多胚柑桔合子胚的显微分离及培养
|
摘要
多胚现象是柑桔研究及育种实践中的一个重要问题，也是柑桔杂交育种最大的障碍，多胚性柑桔品种在杂交育种时，由于珠心胚的干扰抑制，致使有性胚在早期败育死亡，导致育种过程中很难获得真正的杂种苗，使多胚性柑桔杂交育种工作受到很大的限制。因此，探索高效的合子胚分离技术，在合子胚尚未败育之前将其分离出来进行培养，克服珠心胚的干扰，挽救早期败育的合子胚，从而获得杂种苗，提高杂种率，是多胚柑桔杂种实生苗培育的有效途径，具有重要的实践意义。
本研究初步建立了多胚性柑桔早期合子胚的显微分离、离体培养及合子苗的鉴定体系，成功显微分离了柑桔早期合子胚并通过离体培养再生植株，具体结果如下：
1、多胚性柑桔早期合子胚显微分离的最适时期为授粉后50—55d。利用显微操作系统，采用自制钢针(针尖大小控制在50μm左右)代替传统的微细玻璃针，可提高多胚性柑桔早期合子胚的分离效率。
2、MS、White和MT三种基本培养基都适合柑桔早期合子胚的离体培养，柑桔早期合子胚成熟培养的最佳植物生长调节剂浓度配比为IAA1mg／L+BA20mg／L，适当提高培养基中蔗糖浓度(最适浓度为10％)、添加适量水解乳蛋白(最适浓度为400mg／L)有助于柑桔早期合子胚的生长发育。
3、固体培养与悬浮培养方式均适合柑桔早期合子胚的成熟培养，但悬浮培养方式更便于观察调控。
4、培养成熟的柑桔合子胚转入分化培养时，不需改变成熟培养时的植物生长调节剂配方，只需将蔗糖浓度降至2％，由暗培养改为光照培养即可自然再生植株。在此基础上若添加少量ZT(0.2mg／L)，能促进合子胚再生植株的速度。由成熟培养转入分化培养，再生植株的最佳时期为：a、固体培养的合子胚及其次生胚发育至子叶状形态出现时，即可认为是发育成熟，可以转接至分化培养基中再生植株。b、液体培养的合子胚子叶长度大约在1—1.5cm(培养时间约8星期)时，即可认为是发育成熟，可以转接至分化培养基中再生植株。
5、经反复实验，确立了适合柑桔基因组DNA的快速提取方法和PCR反应的优化体系，筛选出6条扩增多态性较高的引物，对再生小苗进行了RAPD鉴定分析，从73株再生小苗中，鉴定出了61株合子苗，合子苗的比例达到83.6％。
综合本研究所有研究结果表明，利用显微操作系统，显微分离多胚性柑桔早期合子胚，并进一步培养再生植株，可获得高比例的合子苗，是排除多胚性柑桔珠心胚的干扰抑制，促进多胚性柑桔有性杂交育种的有效手段。
Polyembryony is a serious handicap in Citrus breeding. It results in the abortion of the zygotic embryo, due to the competition with the nucellar embryos, which are often more vigorous. Within this context, microseparation and in vitro culture of early zygotic embryos from polyembryony Citrus is very important because it makes possible the use of immature hybrid embryos and accelerates the sexual hybridization breeding in Citrus.
The systems of microseparation and in vitro culture of zygotic embryos from polyembryony Citrus and identification of Citrus hybrids were preliminary established in this research. Some early zygotic embryos of Citrus were microseparated and regenerated into seedlings successfully by this way. The results are as follows:
1、The best stage to separate zygotic embryos was 50-55 days after pollination. It can boost the efficiency of zygotic embryos separation to use steel needles with 50μm large diameter needlepoint under the micromanipulator.
2、MS, White and MT basic medium are all suitable for the culture of zygotic embryos. The addition of 1mg/Lof IAA and 20mg/L of BAto the medium provides best conditions for the mature culture of zygotic embryos of Citrus. It is favourable for the development of early zygotic embryos to increase the osmotic pressures of media and supplement lactalbumin hydrolysate (LH). The suitable concentration of sucrose was 10% and LH was 400mg/L.
3、Cultured in solid or liquid medium are all suitable for the development of early zygotic embryos of Citrus. But zygotic embryos cultured in solid medium can be more ease to be observed and controlled.
4、It doesn't need to change the combination of IAA and BA when developed zygotic embryos change to differentiation culture. Seedlings can be regenerated from mature zygotic embryos when the concentration of sucrose decreased to 2% and cultured in illumination, and it can also accelerate the speed of regeneration with addition of 0．2mg/L of ZT in this way. The best stage to change to differentiation culture is the stage when cotyledons came out from the zygotic embryos cultured in solid medium or the cotyledons of the zygotic embryos cultured in liquid medium are 1-1．5 cm long.
5、The method of isolation of genomic DNA from Citrus and the optimal amplification system and program for RAPD-PCR were established after iterative experiments. 61 zygotic seedlings were identificated from 73 regenerated seedlings by random amplified polymorphic DNA(RAPD) with 6 primers of 10 bases. The proportion of zygotic seedlings is 83．6%.
All results above showed that it is a efficiency method to get rid of the inhibition from nucellar embryos and accelerate the sexual hybridization breeding in polyembryony Citrus to microseparate the zygotic embryos of Citrus by micromanipulator and culture them in vitro into regenerated seedlings.
本研究初步建立了多胚性柑桔早期合子胚的显微分离、离体培养及合子苗的鉴定体系，成功显微分离了柑桔早期合子胚并通过离体培养再生植株，具体结果如下：
1、多胚性柑桔早期合子胚显微分离的最适时期为授粉后50—55d。利用显微操作系统，采用自制钢针(针尖大小控制在50μm左右)代替传统的微细玻璃针，可提高多胚性柑桔早期合子胚的分离效率。
2、MS、White和MT三种基本培养基都适合柑桔早期合子胚的离体培养，柑桔早期合子胚成熟培养的最佳植物生长调节剂浓度配比为IAA1mg／L+BA20mg／L，适当提高培养基中蔗糖浓度(最适浓度为10％)、添加适量水解乳蛋白(最适浓度为400mg／L)有助于柑桔早期合子胚的生长发育。
3、固体培养与悬浮培养方式均适合柑桔早期合子胚的成熟培养，但悬浮培养方式更便于观察调控。
4、培养成熟的柑桔合子胚转入分化培养时，不需改变成熟培养时的植物生长调节剂配方，只需将蔗糖浓度降至2％，由暗培养改为光照培养即可自然再生植株。在此基础上若添加少量ZT(0.2mg／L)，能促进合子胚再生植株的速度。由成熟培养转入分化培养，再生植株的最佳时期为：a、固体培养的合子胚及其次生胚发育至子叶状形态出现时，即可认为是发育成熟，可以转接至分化培养基中再生植株。b、液体培养的合子胚子叶长度大约在1—1.5cm(培养时间约8星期)时，即可认为是发育成熟，可以转接至分化培养基中再生植株。
5、经反复实验，确立了适合柑桔基因组DNA的快速提取方法和PCR反应的优化体系，筛选出6条扩增多态性较高的引物，对再生小苗进行了RAPD鉴定分析，从73株再生小苗中，鉴定出了61株合子苗，合子苗的比例达到83.6％。
综合本研究所有研究结果表明，利用显微操作系统，显微分离多胚性柑桔早期合子胚，并进一步培养再生植株，可获得高比例的合子苗，是排除多胚性柑桔珠心胚的干扰抑制，促进多胚性柑桔有性杂交育种的有效手段。
Polyembryony is a serious handicap in Citrus breeding. It results in the abortion of the zygotic embryo, due to the competition with the nucellar embryos, which are often more vigorous. Within this context, microseparation and in vitro culture of early zygotic embryos from polyembryony Citrus is very important because it makes possible the use of immature hybrid embryos and accelerates the sexual hybridization breeding in Citrus.
