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Code Chinese Name Common Name Scientific Name Subculture time 1 Valencia Orange C.sinensis Valencia 15 2 (2x) Jincheng Orange (2x) C. sinensis Jincheng 20 3 (4x) Jincheng Orange (4x) C. sinensis Jincheng 8 4 Anliucheng Orange C. sinensis Anliucheng 8 5 Newhall Navel Orange C. sinensis.newhall 13 6 Bonanza Navel C. sinensis Bonanza 11 Orange 7 Frost Navel orange C. sinensis Frost 8 8 Cara Cara Navel C. sinensis Cara Cara 12 orange 9 Guoqing No.4 C.reticulata Guoqing No.4 8 Satsuma 10 Guoqing No.5 C.reticulata Guoqing No.5 8 Satsuma 11 Goutou sour Orange C. aurantium Goutou 10 12 A Hongju Tangerine C. reticulata Hongju 10 13 B Hongju Tangerine C. reticulata Hongju 14 14 Bendizao Tangerine C. reticulata Bendizao 8 15 Ponkan Mandarin C. reticulata Ponkan 8 16 Red Ruby Grapefruit C. paradisi Red Ruby 10 17 Murcott tangor 17 18 Page tangelo 15 19 Mexican Lime C.aurantifolia Mexican 14 2
20 Meiwa kumquat F. crassifolia Meiwa 11 21 Microcitrus M. papauwana 10 22 + Valencia + Meiwa 14 23 Calamonidn C. reticulata Calamonidn 11 (UVP, Inc., Upland, CA, USA) 3
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Fig. 1 Mitochondrial genetic variation of Citrus calli via long-term subculture revealed by RFLPs with restriction endonuclease/probe combinations of A: Hind/atpA and B: PstI/atp6. Lanes, M: λdna Hind/marker, 1: Valencia orange (C), 2: Valencia orange (L), 3: Jincheng orange (C), 4: Jincheng orange (L), 5: Jincheng orange (4X) (C), 6: Jincheng orange (4X) (L), 7: Anliucheng orange (C), 8: Anliucheng orange (L), 9: Newhall navel orange (C), 10: Newhall navel orange (L), 11: Skagg s Bonanza navel orange (C), 12: Skagg s Bonanza navel orange (L), 13 and 14: Frost navel orange (C), 15: Frost navel orange (L), 16: Cara Cara navel orange (C), 17: Cara Cara navel orange (L), 18: Guoqing No. 4 mandarin (C), 19: Guoqing No. 4 mandarin (L), 20: Guoqing No. 5 mandarin (C), 21: Guoqing No. 5 mandarin (L), 22: Goutoucheng sour orange (C), 23: Goutoucheng sour orange (L), 25 and 26: Hongju tangerine (C), 27: Hongju tangerine (L), 28: Beidizaoju tangerine (C), 29: Beidizaoju tangerine (L), 30: Ponkan mandarin (C), 31: Ponkan mandarin (L), 32: Red Ruby grapefruit (C), 33: Red Ruby grapefruit (L), 34: Murcott tangor (C), 35: Murcott tangor (L), 36: Page tangelo (C), 37: Page tangelo (L), 38: Key lime (C), 39: Key lime (L), 40: Meiwa kumquat (C), 41: Meiwa kumquat (L), 42: Microcitrus (C), 43: Microcitrus (L), 44: Somatic hybrid of Valencia orange + Meiwa kumquat (C), 45: Somatic hybrid of Valencia orange + Meiwa kumquat (L), 46: Calamonidn (C), 47: 46: Calamonidn (L). Note, C: Callus; L: Leaves (UVP, Inc., Upland, CA, USA) 6
2 1 UVP mtdna 22 18 mtdna 1.2 1.4 Figure 2 Rate of callus mtdna abundance increase or decrease compared with that in the control leaves based on the hybridization signal intensive (Hind / atpa in Figure 1) measured by UVP software. mtdna in 18 out of the 22 examined genotypes increased ranged from 1.2 to 6.4 times. 7
Fig.3 Organelle distributions in Newhall orange calluses and mesophyll observed by fluorescence microscopy of Technovit 7100 sections stained with DAPI, thick:. Less numbers and higher DNA contents in callus mitochondria compared with the leaf, and organellea no equal segregation in postmitotic callus cell observed. A: Callus cells; B, Tender leaf cells; C, Postmitotic callus cells. 8
