冰糖橙胚性愈伤组织诱导及无碎叶病植株再生
|
摘要
冰糖橙(Citrus sinensis Osbeck)是20世纪70年代湖南省选育出的优良甜橙品种，它以肉质脆嫩化渣、风味浓甜、少核或无核而深受消费者喜爱，在湖南省黔阳县和永兴县等地重点推广。进入20世纪90年代后，在部分产区由于柑橘碎叶病毒(Citrus Tatter Leaf Virus，CTLV)的感染等原因，使冰糖橙的单位产量下降，果实品质变劣，失去了原有品种的优良特性，市场竞争力下降。目前，由于冰糖橙组织培养方面的基础性研究的缺乏，很大程度上限制了生物技术在其品种改良中的应用。因此，建立冰糖橙胚性愈伤组织诱导及再生体系，并通过胚性愈伤组织培育出无病毒苗木，或进行冰糖橙品种改良，已成为目前冰糖橙生物技术研究中亟待解决的关键问题。
本研究以冰糖橙成熟果实中未发育胚珠为外植体，进行胚性愈伤组织的诱导和植株再生，并对再生植株进行了指示植物与A蛋白酶联免疫吸附法(PAS-ELISA)检测CTLV。建立了冰糖橙未发育胚珠培养获得无CTLV苗木的培养体系。主要的结果如下：
1．在不同的培养基和不同的培养条件下，冰糖橙胚性愈伤组织和胚状体的发生频率不同，麦芽提取物和GA_3有利于胚性愈伤组织的发生，暗培养有利于胚性愈伤组织的诱导。
2．与以往的研究结果不同的是，在诱导培养基中添加AgNO_3并不能促进冰糖橙胚性愈伤组织的形成，反而抑制了胚状体的发生；然而，在继代培养基中添加5mg／L的AgNO_3有利于小而弱的胚状体基部形成胚性愈伤组织，形成胚性愈伤组织的百分率达28％，经过多次继代处理建立了冰糖橙的胚性细胞系。
3．冰糖橙胚状体在EME500(MT+ME500mg／L)、EME1000(NT+ME1000mg／L)与MKBN(MT+KT 0.5mg／L+BA0.5mg／L+NAA 0.1mg／L)三种培养基上再生芽的比率不同，MKBN培养基分化的芽最多(达88.2％)，EME1000次之(80.9％)，第三是EME500(78.7％)。
4．对冰糖橙再生芽比较了试管生根和试管嫁接两种成苗方法。在1／2MT培养基上单独添加NAA(0.5mg／L)有利于再生芽生根，生根率达76.7％；以酸橙(Citrus aurantium)作砧木进行试管嫁接，其嫁接成活率可达88.9％。
5．对再生苗进行了CTLV检测：同时采用了指示植物腊斯克枳橙(Rusk Citrange)法与PAS-ELISA法检测，两种方法检测结果一致，均未检测到CTLV，可初步确定所获得的再生苗为无CTLV的苗木。
对利用未发育胚珠诱导胚性愈伤组织的优势和利用未发育胚珠培养培育无病毒苗木体系与热处理、茎尖培养、茎尖微芽嫁接获得无病毒苗木体系之间的优缺点、CTLV的检测技术以及无病毒苗木在柑橘生产中的应用价值进行了讨论。
Bingtang orange (Citrus sinensis Osbeck) ( BT ), selected out from Hongjiang of Hunan in 1970s, was a very seedless sweet orange cultivar, and had been widely spreaded in the south of China. The yield and quality were getting down since 1990 because of infecting of citrus tatter leaf virus and the other reasons. It was very important to find a way by using biotechnology to overcome these problems. Present, as the lack of the fundamental research about the tissue culture, the application of biotechnology on the improvement of BT had been limited. Therefore, establishing the system of induction of embryogenic calli of BT and regeneration of plantlets, then obtaining the citrus tatter leaf virus-free plantlets or improving this cultivar are significant the sustainable development of the citrus industry.
In this study, the undeveloped ovules of mature fruit of BT were used as explants to induce embryogenic calli and plantlets regeneration. What's more, we identified the regenerated plantlets whether there were citrus tatter leaf virus in them by indicator (Rusk Citrange) and PAS-ELISA. The system of production of citrus tatter leaf virus-free plantlets from undeveloped ovules of BT was established. The main results are as follows:
1. The regenerateratio of embryogenic calli and embryoids of BT varied on different medium and culture conditions (light or darkness). Malt extract, GAs and darkness were beneficial for induction of embryogenic callus.
