Xu Xiao-Yong, Hu Zhi-Yong, Li Jun-Feng, Liu Ji-Hong, Deng Xiu-Xin
National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding, Huazhong Agricultural University, Wuhan 430070, People's Republic of China.
Plant Cell Rep. 2007 Aug;26(8):1263-73. doi: 10.1007/s00299-007-0350-7. Epub 2007 Apr 19.
In the present paper, attempts were made to explore the possibility of employing ultraviolet (UV) irradiation in citrus asymmetric fusion for transfer of limited amount of favorable traits from a desirable cultivar to a target one. Exposure of Satsuma mandarin (Citrus unshiu Marc.) embryogenic protoplasts to UV at an intensity of 300 muW cm(-2) led to reduced viability, especially under long irradiation duration. The protoplasts could not grow during culture when they were irradiated for over 30 s. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay revealed extensive DNA fragmentation in the UV-irradiated protoplasts compared with those without UV treatment. Electrofusion between UV-irradiated protoplasts of Satsuma mandarin (donor) with those of Jincheng (C. sinensis Osbeck, recipient), a local cultivar of superior quality, gave rise to regeneration of several lines of shoots, which failed to root despite enormous endeavors. Ploidy analysis via flow cytometry and chromosome counting showed that four selected shoots were either diploid, triploid or tetraploid. Random amplified polymorphism DNA (RAPD) and amplified fragment length polymorphism (AFLP) confirmed the shoots, irrespective of their ploidy level, as putative somatic hybrids. Cleaved amplified polymorphism sequences (CAPS) demonstrated that the shoots predominantly got their cytoplasmic components, in terms of chloroplast (cp) and mitochondrion DNA, from Jincheng, along with possible recombination of cpDNA in some shoot lines. The current data indicated that UV-based asymmetric fusion could also be employed in citrus somatic hybridization with the intention of creating novel germplasms, which may provide an alternative approach for cultivar improvement.
在本文中，我们尝试探索在柑橘不对称融合中利用紫外线（UV）照射，将优良品种中有限数量的有利性状转移到目标品种的可能性。将温州蜜柑（Citrus unshiu Marc.）胚性原生质体暴露于强度为300 μW cm(-2) 的紫外线下会导致活力降低，尤其是在长时间照射的情况下。当原生质体照射超过30 s时，它们在培养过程中无法生长。末端脱氧核苷酸转移酶生物素-dUTP缺口末端标记（TUNEL）分析显示，与未进行紫外线处理的原生质体相比，紫外线照射的原生质体中存在广泛的DNA片段化。将紫外线照射的温州蜜柑（供体）原生质体与优质地方品种锦橙（C. sinensis Osbeck，受体）的原生质体进行电融合，产生了几株芽苗，但尽管付出了巨大努力，这些芽苗未能生根。通过流式细胞术和染色体计数进行的倍性分析表明，四个选定的芽苗要么是二倍体、三倍体，要么是四倍体。随机扩增多态性DNA（RAPD）和扩增片段长度多态性（AFLP）证实，无论倍性水平如何，这些芽苗都是推定的体细胞杂种。酶切扩增多态性序列（CAPS）表明，就叶绿体（cp）和线粒体DNA而言，这些芽苗的细胞质成分主要来自锦橙，并且在一些芽苗品系中cpDNA可能发生了重组。目前的数据表明，基于紫外线的不对称融合也可用于柑橘体细胞杂交以创造新种质，这可能为品种改良提供一种替代方法。
