【作者】 付春华；

【导师】 邓秀新；

【作者基本信息】 华中农业大学 ， 果树学， 2004， 博士

【摘要】 原生质体融合技术能有效克服柑橘有性杂交不亲合、性器官败育以及珠心胚干扰等问题，是有性杂交的补充。体细胞杂种不仅可以实现核DNA重组，也可使胞质基因实现重组，为遗传改良或创造雄性不育材料提供了一条捷径。本研究旨在根据我国柑橘品种的遗传改良实际，有针对性地选配亲本组合，通过电场诱导开展柑橘原生质体的对称和非对称融合，以期将细胞质雄性不育基因转移到有籽品种中，也为柑橘砧木改良和培育三倍体接穗品种提供可供选择的材料。主要研究结果如下： 1 从建立胚性悬浮系入手，建立起国庆1号(Citrus unshiu ’Guoqing No.1’)、国庆5号(C. unshiu ’Guoqing No.5’)等温州蜜柑品种原生质体培养的胚状体高频再生体系，即首先采用两步法建立起质量好的胚性悬浮系，以获得足够数量状态好、活力强的原生质体用于培养和融合;其次，在培养方法上采用低熔点琼脂糖包埋培养，分裂形成肉眼可见细胞团时及时挑出，放入降压的液体MA上培养;第三，再生的愈伤组织在甘油培养基上诱导出小胚状体后，及时转入液体乳糖培养基上振荡培养20—30 d;最后将胚状体转入固体乳糖培养基上进一步增埴。 2 针对我国柑橘砧木改良，对称融合获得了枸头橙(C. aurantium ’Goutou sour orange’)+枳壳(Poncirus trifoliata ’trifoliate orange’)组合的再生植株，获得了三种类型共30余株植株，经形态学、细胞学、SSR鉴定均为四倍体体细胞杂种。CAPS分析表明，杂种的cpDNA均来源于叶肉亲本，mtDNA均来源于悬浮系亲本。AFLP分析表明，来源于同一融合事件的体细胞杂种，相互间差异不大，单株间的相似系数为0.9835-1.0000。再生植株移入田间2a后出现枯枝现象(26.7％)，可能是由于融合后不亲和所致。 3 对称融合获得了纽荷尔脐橙(C. sinesis ’Newhall navel orange’)与黄皮(Clausena lansium ’Chicken heart sweet wampee’)属间三倍体、四倍体植株。经SSR鉴定均为综合双亲遗传物质的体细胞杂种，且三倍体的产生有可能是因为黄皮有一组染色体发生了丢失。对其胞质基因组分析表明，cpDNA均来源于叶肉亲本，mtDNA均来源于悬浮系亲本。该组合难以生根，嫁接在伏令夏橙上的成活率达80％，这些嫁接菌在田间初期生长正常，但生长至一定高度后即出现落叶现象，最后发生整个植株死亡现象。 4 开展了转移温洲蜜柑CMS性状到有籽品种的研究。通过对称和非对称融合，获得了国庆1号(C.onshiu‘Guoqing No.1’)+439橘橙(C reticulata‘Ougan’X C.sinensis‘Gailia月gche塔’‘tangor 439’)、国庆l号(C unshiu‘Guoqing No.1’)+沙田柏(C岁andis‘shatian pununelo’)、国庆l号(C unshiu‘Guoqing No.1’)+冰糖橘(C reticulata‘bingtang tangerine’)、国庆5号(C.unshiu‘Guoqing No.5’)+黄岩本地早(C Suecosa ‘Huangyanbendizao tangerine’)、国庆1号(C unshiu‘Guoqing No.l’)+四季橘(Crericolaza‘Calamondin’)、国庆l号(C unshiu‘Guoqing No.1’)+LIJ金柑(凡rtunellahindsii‘Hong Kong Kumquat’)6个组合的再生愈伤组织和胚状体。经细胞学和SSR鉴定,国庆1号+四季橘组合获得的4个四倍体细胞系和1个六倍体两个细胞系均为体细胞杂种;国庆5号十黄岩本地早为二倍体细胞和非整倍体细胞混合的二倍体体细胞杂种;国庆1号十山金柑的一个细胞系为四倍体体细胞杂种,其余为亲本国庆1号同源融合后再生的四倍体细胞系:国庆1号十439橘橙、国庆1号十沙田袖、国庆1号+冰糖橘三个组合再生的细胞系是国庆1号原生质体培养再生而来的。 5本研究对柑桔亚原生质的分离进行了尝试。结果表明,柑橘原生质体的直径为20一30协m,采用蔗糖梯度超速离心分离亚原生质体,其适宜离心力是100,000一140,0009;利用DAPI染色效果最好,可明显区分开细胞核和细胞质;愈伤组织经CB处理36h后再分离亚原生质体,可显著提高核质体的产量,分离的核质体直径为1.8一3.5林m,但产量比较低,仅为2一3xl扩个,/g鲜重。本实验未能分离到胞质体,尚需从梯度等方面加以优化,以获得足够的胞质体和核质体用于融合。 本文还对远缘体细胞杂交中的遗传变异、利用原生质体融合转移温州蜜柑的CMS于有籽品种、转基因材料在体细胞杂交中的应用潜力、体细胞杂种生长优势的存在及利用等问题进行了探讨。关键词:相一橘体细胞杂交原生质体培养分子标记胞质雄性不育遗传变异更多还原

