38的杂种群体中，结实率差异很大，自交每荚仅0.11粒种子，开放授粉为0.23 - 0.76粒，与大白菜回交为0.02 - 0.04粒。自交获得的后代株系叶片较大，叶形介于亲本之间。花粉育性在第一代和第二代后代中逐渐恢复。回交后代株系总体上表现出与大白菜相似的形态。在体细胞杂种及其后代中观察到了形态变异。