2. Silver nitrate was not beneficial to the induction of embryogenic callus, and it controlled the formation of embryoids. During the subculture, silver nitrate (5mg/L) was suitable for embryogenic callus from the embryoids, and 28 percent could be obtained. The embryogenic cell line of BT was established by several subcultures.
3. The regeneration rate of plantlets from embryoids varied on different medium. The rate was higher (88.2%) on the MKBN (MT+ KT 0.5mg/L + BA0.5mg/L + NAA0.1mg/L) medium than on the EME1000 (MT+ ME1000mg/L)(80.9%) and EME500 (MT+ ME500mg/L)(78.7%) media.
4. The rooting rate was 76.7 percent on the medium with NAA (0.5mg/L). Grafted on sour orange in vitro, the survival rate was 88.9 percent.
5. The regenerated plantlets were identified whether there were citrus tatter leaf virus in them by using Rusk citrange as indicator and by PAS-ELISA. The results showed that the citrus tatter leaf virus-free plantlets of BT were obtained. Of course, it needs further study to examine citrus
tatter leaf virus by RT-PCR.
The advantage and disadvantage of this system of virus-free plantlets from undeveloped ovules of citrus, the examining technique of citrus tatter leaf virus and the application of the virus-free plantlets in citrus industry were also discussed at the end of this paper.
本研究以冰糖橙成熟果实中未发育胚珠为外植体，进行胚性愈伤组织的诱导和植株再生，并对再生植株进行了指示植物与A蛋白酶联免疫吸附法(PAS-ELISA)检测CTLV。建立了冰糖橙未发育胚珠培养获得无CTLV苗木的培养体系。主要的结果如下：
1．在不同的培养基和不同的培养条件下，冰糖橙胚性愈伤组织和胚状体的发生频率不同，麦芽提取物和GA_3有利于胚性愈伤组织的发生，暗培养有利于胚性愈伤组织的诱导。
2．与以往的研究结果不同的是，在诱导培养基中添加AgNO_3并不能促进冰糖橙胚性愈伤组织的形成，反而抑制了胚状体的发生；然而，在继代培养基中添加5mg／L的AgNO_3有利于小而弱的胚状体基部形成胚性愈伤组织，形成胚性愈伤组织的百分率达28％，经过多次继代处理建立了冰糖橙的胚性细胞系。
3．冰糖橙胚状体在EME500(MT+ME500mg／L)、EME1000(NT+ME1000mg／L)与MKBN(MT+KT 0.5mg／L+BA0.5mg／L+NAA 0.1mg／L)三种培养基上再生芽的比率不同，MKBN培养基分化的芽最多(达88.2％)，EME1000次之(80.9％)，第三是EME500(78.7％)。
4．对冰糖橙再生芽比较了试管生根和试管嫁接两种成苗方法。在1／2MT培养基上单独添加NAA(0.5mg／L)有利于再生芽生根，生根率达76.7％；以酸橙(Citrus aurantium)作砧木进行试管嫁接，其嫁接成活率可达88.9％。
5．对再生苗进行了CTLV检测：同时采用了指示植物腊斯克枳橙(Rusk Citrange)法与PAS-ELISA法检测，两种方法检测结果一致，均未检测到CTLV，可初步确定所获得的再生苗为无CTLV的苗木。
对利用未发育胚珠诱导胚性愈伤组织的优势和利用未发育胚珠培养培育无病毒苗木体系与热处理、茎尖培养、茎尖微芽嫁接获得无病毒苗木体系之间的优缺点、CTLV的检测技术以及无病毒苗木在柑橘生产中的应用价值进行了讨论。
Bingtang orange (Citrus sinensis Osbeck) ( BT ), selected out from Hongjiang of Hunan in 1970s, was a very seedless sweet orange cultivar, and had been widely spreaded in the south of China. The yield and quality were getting down since 1990 because of infecting of citrus tatter leaf virus and the other reasons. It was very important to find a way by using biotechnology to overcome these problems. Present, as the lack of the fundamental research about the tissue culture, the application of biotechnology on the improvement of BT had been limited. Therefore, establishing the system of induction of embryogenic calli of BT and regeneration of plantlets, then obtaining the citrus tatter leaf virus-free plantlets or improving this cultivar are significant the sustainable development of the citrus industry.