The systems of microseparation and in vitro culture of zygotic embryos from polyembryony Citrus and identification of Citrus hybrids were preliminary established in this research. Some early zygotic embryos of Citrus were microseparated and regenerated into seedlings successfully by this way. The results are as follows:
1、The best stage to separate zygotic embryos was 50-55 days after pollination. It can boost the efficiency of zygotic embryos separation to use steel needles with 50μm large diameter needlepoint under the micromanipulator.
2、MS, White and MT basic medium are all suitable for the culture of zygotic embryos. The addition of 1mg/Lof IAA and 20mg/L of BAto the medium provides best conditions for the mature culture of zygotic embryos of Citrus. It is favourable for the development of early zygotic embryos to increase the osmotic pressures of media and supplement lactalbumin hydrolysate (LH). The suitable concentration of sucrose was 10% and LH was 400mg/L.
3、Cultured in solid or liquid medium are all suitable for the development of early zygotic embryos of Citrus. But zygotic embryos cultured in solid medium can be more ease to be observed and controlled.
4、It doesn't need to change the combination of IAA and BA when developed zygotic embryos change to differentiation culture. Seedlings can be regenerated from mature zygotic embryos when the concentration of sucrose decreased to 2% and cultured in illumination, and it can also accelerate the speed of regeneration with addition of 0．2mg/L of ZT in this way. The best stage to change to differentiation culture is the stage when cotyledons came out from the zygotic embryos cultured in solid medium or the cotyledons of the zygotic embryos cultured in liquid medium are 1-1．5 cm long.
5、The method of isolation of genomic DNA from Citrus and the optimal amplification system and program for RAPD-PCR were established after iterative experiments. 61 zygotic seedlings were identificated from 73 regenerated seedlings by random amplified polymorphic DNA(RAPD) with 6 primers of 10 bases. The proportion of zygotic seedlings is 83．6%.
All results above showed that it is a efficiency method to get rid of the inhibition from nucellar embryos and accelerate the sexual hybridization breeding in polyembryony Citrus to microseparate the zygotic embryos of Citrus by micromanipulator and culture them in vitro into regenerated seedlings.
引文
[1] Soost RK, Willians TE, Torres AW, et al. Identification of nucellar and zygotic seedlings of Citrus with leaf isozymes[J]. HortScience, 1980, 15(6): 728～729
[2] Sharma DR, Kaur R, KumarK. Embryo rescue in plants-areview[J]. Euphytica, 1996, 89: 325～337
[3] 梁青，陈学森，刘文，等．胚抢救在果树育种上的研究及应用[J]．园艺学报，2006，33(2)：445～452
[4] 王大元，张进仁．柑桔胚的人工培养[J]．植物学报，1975，17(2)：149-152
[5] Dubey KC, Rishi N. Studies on the nucellar embryos of some cultivated citrus species[J]. Horticultural Research, 1976, 15(2): 49～52
[6] 王元裕，周碧英，高士贤．柑桔胚培养技术的研究Ⅰ[J]．园艺学报，1979，6(1)：2～8
[7] Rangwany NS. Experimental studies on female reproductive turctures of citrus microcarpa Bange[J]. Phytomorphology, 1961, 11: 109～127
[8] Rangwany TS, Murarhige T, Bitters WP. In vitro studies of zygotic and nucellar embryogenesis in citrus In: Chapme H D(Ed): Proc Int citrus Symp[J]. Riverside Univ. California, 1969,225～229
[9] 屈内昭作，汤田英二，中川昌一．关于柑桔胚培养的研究(第1报)[J]．Japan Sol Hort Sci，1976，45(3)：253～260
[10] 王元裕，周碧英，高士贤．柑桔胚培养技术的研究Ⅱ[J]．园艺学报，1981，8(1)：13～18
[11] 陈振光，王家福．柑桔合子胚早期离体培养获得植株[J]．福建农学院学报，1986，15(4)：271～276
[12] 陈振光，王家福．柑桔球形合子胚的离体培养[J]．福建农学院学报，1991，20(3)：297～304
[13] 黄仕周，熊德华．建水无籽蜜桔德幼胚培养和无性系苗德繁殖[J]．云南植物研究，1992，14(1)：73～79
[14] 赵琦，韦党扬，李兴忠，等．哈姆林Ⅱ╳路比血橙幼胚组培植株高接结果观察[J]．浙江柑桔，1996，13(3)：26～27
[15] 韦党扬，赵琦，李向林，等．哈姆林Ⅱ╳路比血橙组培植株的培育及鉴定[J]．贵州农学院学报，1997，16(1)：50～54
[16] 伊华林，邓秀新，史永忠，等．三倍体柑桔幼胚离体培养研究[J]．园艺学报，1997．24(3)：289～290
[17] Carimi F, Depasquale F, Puglia AM. In vitrorescue of zygotic embryos of sour orange, Citrus aurantium L., and their detection based on RFLP analysis[J]. Plant breeding, 1998, 117(3): 261～266(28ref.)
[18] Soares F, Walter DS, Medrado, etal. Frequency of citrus hybrids from controlled crossings: seed germination in seedbeds versus in vitroembryo cultivation[J]. Pesq. agropec, bras., 2002, 37(7):981～988
[19] 吴金寿，王平，吕柳新，等．柚杂交胚离体培养再生植株的研究[J]．福建农林大学学报(自然科学版)，2003，32(4)：474～477
[20] Viloria Z, Grosser JW, Bracho B. Immature embryo rescue, culture and seedling development of acid citrus fruit derived from interploid hybridization[J]. Plant cell, Tissue and Organ culture, 2005, 82(2): 159～167
[21] Hu CY, Ferreira AG, Cultura de E. Cultura de tecidos e transformacaos genetica de plantas. Brasilia: EMBRAPA/CNPH/CBAB, 1998, 2: 371～393
[22] 王家福，陈振光．影响柑桔球形合子胚离体培养的若干因素[J]．热带作物学报，1999，20(2)：27～30
[23] Edvan AC, Moacir P, Jose DR, et al. Development of globular embryos from the hybridization between 'Pera Rio' sweet orange and 'Ponca' mandarin. Rev. Bras. Frutic, 2003, 25(3): 483～488
[24] Das A, Paul AK, Chaudhuris S. Micropropagation of sweet orange Citrus sinensis Osbeck for the development of nucella seedling[J]. Indian Journal of Experimental Botany, 2000, 38 (3): 269～272
[25] Edvan AC, Moacir P, Leonardo F D, et al. Performance of different embryo stage of sweet orange x mandarin cultivated in vitro[J], Rev. Bras. Frutic, 2003, 25(3): 523～525
[26] 王兰岚，陆仲康，黄力全，等．激光微束显微切割植物染色体的研究[J]．中国激光，1991，18(3)：313～316
[27] Fukui K, Mnezawa M, Kamisugi Y, et al. Microdissection of plant chromosomes by argon-ion laser beam[J]. Theor Appl Genet, 1992, 84(7): 787～791
[28] Houben A, Kynast R G, Helm U, et al. Molecular cytogenetic characterization of the terminal heterochromatic segment of the B-chromosome of rye (Secale cereale) [J]. Chromosoma, 1996, 105(4): 97～103