【Abstract】 The protoplast fusion provides an effectively complementary technique in citrus which can overcome efficiently the breeding barriers of sexual crossing e.g. incomparability, male or female sterile, polyembryo etc. Futhermore, it may be possible to combine and exchange both the nuclear and cytoplasm genomes in the hybrid progeny, especially for creation and improvement of CMS traits. To obtain more novel germplasms for the rootstock and scion improvement of citrus, symmetric and asymmetric fusions mediated by electricity were carried out in this research. Our purpose was also extending to transfer the CMS trait of Satsuma mandarin to superior quality but seedy cultivars. The main results of this research are as follows:1 An efficient embryoid differentiation system from Satsuma mandarin protoplast culture was firstly established. The procedures were as follows: 1) the embryogenic calli suspension culture was established by two-steps method; 2) the protoplasts were cultured using solid-embedding method, followed by transfering the visible mini-calli to MA medium with reduced osmotic pressure for multiplication, and the mini-embryoids were induced on the Glycerol medium; and 3) the embryoids should be cultured in the liquid lactose medium for about 20-30 d, and followed by culturing on solid lactose medium for multiplication.2 The fusion combination between Goutou sour orange (C. aurantium) and trifoliate orange (Poncirus trifoliata) for rootstock improvement regenerated into plantlets. According to leaf morphology and growth vigor, the 30 regenerated plants were divided into three types. They had been identified to be tetrapolid somatic hybrids by morphology, cytology, ploidy and SSR analysis. AFLP analysis showed that the similarity coefficient among the somatic hybrids obtained from one fusion effect was 0.9835-1.000, which indicted that the differences among them were very small. Unfortunately, 26.7% of the plants were dieback after being transferred into field for 2 a, possibly due to the incomparability between the two fusion parents.3 Triploid and tetraploid shoots were obtained from the fusion combination between Newhall navel orange (Citrus sinesis) and "Chicken heart " sweet wampee (Clausena lansium). The shoots were recalcitrant to root and all the plantlets were obtained viamini-grafting on Valencia sweet orange. SSR analysis showed that all those were somatic hybrids, and the production of triploid plantlets maybe due to the chromosome elimination of "chicken heart " sweet wampee. Analysis of cytoplasm genomes using universal primers revealed that their cpDNA band pattern was identical to the mesophyll parent-"chicken heart " sweet wampee, while their mitochondrial genomes were from the Newhall navel orange. These plants grew well in the field firstly, but when reaching a certain height, they were all dieback.4 Embryoids or calli were obtained from six fusion combinations between Satsuma mandarin and seedy cultivars: Guoqing No.l (C. unshiu) + tangor 439 (C. reticulata ’Ougan’ X C. sinensis ’Gailiangcheng’), Guoqing No.l (C. unshiu) + Bingtang tangerine (C. reticulata), Guoqing No.l (C. unshiu) + Shatian pummelo (C. grandis), Guoqing No.l (C. unshiu) + Huanyanbendizao (C. Succosa), Guoqing No.l (C. unshiu) + calamondin (C. reticulata) and Guoqing No.l (C. unshiu) +HongKong Kumquat (C. reticulata). Among them, some cell lines from Guoqing No.l+ Huanyanbendizao, Guoqing No.l+ calamondin, Guoqing No.l+ HongKong Kumquat had been identified to be somatic hybrids by SSR analysis. For the Guoqing No.l+ calamondin, four tetraploid and one hexaploid somatic cell lines were obtained, and embryoids obtained from Guoqing No.l+ Huanyanbendizao combination were mixoploid somatic hybrids, mainly were 2n = 2x = 18, but some were aneuploidy (2n = 19,20), only one tetraploid cell lines from Guoqing No.l + HongKong Kumquat were somatic hybrids, and the others were formed from the homokaryon of Guoqing No.l. The rest three combinations Guoqing No.l + tangor 439, Guoqing No.l + Bingtang tangerine,更多还原