Xu X Y, Liu J H, Deng X X
National Key Laboratory of Crop Genetic Improvement, National Center of Crop Molecular Breeding, Huazhong Agricultural University, Wuhan 430070, P.R. China.
Plant Cell Rep. 2006 Jun;25(6):533-9. doi: 10.1007/s00299-005-0057-6. Epub 2006 Feb 14.
Cytoplasm of Satsuma mandarin (Citrus unshiu Marc.) is known to influence seedlessness. Transfer of cytoplasm to a seedy cultivar could possibly lead to the production of seedless citrus fruits. In the present paper cytoplasts were isolated from cell suspension-derived protoplasts of Satsuma mandarin via ultra-centrifugation in a discontinuous gradient. No nucleus could be detected in the cytoplasts by DAPI (4', 6-diamidino-2-phenylindole) staining compared with normal protoplasts. The cytoplasts, with high viability and small size, did not divide during solid embedding culture. Cytoplasts of Satsuma mandarin were electrically fused with embryogenic protoplasts of Murcott tangor (C. reticulata x C. sinensis), which led to regeneration of several cell lines. Flow cytometry (FCM) indicated that the cell lines were diploids. Simple sequence repeats (SSR) and cleaved amplified polymorphism sequence (CAPS) showed that the cell lines got their nuclear DNA from the protoplast parent, whereas the cytoplast parent donated the mtDNA, confirming transfer of mtDNA from Satsuma mandarin into Murcott tangor via cytoplast-protoplast fusion though no polymorphism was detected in chloroplast DNA between the fusion partners. This is the first report on isolation and characterization of cytoplasts, together with cytoplast-protoplast fusion in Citrus, which has a potential for citrus cultivar improvement involving cytoplasm transfer via cytoplast-protoplast fusion.
温州蜜柑（Citrus unshiu Marc.）的细胞质已知会影响无核性。将细胞质转移到有籽品种中可能会导致无核柑橘果实的产生。在本文中，通过在不连续梯度中进行超速离心，从温州蜜柑细胞悬浮液来源的原生质体中分离出了细胞质体。与正常原生质体相比，用4',6-二脒基-2-苯基吲哚（DAPI）染色在细胞质体中未检测到细胞核。这些细胞质体活力高、体积小，在固体包埋培养过程中不分裂。将温州蜜柑的细胞质体与默科特橘橙（C. reticulata x C. sinensis）的胚性原生质体进行电融合，从而获得了多个细胞系的再生植株。流式细胞术（FCM）表明这些细胞系为二倍体。简单序列重复（SSR）和酶切扩增多态性序列（CAPS）分析表明，这些细胞系的核DNA来自原生质体亲本，而细胞质体亲本则提供了线粒体DNA（mtDNA），这证实了通过细胞质体-原生质体融合将温州蜜柑的mtDNA转移到了默科特橘橙中，尽管在融合亲本之间的叶绿体DNA中未检测到多态性。这是关于柑橘细胞质体的分离与鉴定以及细胞质体-原生质体融合的首次报道，该技术在通过细胞质体-原生质体融合进行细胞质转移以改良柑橘品种方面具有潜力。