[29] 马有志，徐琼芳，辛志勇，等．小麦染色体的显微激光分离[J]．遗传学报，1999，26(1)：43～48
[30] Sandery M J, Forster J, MacadamS R, et al. Isolation of a sequence common to A- and B- chromosmes of rye(Senale cerale) by microcloning[J]. Plant Mol Biol Rep, 1991, 9(1): 21～30
[31] Jnng C, Claussen U, Horsthemke B, et al. A DNA library from an individual Beta patellaris chromosome conferring nematode resistance obtained by microdissection of meiotic metaphase chromosomes[J]. Plant Molecular Biology, 1992, 20(3): 503～511
[32] Schondelmaier J, Martin R, Jahoor A, et al. Microdissection and microcloning of the barley (Hordeum vulgare L.) chromosome 1HS[J]. Theor Appl Genet, 1993, 86(5): 629～636
[33] 田革叉，卢一凡，邓继先，等．小麦染色体显微切割及HMW-GSIDx5亚基因克隆[J]．生物工程学报，1999，15(2)：255～258
[34] Chen O, Armstrong G K. Characterization of a library from a single microdissected oat (A venasativa L.) chromosome[J]. Genome, 1995, 38(4): 706～714
[35] 胡赞民，党本元，周奕华，等．玉米单染色体的分离和体外扩增[J]．遗传学报，1998，25(6)：545～550
[36] 周奕华，党本元，胡赞民，等．大豆单染色体的显微分离及体外扩增[J]．植物学报，1998，40(2)：144～150
[37] 程祝宽，颜辉煌，党本元，等．水稻第5染色体短臂四体在染色体臂微分离中的应用[J]．科学通报，1998，43(3)：272～276
[38] 崔丽华，胡赞民，党本元，等．蚕豆大M染色体的显微分离与DNA文库的构建[J]．实验 生物学报，1999，32(3)：313～319
[39] 郭东林，王同昌，王宁，等．斯卑尔脱小麦1B染色体的显微分离及其DNA的PCR扩增[J]．植物研究，2002，22(4)：487～491
[40] 胡赞民，崔丽华，王兰凤，等．王百合单条染色体和染色体片断的分离[J]．遗传学报，1997，24(3)：278～281
[41] 吴菁华，黄代青，吕柳新，等．水仙(Narcissus tazetta L．)单染色体的显微分离[J]．福建农林大学学报：自然科学版，2003，32(4)：471～473
[42] 邓传良，李湘阳，周坚．石蒜单染色体的显微分离及体外扩增[J]．南京林业大学学报，2002，26(3)：72～74
[43] 黄代青，吕柳新，周奕华，柚(Citrus grandis Osbeck)第一染色体的显微分离，农业生物技术学报，2002，10(1)：53～55
[44] 陈晓静，申艳红，卢秉国．番木瓜单染色体的显微分离与克隆[J]．热带业热带植物学报，2005，13(4)：310～314
[45] 黄代青，吕柳新，王平．银杏第1染色体DNA文库的构建[J]．福建农林大学学报：自然科学版，2002，31(4)：490～494
[46] 李湘阳，周坚．杉木单染色体的显微分离及体外扩增[J]．林业科学研究，2002，15(6)：746～750
[47] Quon KC, Marczynmski G T, Shapiro L. Cell cycle control by an essential bacterial two-component signal transducoon Protein[J]. Cell, 1996, 84(1): 83～93
[48] Wuann J, Nurse P. Regulation sphase: CDKs, licensing and proteolysis[J]. Cell, 1996, 85 (6): 785～787
[49] Peter M, Herskowitz I. Joining the complex: cyclin-dependent kinase inhibitory protein and the cell cycle[J]. Cell, 1994, 79(2): 181～184
[50] Sherr C J. G_1 phase progression: cyclin on cue[J]. Cell, 1994, 79(4): 551～555
[51] Sherr C J. Mammalian G_1 cyclins[J]. Cell, 1993, 73(6): 1059～1065
[52] 于建春，金绍波，赵建，等．一种分析有丝分裂期蛋白的新方法[J]．天津师范大学学报，2001．21(3)：65～66，70
[53] 于建春，彭永康，赵建，等．大蒜有丝分裂不同时期蛋白质的变化[J]．植物学报，1998，40(11)：1075～1078
[54] 彭永康，于玲，霍昕．用显微分离和超敏感银染电泳方法研究百合减数第一分裂周期蛋 白质变化[J]．植物研究，2001，21(2)：313～316
[55] Cass DD. An ultrastructural and Nomarski-interference study of the sperms of barley[J]. Can J Bot, 1973,51(3): 601～605
[56] Russell SD. Isolation and characterization of sperm cells in flowering plants[J]. Annu Rev Plant Physiol Plant Mol Biol, 1991,42: 189～204
[57] Chaboud A, Perez R. Generative cells and male gametes: isolation, physiology, and biochemistry[J]. Int Rev Cytof, 1992, 140(2): 205～232
[58] Shivanna KR, Xu H, Taylor P, et al. Isolation of sperms from the pollen tubes of flowering plants during fertilization[J]. Plant Physiol, 1988, 87(3): 647～650
[59] Tian HQ, Zhang J, Russell SD. Isolation of the male germ unit: origination and function in tabacco (Nicotiana tabacum L. ) [J]. Plant Cell Rep, 1998, 18(1): 143～147
[60] 陈素红，杨延红，廖景平，等．蓝猪耳精细胞的分离及两个精细胞群体的收集[J]．热带亚热带植物学报，2004，12(6)：557～561
[61] 胡适宜，李乐工，朱澈．烟草生活胚囊及胚囊原生质体的分离[J]．植物学报，1985，27(2)：337～344
[62] Kranz E, Bautor J. In vitro fertilization of single, isolated gametes of maize mediated by electrofusion[J]. Sex Plant Reprod, 1991, 4(1): 12～16
[63] Kovacs M, Barnabas B, Kranz E. Electro-fused isolated wheat(Triticum aestivum L.)gametes develop into multicellular structures[J]. Plant Cell Reports, 1995, 15(3): 178～180
[64] Holm PB, inudsen S, Mouritzen P, et al. Regeneration of fertile barley plants from mechanically isolated protoplasts of the fertilizedegg cell[J]. Plant cell, 1994, 6(4): 531～543
[65] 韩红梅，赵洁，施华中，等．水稻卵细胞与合子的分离[J]．植物学报，1998，40(2)：186～188
[66] Cao YJ, Russell SD. Mechanical isolation and ultrastructural characterization of viable egg cells in Plumbago zeylanica[J]. Sex Plant Reprod, 1997, 10(6): 368～373
[67] 陈素红，扬延红，廖景平，等．蓝猪耳卵细胞和合子的分离[J]．植物生理与分子生物学 学报，2005，31(4)：383～388
[68] Zhang J, DongWH, Galli A, et al. Regeneration of fertileplants from isolated zygotes of rice(Oryza sativa) [J]. Plant Cell Rep, 1999, 19(2): 128～132
[69] Sun MX, Moscatelli A, Yang HY, Cresti M. In vitrodouble fertilization in Nicotiana tabacum: the role of cell volume in cell fusion[J]. Sex Plant Reprod, 2001, 13(4): 225～229
[70] Dresselhaus T, Lorz H, Kranz E. Representative cDNA libraries from few plant cells[J]. Plant J, 1994, 5(4): 605～610
[71] Dresselhaus T, Hagel C, Lorz H, et al. Isolation of a full-length cDNA encoding calreticulin from a PCR library of in vitro zygotes of maize[J]. Plant Mol Biol, 1996, 31(1): 23～34
[72] Dresselhaus T, Cordts S, Lorz H. A transcript encoding translation initiation factor elF-5A is stored in unfertilized egg ce].Is of maize[J]. Plant Mol Biol, 1999, 39(5): 1063～1071
[73] Dumas C, Mogensen HL. Gametes and fertilization: maize as a model system for experimental embryogenesis in floweringplants[J]. Plant Cell, 1993, 5(10): 1337～1348
[74] Xu HP, Weterings K, Vriezen W, et al. Isolation and characterization of male-germ-cell transcripts in Nicotiana tabacum[J]. Sex Plant Reprod, 2002, 14(6): 339～346
[75] Singh MB, Xu HL, Bhalla PL. Developmental expression of polyubiquitin genes and distribution of ubiquitinated proteins in generativeand sperm cells[J]. Sex Plant Reprod, 2002, 14(6): 325～329
