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Creation And Genetic Analysis Of New Citrus Germplasms Via Cell Fusion

Posted on:2005-02-19Degree:DoctorType:Dissertation
Country:ChinaCandidate:C H FuFull Text:PDF
GTID:1103360125469108Subject:Pomology
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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,...
Keywords/Search Tags:Citrus, CMS, genetic variation, molecular markers, protoplast culture, somatic hybridization
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