4 A DAPI 40 B DNA 0.8%M λdna/hind marker12 3 4 C DNA N DNAT DNA NcNL Pt Fig. 4 A Intact nuclei of Newhall navel orange callus stained by DAPI and photographed under Olympus microscope with 40 X objective lens. B Nuclear DNA tested on 0.8% agarose gellanes, M: λdna/hind marker, 1: Newhall navel orange callus, 2: Newhall navel orange leaves, 3: Page tangelo callus, 4: Trifoliate orange leaves. CNuclear DNA dot blotting hybridized by mixed mitochondrial probes. Samples, N (up row): nuclear DNA, T: (down row), total DNA, Nc: Newhall navel orange callus, NL: Newhall navel orange leaves; Pt, Trifoliate orange 9
1.. 200330 169174 2.. 200633647654 3.. DNA19994111 1204-1207 4. [] 2000 5.. DNA [] 2003 6.. + 200229 (4): 364-369 7. Adams K L, Qiu Y L, Stoutemyer M, Palmer J D. Punctuated evolution of mitochondrial gene content: High and variable rates of mitochondrial gene loss and transfer to the nucleus during angiosperm evolution. Proc. Natl. Acad. Sci. USA, 2002, 99 (15): 9905-9912 8. Adams K L, Rosenblueth M, Qiu Y L, Palmer J D. Multiple losses and transfers to the nucleus of two mitochondrial succinate dehydrogenase genes during angiosperm evolution genetics. Genetics, 2001, 158: 1289-1300 9. Bergthorsson U, Adams K, RENDAN Thomason P, Palmer J D. Widespread horizontal transfer of mitochondrial genes in flowering plants. Nature, 2003, 424: 197-201 10. Cheng Y J, Guo W W, Deng X X. Molecular characterization of cytoplasmic and nuclear genomes in phenotypically abnormal Valencia orange (Citrus sinensis) + Meiwa kumquat (Fortunella crassifolia) intergeneric somatic hybrids. Plant Cell Rep, 2003a, 21 (5): 445-451 11. Consortium Members. The Rice Chromosome 10 Sequencing Consortium. Science, 2003, 300: 1566 1569 12. Daley D O, Adams K L, Clifton R, Qualmann S, Millar A H, Palmer J D, Pratje E, Whelan J. Gene transfer from mitochondrion to nucleus: novel mechanisms for gene activation from Cox2. Plant J, 2002, 30(1): 11-21 13. Guo WW, Grosser JW (2005) Somatic hybrid vigor in Citrus: direct evidence from protoplast fusion of an embryogenic callus line with a transgenic mesophyll parent expressing the GFP gene. Plant Science 168(6): 1541-1545 14. Maliga P. Mobile plastid genes. Nature, 2003, 422: 31-32 15. Nagata N, Saito C, Sakai A, Kuroiwa H, Kuroiwa T. The selective increase or decrease of 10
organellar DNA in generative cells just after pollen mitosis one controls cytoplasmic inheritance. Planta, 1999, 209: 53-65 16. Stupar RM, Lilly JW, Town DC, Cheng Z, Kaul S, Buell CR, Jiang J. Complex mtdna constitutes an approximate 620-kb insertion on Arabidopsis thaliana chromosome 2: implication of potential sequencing errors caused by large-unit repeats. Proc Natl Sci USA, 2001, 98: 5099-5103 17. Yamamoto M, Matsumoto R. Okudai N, Yamasa Y. Aborted anthers of Citrus result from gene-cytoplasmic male sterility. Sci Hort, 1997, 70: 9-14 Mitochondrial DNA increased in long-term sub-cultured Citrus calluses Cheng Yunjiang Guo Wenwu and Deng Xiuxin National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070 Abstract Mitochondrial genome (mtdna) genetic stability was investigated in long-term sub-cultured Citrus callus by restriction fragment length polymorphism (RFLP) and histological observations. The results revealed that mtdna performed high stabilities in old-cultivated species, and variations were observed among several samples with hybrid-origin. However, mtdna abundance increased among eighteen out the twenty-three calluses, the ratios ranged from 1.2 to 6.4 times. Dot blotting of the nuclei DNA with mitochondrial specific probes showed that more intensive hybridization signals in callus than those in the corresponding control leaves, that indicated that mtdna transfer to the nuclei probably be accelerated in callus. Key words: Citrus, callus, germplasm conservation, mitochondrial genome, mtdna 11