[76] 苟小平，苗琛，陈放，等．水稻精细胞cDNA文库的构建与分析[J]．植物学报，2001，32(2)：778～782
[77] Xu HL, Swoboda I, Bhalla PL, et al. Male gametic cell-specific gene expression in flowering plants[J]. Proc Natl Acad Sci USA, 1999b, 96(5): 2554～2558
[78] Ueda K, Kinoshita Y, Xu ZJ, et al. Unusual core histones specifically expressed in male gametic cells of Lilium longiflorum[J]. Chromosoma, 2000, 108(8): 491～500
[79] 白或，苟小平，徐莺，等．水稻精细胞差异表达基因RSG6的克隆和抗体制备[J]．应用与环境生物学报，2002，8(4)：378～382
[80] 苗琛，苟小平，兰利琼，等．水稻精细胞基因RSSG58的分子克隆和表达[J]．植物学报，2003，45(2)：234～241
[81] Campenot M K, ZhangGC, Cutler AJ, et al. Zea mays embryo sacs in culture. I. Plant regeneration from 1 day after pollination embryos[J]. American Journal of Botany, 1992, 79(10): 1368～1373
[82] Leduc N, Matthys-Rochon E, Rougier M, et al. Isolated maize zygotes mimic in vivo embryonic development and express microinjected genes when cultured in vitro[J]. Dev Biol, 1996, 177(1): 190～203
[83] 何玉池，孙蒙祥，杨弘远．烟草合子离体培养再生可育植株[J]．科学通报，2004，49(5)：457～461
[84] Ponya Z, Finy P, Feher A, et al. Optimisation of introducing foreign genes into egg ceils and zygotes of wheat (Triticum aestivum L.) via microinjection[J]. Protoplasma, 1999, 208(2): 163～172
[85] Holm P B, Olsen O, Schnorf M, et al. Transformation of barley by microinjection into isolated zygote protoplast[J]. Transgenic Research, 2000, 9(1): 21～32
[86] Li ST, Yang HY. Gene transfer into isolated and cultured tobacco zygotes by a specially designed device for electroporation[J]. Plant Cell Reports, 2000, 19(12): 1184～1187
[87] 刘继红，邓秀新．柑桔有性杂种鉴定研究进展[J]．中国南方果树，2002，31(3)：6～8
[88] Mendel K. Bud mutation in citrus and their potential commercial value[J]. Proc Int Soc Citriculture, 1981, 1: 86～89
[89] 简令成，刘庚峰，曾南浦．鉴别柑桔杂种苗与珠心苗的一个新指标[J]．遗传学报，1985，12：26～28
[90] Spiegel-Roy P, Teich A H. Thorn as a possible genetic marker to distinguish zygotic from nucellar seedlings in citrus[J]. Euphytica, 1972, 21(3): 534～537
[91] Teich A H, Spiege-Roy P. Differentiation between nucellar and zygotic Citrus seedlings by leaf shape[J]. Theor Appl Genet, 1972, 42 (7): 314～315
[92] Antonio CO, Aristides NG, Mariangela C, et al. Identification of citrus hybrids through the combination of leaf apex morphology and SSR markers[J]. Euphytica, 2002, 128(3): 397～403
[93] Esen A, Scorn RW, Soost RK. A simple and rapid screening procedure for identification of zygotic Citrus seedlings among cross of certain taxa[J]. J Amer Soc Hort Sci, 1975, 100: 558～564
[94] Tatum JH, Berry RE, Hearn CJ. Characterization of citrus cultivars and separation of nuclear and zygotic seedlings by thinlayer chromatography[J]. Proc Fla State Hort Sci, 1974, 87: 75～82
[95] Weinbum SA, Cohen E, Spiegel-Roy P. Rapid screening of satsuma mandarin progeny to distinguish nucellar and zygotic seedlings[J]. Hort Sci, 1982, 17: 239～240
[96] Soost RK, William TE. Identification of zygotic and nucellar seedlings of citrus with leaf enzyme[J]. Hort Sci, 1980, 15: 728～729
[97] Torres AM, Soost RK, Mau-Lastovi cka T. Citrus enzymes—genetics and di stinguishing zygotic form nucellar seedlings[J]. J Hered, 1982, 73(5): 335～338
[98] 陈振光，王家福．柑桔合子胚早期离体培养获得植株[J]．福建农学院学报，1986，15(4)：271～276
[99] 削顺元，章文才，陈吉笙，等．利用同工酶鉴定柑桔杂种与遗传分析的研究[J]．园艺学报，1989，16(4)：255～260
[100] King BJ, Lee LS, Scott PT. Identification of triploid Citrus by isozyme analysis[J]. Euphytica, 1996, 90(2): 223～231
[101] Bastianel M, Schwards SF, Coleta-Filho HD, et al. Identification of zygotic and nucellar tangerine seedlings(Citrus spp.) using RAPD[J]. Genet and Mol Biol, 1998, 21(1): 123～127
[102] Elisiario PJ, Justo EM, Leitao JM. Identification of mandarin hybrids by isozyme and RAPD analysis[J]. Sci Hort, 1999, 81: 287～299
[103] Cristofani M, Machado MA. Identification of zygotic and nucellar seedlings of Rangpur lime cv. Cravo in the nursery using molecular markers[J]. Laranja, 1998, 19: 159～164
[104] Maria AR, Angel VM, Guillermo CC, et al. Polyembryony and identification of Volkamerian lemon zygotic and nucellar seedlings using RAPD[J]. Pesq. Agropec. Bras., 2004, 39(6): 551-559
[105] Cai Q, Guy CL , Morre GA. Extensive of the linkage map in citrus using RAPD makers and RFLP mapping of cold-acclimation-responsive loci [J]. Theor Appl Genet, 1994, 89: 606~614
[106] Vilanova S, Romero C, Abbott AG, et al. An apricot (Prunus armeniaca L. )F_2 progeny linkage map based on SSR and AFLP markers, mapping plum pox virus resistance and self-incompatibility traits[J]. Theor Appl Genet, 2003, 107: 239-247
[107] Gmitter. A localized linkage map of the citrus tristeza virus resistance generegion EJ3[J]. Theor Appl Genet, 1996, 92 (6) : 688-695.
[108] Huaracha E, Xu M, Korban SS. Narrowing down the region of the Vf locus for scab resistance in apple using AFLP-derived SCARs[J]. Theor Appl Genet, 2004. 108: 274-279.
[109] Koller BA, Lehmann JM, Dermott MC. Identification of apple cultivars using RAPD markers [J]. Theor Appl Genet, 1993, 85: 901— 904
[110] Maria TS, Nicasio T, Loredana A, et al. Flow cytometry, SSR and modified AFLP markers for the indentification of zygotic plantlets in backcrosses between 'Femminello' lemon cybrids(2n and 4n)and a diploid clone of 'Femminello' lemon (Citrus limon L. Burm. F. ) tolerant to mal secco disease [J]. Plant Science, 2003, 164: 1009-1017
[111] U r-Rahman H, James DJ , Hadonou AM , et al. The use of RAPD for verifying the apomictic status of seedlings of Malus species [J]. Theor Appl Genet, 1997, 95: 1080-1083
[112] Breto MP, Ruiz C, Pina jA, et al. The Diversification of Citrus Clementina Hort. ex Tan.. A Vegetatively Propagated Crop Species [J]. Molecular Phylogenetics and Evolution, 2001, 11(2): 285-293.
[113] Collins GG, Symons RH. Polymorphisms in grapevine DNA detected by the RAPD-PCR technique [J]. Plant Mol Bio Rep, 1993, 11 (2) : 105-112
[114] Carimi F, Pasqualef DE, Puhliss M. In vitro rescue of zygotic of sour orange, Citrus aurantium L., and their detection based on RFLP analysis. Plant breeding, 1998, 117(3): 261-266 (28ref.)
[115] Williams J G. K, Kubelik A R, Livak. K J, et al. DNA polymorphisms amplified by arbitrary primers are useful as genetic marker[J], Nucleic Acida Res, 1990, 18 (22): 6531～6535
[116] 汪小全，邹喻苹，张大明，等．RAPD应用于遗传多样性和系统学研究中的问题[J]．植物学报．1996，38(12)：954～962
[117] Wilkie SE, Isaac PG, Slater RJ. Random amplified polymorphic DNA markers for genetic analysis in Allinm[J], TAG, 1993, 86:497～504
[118] Luro F, Laigrct F, Bover J M. DNA amplified fingerprinting, a useful tool for determination of genetic origin and diversity analysis in citrus[J]. Hot science, 1995, 30(5): 1063～1067
[119] 王三红，陈力耕，陈大明．RAPD在柑橘珠心苗鉴定上的应用[J]．南京农业大学学报，1999，22(3)：17～20
[120] 洪棋斌，李喜庆，江东，等．单胚清见橘橙×椪柑产生高比例多胚种子及多胚有性的分子检测[J]．中国农业科学，2003，36(7)：829～833
[121] Gilmar S, Marines B, Ana LCD. Identification of zygotic trifoliate orange seedlings with RAPD molecular markers[J]. Pesq. agropec. Bras., 2004, 39(2): 167～172
[122] Maria AR, Angel VM, Guillermo CC, et al. Polyembryony and identification of Volkamerian lemon zygotic and nucellar seedlings using RAPD[J]. Pesq. agropec. bras., 2004, 39(6): 551～559
[123] Roberto PO, Carlos IAY, Marcos AM. Selection processes in a citrus hybrid population using RAPD markers[J]. Pesq. agropec. Bras., 2003, 38(11): 1297～1302
[124] Maria AR, Angel VM, Alejandra GE, et al. Poliembryony and RAPB markers for identification of zygotic and nucellar seedlings in Citrus[J]. Agrociencia, 2005, 39: 371～383
[125] 李华容．RAPD技术在果树上的应用及问题与展望：广西园艺，2004，15(3)：48～49
[126] 蒋曹德，邓昌彦．随机扩增多态DNA规范化反应体系探讨[J]．生物技术通报，2002，158(3)：40～43
[127] Wang JF, Chen ZG, Lin TX. Observation on the Embryonic Development in Citrus after Cross Pollination[J]. Development & Reproductive Biology, 1999, 8(2): 57～63
[128] 邢建民，赵德修，李茂寅，等．水母雪莲悬浮培养细胞生长和黄酮类活性成分合成[J]．植物学报．1998，40(9)：836～841．
[129] 侯学文，郭勇．接种量及蔗糖浓度对悬浮培养玫瑰茄细胞生长的影响[J]．华南农业大学学报，2000，21(4)：51～54．
[130] 李浚明编译．植物组织学教程(第2版)[M]．北京：中国农业大学出版社，2002．60
[131] 闭秀静，欧阳潘，刘德华，等．悬浮培养中旱芹体细胞胚胎发生的工艺条件研究[J]．植物学报，1996，38(6)：451～456．
[132] 迟吉娜，马峙英，韩改英，等．陆地棉组织培养体细胞胚发生技术改进[J]．棉花学报，2005，17(4)：195～200
[133] 许耀祖，王晓军，赵民安，等．几种生物学因子对熏衣草胚性愈伤组织悬浮培养生长的影响[J]．生物技术通讯，2005，16(1)：34～36
[134] 何丽君，慈忠玲．濒危植物四合木(Tetraena mongolica Maxim．)悬浮培养下体细胞胚胎发生[J]．内蒙古农业大学学报，2001，22(2)：16～22
[135] Dellaporta S L, Wood J, Hick J B. A plant DNA minipreparation: version Ⅱ[J]. Plant Molecular Biology Reporter, 1983, 1(4): 19～21
[136] Ausubel F M，Brent R，Kingston R E，et al．精编分子生物学实验指南[M]．北京：科学出版社，2002：37～38．
[137] 陈大明，张上隆，金勇丰．一种木本果树基因组DNA提取方法研究[J]．浙江农业大学学报，1997，23(6)：621～624
[138] 肖顺元，Frederick G G，Jude W G，等．RAPD分析——鉴定柑桔体细胞杂种的快速方法[J]．遗传，1995，17(4)：40～42
[139] 程运江，伊华林，庞晓明，等．几种木本果树DNA的有效提取[J]．华中农业大学学报，2001，20(5)：481～483
[140] Lu Z X, Reighard G L, Baird W V, et al. Identification of peach rootstock cultivars by RAPD markers[J]. Hortscience, 1996, 31(1): 127～129
[141] Gidoni D, Rom M, Kunik T, et al. Strawberry-cultivar identification using randomly amplified polymorphic DNA (RAPD) markers[J]. Plant Breeding, 1994, 113(4): 339～342
[2] Sharma DR, Kaur R, KumarK. Embryo rescue in plants-areview[J]. Euphytica, 1996, 89: 325～337
[3] 梁青，陈学森，刘文，等．胚抢救在果树育种上的研究及应用[J]．园艺学报，2006，33(2)：445～452
[4] 王大元，张进仁．柑桔胚的人工培养[J]．植物学报，1975，17(2)：149-152
[5] Dubey KC, Rishi N. Studies on the nucellar embryos of some cultivated citrus species[J]. Horticultural Research, 1976, 15(2): 49～52
[6] 王元裕，周碧英，高士贤．柑桔胚培养技术的研究Ⅰ[J]．园艺学报，1979，6(1)：2～8
[7] Rangwany NS. Experimental studies on female reproductive turctures of citrus microcarpa Bange[J]. Phytomorphology, 1961, 11: 109～127
[8] Rangwany TS, Murarhige T, Bitters WP. In vitro studies of zygotic and nucellar embryogenesis in citrus In: Chapme H D(Ed): Proc Int citrus Symp[J]. Riverside Univ. California, 1969,225～229
[9] 屈内昭作，汤田英二，中川昌一．关于柑桔胚培养的研究(第1报)[J]．Japan Sol Hort Sci，1976，45(3)：253～260
[10] 王元裕，周碧英，高士贤．柑桔胚培养技术的研究Ⅱ[J]．园艺学报，1981，8(1)：13～18
[11] 陈振光，王家福．柑桔合子胚早期离体培养获得植株[J]．福建农学院学报，1986，15(4)：271～276
[12] 陈振光，王家福．柑桔球形合子胚的离体培养[J]．福建农学院学报，1991，20(3)：297～304
[13] 黄仕周，熊德华．建水无籽蜜桔德幼胚培养和无性系苗德繁殖[J]．云南植物研究，1992，14(1)：73～79
[14] 赵琦，韦党扬，李兴忠，等．哈姆林Ⅱ╳路比血橙幼胚组培植株高接结果观察[J]．浙江柑桔，1996，13(3)：26～27
[15] 韦党扬，赵琦，李向林，等．哈姆林Ⅱ╳路比血橙组培植株的培育及鉴定[J]．贵州农学院学报，1997，16(1)：50～54
[16] 伊华林，邓秀新，史永忠，等．三倍体柑桔幼胚离体培养研究[J]．园艺学报，1997．24(3)：289～290
[17] Carimi F, Depasquale F, Puglia AM. In vitrorescue of zygotic embryos of sour orange, Citrus aurantium L., and their detection based on RFLP analysis[J]. Plant breeding, 1998, 117(3): 261～266(28ref.)
[18] Soares F, Walter DS, Medrado, etal. Frequency of citrus hybrids from controlled crossings: seed germination in seedbeds versus in vitroembryo cultivation[J]. Pesq. agropec, bras., 2002, 37(7):981～988
[19] 吴金寿，王平，吕柳新，等．柚杂交胚离体培养再生植株的研究[J]．福建农林大学学报(自然科学版)，2003，32(4)：474～477
[20] Viloria Z, Grosser JW, Bracho B. Immature embryo rescue, culture and seedling development of acid citrus fruit derived from interploid hybridization[J]. Plant cell, Tissue and Organ culture, 2005, 82(2): 159～167
[21] Hu CY, Ferreira AG, Cultura de E. Cultura de tecidos e transformacaos genetica de plantas. Brasilia: EMBRAPA/CNPH/CBAB, 1998, 2: 371～393
[22] 王家福，陈振光．影响柑桔球形合子胚离体培养的若干因素[J]．热带作物学报，1999，20(2)：27～30
[23] Edvan AC, Moacir P, Jose DR, et al. Development of globular embryos from the hybridization between 'Pera Rio' sweet orange and 'Ponca' mandarin. Rev. Bras. Frutic, 2003, 25(3): 483～488
[24] Das A, Paul AK, Chaudhuris S. Micropropagation of sweet orange Citrus sinensis Osbeck for the development of nucella seedling[J]. Indian Journal of Experimental Botany, 2000, 38 (3): 269～272
[25] Edvan AC, Moacir P, Leonardo F D, et al. Performance of different embryo stage of sweet orange x mandarin cultivated in vitro[J], Rev. Bras. Frutic, 2003, 25(3): 523～525
[26] 王兰岚，陆仲康，黄力全，等．激光微束显微切割植物染色体的研究[J]．中国激光，1991，18(3)：313～316
[27] Fukui K, Mnezawa M, Kamisugi Y, et al. Microdissection of plant chromosomes by argon-ion laser beam[J]. Theor Appl Genet, 1992, 84(7): 787～791
[28] Houben A, Kynast R G, Helm U, et al. Molecular cytogenetic characterization of the terminal heterochromatic segment of the B-chromosome of rye (Secale cereale) [J]. Chromosoma, 1996, 105(4): 97～103
[29] 马有志，徐琼芳，辛志勇，等．小麦染色体的显微激光分离[J]．遗传学报，1999，26(1)：43～48
[30] Sandery M J, Forster J, MacadamS R, et al. Isolation of a sequence common to A- and B- chromosmes of rye(Senale cerale) by microcloning[J]. Plant Mol Biol Rep, 1991, 9(1): 21～30
[31] Jnng C, Claussen U, Horsthemke B, et al. A DNA library from an individual Beta patellaris chromosome conferring nematode resistance obtained by microdissection of meiotic metaphase chromosomes[J]. Plant Molecular Biology, 1992, 20(3): 503～511
[32] Schondelmaier J, Martin R, Jahoor A, et al. Microdissection and microcloning of the barley (Hordeum vulgare L.) chromosome 1HS[J]. Theor Appl Genet, 1993, 86(5): 629～636
[33] 田革叉，卢一凡，邓继先，等．小麦染色体显微切割及HMW-GSIDx5亚基因克隆[J]．生物工程学报，1999，15(2)：255～258
[34] Chen O, Armstrong G K. Characterization of a library from a single microdissected oat (A venasativa L.) chromosome[J]. Genome, 1995, 38(4): 706～714
[35] 胡赞民，党本元，周奕华，等．玉米单染色体的分离和体外扩增[J]．遗传学报，1998，25(6)：545～550
[36] 周奕华，党本元，胡赞民，等．大豆单染色体的显微分离及体外扩增[J]．植物学报，1998，40(2)：144～150
[37] 程祝宽，颜辉煌，党本元，等．水稻第5染色体短臂四体在染色体臂微分离中的应用[J]．科学通报，1998，43(3)：272～276
[38] 崔丽华，胡赞民，党本元，等．蚕豆大M染色体的显微分离与DNA文库的构建[J]．实验 生物学报，1999，32(3)：313～319
[39] 郭东林，王同昌，王宁，等．斯卑尔脱小麦1B染色体的显微分离及其DNA的PCR扩增[J]．植物研究，2002，22(4)：487～491
[40] 胡赞民，崔丽华，王兰凤，等．王百合单条染色体和染色体片断的分离[J]．遗传学报，1997，24(3)：278～281
[41] 吴菁华，黄代青，吕柳新，等．水仙(Narcissus tazetta L．)单染色体的显微分离[J]．福建农林大学学报：自然科学版，2003，32(4)：471～473
[42] 邓传良，李湘阳，周坚．石蒜单染色体的显微分离及体外扩增[J]．南京林业大学学报，2002，26(3)：72～74
[43] 黄代青，吕柳新，周奕华，柚(Citrus grandis Osbeck)第一染色体的显微分离，农业生物技术学报，2002，10(1)：53～55
[44] 陈晓静，申艳红，卢秉国．番木瓜单染色体的显微分离与克隆[J]．热带业热带植物学报，2005，13(4)：310～314
[45] 黄代青，吕柳新，王平．银杏第1染色体DNA文库的构建[J]．福建农林大学学报：自然科学版，2002，31(4)：490～494
[46] 李湘阳，周坚．杉木单染色体的显微分离及体外扩增[J]．林业科学研究，2002，15(6)：746～750
[47] Quon KC, Marczynmski G T, Shapiro L. Cell cycle control by an essential bacterial two-component signal transducoon Protein[J]. Cell, 1996, 84(1): 83～93
[48] Wuann J, Nurse P. Regulation sphase: CDKs, licensing and proteolysis[J]. Cell, 1996, 85 (6): 785～787
[49] Peter M, Herskowitz I. Joining the complex: cyclin-dependent kinase inhibitory protein and the cell cycle[J]. Cell, 1994, 79(2): 181～184
[50] Sherr C J. G_1 phase progression: cyclin on cue[J]. Cell, 1994, 79(4): 551～555
[51] Sherr C J. Mammalian G_1 cyclins[J]. Cell, 1993, 73(6): 1059～1065
[52] 于建春，金绍波，赵建，等．一种分析有丝分裂期蛋白的新方法[J]．天津师范大学学报，2001．21(3)：65～66，70
[53] 于建春，彭永康，赵建，等．大蒜有丝分裂不同时期蛋白质的变化[J]．植物学报，1998，40(11)：1075～1078
[54] 彭永康，于玲，霍昕．用显微分离和超敏感银染电泳方法研究百合减数第一分裂周期蛋 白质变化[J]．植物研究，2001，21(2)：313～316
[55] Cass DD. An ultrastructural and Nomarski-interference study of the sperms of barley[J]. Can J Bot, 1973,51(3): 601～605
[56] Russell SD. Isolation and characterization of sperm cells in flowering plants[J]. Annu Rev Plant Physiol Plant Mol Biol, 1991,42: 189～204
[57] Chaboud A, Perez R. Generative cells and male gametes: isolation, physiology, and biochemistry[J]. Int Rev Cytof, 1992, 140(2): 205～232
[58] Shivanna KR, Xu H, Taylor P, et al. Isolation of sperms from the pollen tubes of flowering plants during fertilization[J]. Plant Physiol, 1988, 87(3): 647～650
[59] Tian HQ, Zhang J, Russell SD. Isolation of the male germ unit: origination and function in tabacco (Nicotiana tabacum L. ) [J]. Plant Cell Rep, 1998, 18(1): 143～147
[60] 陈素红，杨延红，廖景平，等．蓝猪耳精细胞的分离及两个精细胞群体的收集[J]．热带亚热带植物学报，2004，12(6)：557～561
[61] 胡适宜，李乐工，朱澈．烟草生活胚囊及胚囊原生质体的分离[J]．植物学报，1985，27(2)：337～344
[62] Kranz E, Bautor J. In vitro fertilization of single, isolated gametes of maize mediated by electrofusion[J]. Sex Plant Reprod, 1991, 4(1): 12～16
[63] Kovacs M, Barnabas B, Kranz E. Electro-fused isolated wheat(Triticum aestivum L.)gametes develop into multicellular structures[J]. Plant Cell Reports, 1995, 15(3): 178～180
[64] Holm PB, inudsen S, Mouritzen P, et al. Regeneration of fertile barley plants from mechanically isolated protoplasts of the fertilizedegg cell[J]. Plant cell, 1994, 6(4): 531～543
[65] 韩红梅，赵洁，施华中，等．水稻卵细胞与合子的分离[J]．植物学报，1998，40(2)：186～188
[66] Cao YJ, Russell SD. Mechanical isolation and ultrastructural characterization of viable egg cells in Plumbago zeylanica[J]. Sex Plant Reprod, 1997, 10(6): 368～373
[67] 陈素红，扬延红，廖景平，等．蓝猪耳卵细胞和合子的分离[J]．植物生理与分子生物学 学报，2005，31(4)：383～388
[68] Zhang J, DongWH, Galli A, et al. Regeneration of fertileplants from isolated zygotes of rice(Oryza sativa) [J]. Plant Cell Rep, 1999, 19(2): 128～132
[69] Sun MX, Moscatelli A, Yang HY, Cresti M. In vitrodouble fertilization in Nicotiana tabacum: the role of cell volume in cell fusion[J]. Sex Plant Reprod, 2001, 13(4): 225～229
[70] Dresselhaus T, Lorz H, Kranz E. Representative cDNA libraries from few plant cells[J]. Plant J, 1994, 5(4): 605～610
[71] Dresselhaus T, Hagel C, Lorz H, et al. Isolation of a full-length cDNA encoding calreticulin from a PCR library of in vitro zygotes of maize[J]. Plant Mol Biol, 1996, 31(1): 23～34
[72] Dresselhaus T, Cordts S, Lorz H. A transcript encoding translation initiation factor elF-5A is stored in unfertilized egg ce].Is of maize[J]. Plant Mol Biol, 1999, 39(5): 1063～1071
[73] Dumas C, Mogensen HL. Gametes and fertilization: maize as a model system for experimental embryogenesis in floweringplants[J]. Plant Cell, 1993, 5(10): 1337～1348
[74] Xu HP, Weterings K, Vriezen W, et al. Isolation and characterization of male-germ-cell transcripts in Nicotiana tabacum[J]. Sex Plant Reprod, 2002, 14(6): 339～346
[75] Singh MB, Xu HL, Bhalla PL. Developmental expression of polyubiquitin genes and distribution of ubiquitinated proteins in generativeand sperm cells[J]. Sex Plant Reprod, 2002, 14(6): 325～329
[76] 苟小平，苗琛，陈放，等．水稻精细胞cDNA文库的构建与分析[J]．植物学报，2001，32(2)：778～782
[77] Xu HL, Swoboda I, Bhalla PL, et al. Male gametic cell-specific gene expression in flowering plants[J]. Proc Natl Acad Sci USA, 1999b, 96(5): 2554～2558
[78] Ueda K, Kinoshita Y, Xu ZJ, et al. Unusual core histones specifically expressed in male gametic cells of Lilium longiflorum[J]. Chromosoma, 2000, 108(8): 491～500
[79] 白或，苟小平，徐莺，等．水稻精细胞差异表达基因RSG6的克隆和抗体制备[J]．应用与环境生物学报，2002，8(4)：378～382
[80] 苗琛，苟小平，兰利琼，等．水稻精细胞基因RSSG58的分子克隆和表达[J]．植物学报，2003，45(2)：234～241
[81] Campenot M K, ZhangGC, Cutler AJ, et al. Zea mays embryo sacs in culture. I. Plant regeneration from 1 day after pollination embryos[J]. American Journal of Botany, 1992, 79(10): 1368～1373
[82] Leduc N, Matthys-Rochon E, Rougier M, et al. Isolated maize zygotes mimic in vivo embryonic development and express microinjected genes when cultured in vitro[J]. Dev Biol, 1996, 177(1): 190～203
[83] 何玉池，孙蒙祥，杨弘远．烟草合子离体培养再生可育植株[J]．科学通报，2004，49(5)：457～461
[84] Ponya Z, Finy P, Feher A, et al. Optimisation of introducing foreign genes into egg ceils and zygotes of wheat (Triticum aestivum L.) via microinjection[J]. Protoplasma, 1999, 208(2): 163～172
[85] Holm P B, Olsen O, Schnorf M, et al. Transformation of barley by microinjection into isolated zygote protoplast[J]. Transgenic Research, 2000, 9(1): 21～32
[86] Li ST, Yang HY. Gene transfer into isolated and cultured tobacco zygotes by a specially designed device for electroporation[J]. Plant Cell Reports, 2000, 19(12): 1184～1187
[87] 刘继红，邓秀新．柑桔有性杂种鉴定研究进展[J]．中国南方果树，2002，31(3)：6～8
[88] Mendel K. Bud mutation in citrus and their potential commercial value[J]. Proc Int Soc Citriculture, 1981, 1: 86～89
[89] 简令成，刘庚峰，曾南浦．鉴别柑桔杂种苗与珠心苗的一个新指标[J]．遗传学报，1985，12：26～28
[90] Spiegel-Roy P, Teich A H. Thorn as a possible genetic marker to distinguish zygotic from nucellar seedlings in citrus[J]. Euphytica, 1972, 21(3): 534～537
[91] Teich A H, Spiege-Roy P. Differentiation between nucellar and zygotic Citrus seedlings by leaf shape[J]. Theor Appl Genet, 1972, 42 (7): 314～315
[92] Antonio CO, Aristides NG, Mariangela C, et al. Identification of citrus hybrids through the combination of leaf apex morphology and SSR markers[J]. Euphytica, 2002, 128(3): 397～403
[93] Esen A, Scorn RW, Soost RK. A simple and rapid screening procedure for identification of zygotic Citrus seedlings among cross of certain taxa[J]. J Amer Soc Hort Sci, 1975, 100: 558～564
[94] Tatum JH, Berry RE, Hearn CJ. Characterization of citrus cultivars and separation of nuclear and zygotic seedlings by thinlayer chromatography[J]. Proc Fla State Hort Sci, 1974, 87: 75～82
[95] Weinbum SA, Cohen E, Spiegel-Roy P. Rapid screening of satsuma mandarin progeny to distinguish nucellar and zygotic seedlings[J]. Hort Sci, 1982, 17: 239～240
[96] Soost RK, William TE. Identification of zygotic and nucellar seedlings of citrus with leaf enzyme[J]. Hort Sci, 1980, 15: 728～729
[97] Torres AM, Soost RK, Mau-Lastovi cka T. Citrus enzymes—genetics and di stinguishing zygotic form nucellar seedlings[J]. J Hered, 1982, 73(5): 335～338
[98] 陈振光，王家福．柑桔合子胚早期离体培养获得植株[J]．福建农学院学报，1986，15(4)：271～276
[99] 削顺元，章文才，陈吉笙，等．利用同工酶鉴定柑桔杂种与遗传分析的研究[J]．园艺学报，1989，16(4)：255～260
[100] King BJ, Lee LS, Scott PT. Identification of triploid Citrus by isozyme analysis[J]. Euphytica, 1996, 90(2): 223～231
[101] Bastianel M, Schwards SF, Coleta-Filho HD, et al. Identification of zygotic and nucellar tangerine seedlings(Citrus spp.) using RAPD[J]. Genet and Mol Biol, 1998, 21(1): 123～127
[102] Elisiario PJ, Justo EM, Leitao JM. Identification of mandarin hybrids by isozyme and RAPD analysis[J]. Sci Hort, 1999, 81: 287～299
[103] Cristofani M, Machado MA. Identification of zygotic and nucellar seedlings of Rangpur lime cv. Cravo in the nursery using molecular markers[J]. Laranja, 1998, 19: 159～164
[104] Maria AR, Angel VM, Guillermo CC, et al. Polyembryony and identification of Volkamerian lemon zygotic and nucellar seedlings using RAPD[J]. Pesq. Agropec. Bras., 2004, 39(6): 551-559
[105] Cai Q, Guy CL , Morre GA. Extensive of the linkage map in citrus using RAPD makers and RFLP mapping of cold-acclimation-responsive loci [J]. Theor Appl Genet, 1994, 89: 606~614
[106] Vilanova S, Romero C, Abbott AG, et al. An apricot (Prunus armeniaca L. )F_2 progeny linkage map based on SSR and AFLP markers, mapping plum pox virus resistance and self-incompatibility traits[J]. Theor Appl Genet, 2003, 107: 239-247
[107] Gmitter. A localized linkage map of the citrus tristeza virus resistance generegion EJ3[J]. Theor Appl Genet, 1996, 92 (6) : 688-695.
[108] Huaracha E, Xu M, Korban SS. Narrowing down the region of the Vf locus for scab resistance in apple using AFLP-derived SCARs[J]. Theor Appl Genet, 2004. 108: 274-279.
[109] Koller BA, Lehmann JM, Dermott MC. Identification of apple cultivars using RAPD markers [J]. Theor Appl Genet, 1993, 85: 901— 904
[110] Maria TS, Nicasio T, Loredana A, et al. Flow cytometry, SSR and modified AFLP markers for the indentification of zygotic plantlets in backcrosses between 'Femminello' lemon cybrids(2n and 4n)and a diploid clone of 'Femminello' lemon (Citrus limon L. Burm. F. ) tolerant to mal secco disease [J]. Plant Science, 2003, 164: 1009-1017
[111] U r-Rahman H, James DJ , Hadonou AM , et al. The use of RAPD for verifying the apomictic status of seedlings of Malus species [J]. Theor Appl Genet, 1997, 95: 1080-1083
[112] Breto MP, Ruiz C, Pina jA, et al. The Diversification of Citrus Clementina Hort. ex Tan.. A Vegetatively Propagated Crop Species [J]. Molecular Phylogenetics and Evolution, 2001, 11(2): 285-293.
[113] Collins GG, Symons RH. Polymorphisms in grapevine DNA detected by the RAPD-PCR technique [J]. Plant Mol Bio Rep, 1993, 11 (2) : 105-112
[114] Carimi F, Pasqualef DE, Puhliss M. In vitro rescue of zygotic of sour orange, Citrus aurantium L., and their detection based on RFLP analysis. Plant breeding, 1998, 117(3): 261-266 (28ref.)
[115] Williams J G. K, Kubelik A R, Livak. K J, et al. DNA polymorphisms amplified by arbitrary primers are useful as genetic marker[J], Nucleic Acida Res, 1990, 18 (22): 6531～6535
[116] 汪小全，邹喻苹，张大明，等．RAPD应用于遗传多样性和系统学研究中的问题[J]．植物学报．1996，38(12)：954～962
[117] Wilkie SE, Isaac PG, Slater RJ. Random amplified polymorphic DNA markers for genetic analysis in Allinm[J], TAG, 1993, 86:497～504
[118] Luro F, Laigrct F, Bover J M. DNA amplified fingerprinting, a useful tool for determination of genetic origin and diversity analysis in citrus[J]. Hot science, 1995, 30(5): 1063～1067
[119] 王三红，陈力耕，陈大明．RAPD在柑橘珠心苗鉴定上的应用[J]．南京农业大学学报，1999，22(3)：17～20
[120] 洪棋斌，李喜庆，江东，等．单胚清见橘橙×椪柑产生高比例多胚种子及多胚有性的分子检测[J]．中国农业科学，2003，36(7)：829～833
[121] Gilmar S, Marines B, Ana LCD. Identification of zygotic trifoliate orange seedlings with RAPD molecular markers[J]. Pesq. agropec. Bras., 2004, 39(2): 167～172
[122] Maria AR, Angel VM, Guillermo CC, et al. Polyembryony and identification of Volkamerian lemon zygotic and nucellar seedlings using RAPD[J]. Pesq. agropec. bras., 2004, 39(6): 551～559
[123] Roberto PO, Carlos IAY, Marcos AM. Selection processes in a citrus hybrid population using RAPD markers[J]. Pesq. agropec. Bras., 2003, 38(11): 1297～1302
[124] Maria AR, Angel VM, Alejandra GE, et al. Poliembryony and RAPB markers for identification of zygotic and nucellar seedlings in Citrus[J]. Agrociencia, 2005, 39: 371～383
[125] 李华容．RAPD技术在果树上的应用及问题与展望：广西园艺，2004，15(3)：48～49
[126] 蒋曹德，邓昌彦．随机扩增多态DNA规范化反应体系探讨[J]．生物技术通报，2002，158(3)：40～43
[127] Wang JF, Chen ZG, Lin TX. Observation on the Embryonic Development in Citrus after Cross Pollination[J]. Development & Reproductive Biology, 1999, 8(2): 57～63
[128] 邢建民，赵德修，李茂寅，等．水母雪莲悬浮培养细胞生长和黄酮类活性成分合成[J]．植物学报．1998，40(9)：836～841．
[129] 侯学文，郭勇．接种量及蔗糖浓度对悬浮培养玫瑰茄细胞生长的影响[J]．华南农业大学学报，2000，21(4)：51～54．
[130] 李浚明编译．植物组织学教程(第2版)[M]．北京：中国农业大学出版社，2002．60
[131] 闭秀静，欧阳潘，刘德华，等．悬浮培养中旱芹体细胞胚胎发生的工艺条件研究[J]．植物学报，1996，38(6)：451～456．
[132] 迟吉娜，马峙英，韩改英，等．陆地棉组织培养体细胞胚发生技术改进[J]．棉花学报，2005，17(4)：195～200
[133] 许耀祖，王晓军，赵民安，等．几种生物学因子对熏衣草胚性愈伤组织悬浮培养生长的影响[J]．生物技术通讯，2005，16(1)：34～36
[134] 何丽君，慈忠玲．濒危植物四合木(Tetraena mongolica Maxim．)悬浮培养下体细胞胚胎发生[J]．内蒙古农业大学学报，2001，22(2)：16～22
[135] Dellaporta S L, Wood J, Hick J B. A plant DNA minipreparation: version Ⅱ[J]. Plant Molecular Biology Reporter, 1983, 1(4): 19～21
[136] Ausubel F M，Brent R，Kingston R E，et al．精编分子生物学实验指南[M]．北京：科学出版社，2002：37～38．
[137] 陈大明，张上隆，金勇丰．一种木本果树基因组DNA提取方法研究[J]．浙江农业大学学报，1997，23(6)：621～624
[138] 肖顺元，Frederick G G，Jude W G，等．RAPD分析——鉴定柑桔体细胞杂种的快速方法[J]．遗传，1995，17(4)：40～42
[139] 程运江，伊华林，庞晓明，等．几种木本果树DNA的有效提取[J]．华中农业大学学报，2001，20(5)：481～483
[140] Lu Z X, Reighard G L, Baird W V, et al. Identification of peach rootstock cultivars by RAPD markers[J]. Hortscience, 1996, 31(1): 127～129
[141] Gidoni D, Rom M, Kunik T, et al. Strawberry-cultivar identification using randomly amplified polymorphic DNA (RAPD) markers[J]. Plant Breeding, 1994, 113(4): 339～342