In this study, the undeveloped ovules of mature fruit of BT were used as explants to induce embryogenic calli and plantlets regeneration. What's more, we identified the regenerated plantlets whether there were citrus tatter leaf virus in them by indicator (Rusk Citrange) and PAS-ELISA. The system of production of citrus tatter leaf virus-free plantlets from undeveloped ovules of BT was established. The main results are as follows:
1. The regenerateratio of embryogenic calli and embryoids of BT varied on different medium and culture conditions (light or darkness). Malt extract, GAs and darkness were beneficial for induction of embryogenic callus.
2. Silver nitrate was not beneficial to the induction of embryogenic callus, and it controlled the formation of embryoids. During the subculture, silver nitrate (5mg/L) was suitable for embryogenic callus from the embryoids, and 28 percent could be obtained. The embryogenic cell line of BT was established by several subcultures.
3. The regeneration rate of plantlets from embryoids varied on different medium. The rate was higher (88.2%) on the MKBN (MT+ KT 0.5mg/L + BA0.5mg/L + NAA0.1mg/L) medium than on the EME1000 (MT+ ME1000mg/L)(80.9%) and EME500 (MT+ ME500mg/L)(78.7%) media.
4. The rooting rate was 76.7 percent on the medium with NAA (0.5mg/L). Grafted on sour orange in vitro, the survival rate was 88.9 percent.
5. The regenerated plantlets were identified whether there were citrus tatter leaf virus in them by using Rusk citrange as indicator and by PAS-ELISA. The results showed that the citrus tatter leaf virus-free plantlets of BT were obtained. Of course, it needs further study to examine citrus
tatter leaf virus by RT-PCR.
The advantage and disadvantage of this system of virus-free plantlets from undeveloped ovules of citrus, the examining technique of citrus tatter leaf virus and the application of the virus-free plantlets in citrus industry were also discussed at the end of this paper.
引文
1．李红叶，陈力耕，周雪平．柑橘病毒与类似病毒病分子生物学和抗病毒基因工程研究进展，果树科学，2000，17(2)：131-137
2．张映南，李润唐，谷洪源．冰糖橙低产劣质的原因和对策．中国南方果树，2001，30(1)：8-10
3．吴德喜，舒广平，宋顺华．柑橘碎叶病毒生物鉴定研究．中国柑橘，1990，1：47-49
4. Navarro L. The citrus variety improvement program in Spain [A]. In. Proe. 7th conf. IOCV. Riverside. 1976: 198-202
5．何天富．柑橘学．中国农业出版社，1999
6．董高峰，李耿光，张兰英等．柑橘无病原体苗培育研究进展．广西植物，2001，21(1)：53-58
7. Robacker CD, Chang CJ. Shoot-tip culture of Muscadine grape to eleiminate pierce's disease bacteria [J]. Hortscience, 1992, 27 (5): 449-450
8．董雅凤，张尊平，张少瑜等．苹果和梨树茎尖培养结合热处理脱病毒研究．北方果树，2002，2：9-1
9．薛光荣，杨振英，洪霓等．茎尖培养等处理脱除梨病毒的技术研究[J]．中国果树，1996，3：9-11
10．洪霓，王国平，张尊平等．梨病毒脱除技术研究[J]．中国果树，1995，4：5-7
11．陈青瑛，陈景耀．龙眼茎尖离体培养及其脱毒效果[J]．植物生理学通讯，1996，32(2)：126-131
12．谢东，许仁林，魏书．草莓生物技术育种研究进展．果树科学，1996，13(3)：195-198
13．林伯年，胡春根，沈德绪等．甜橙成年树侧芽离体繁殖诱导成功研究．园艺学报，1992，19(3)：209-214
14．宋瑞琳，吴如健，陈青瑛等．脱除柑橘碎叶病毒的初步研究．福建省农科院学报，1994，9(1)：57-59
15．何新华，蒋元晖，赵学源．柑橘碎叶病毒的脱毒．云南农业大学学报，1995，10(2)：100-101
16. Murashinge T, Bitters P, Rangan TS, et al. A technique of shoot apex grafting and its utilization towards recovering virus-free citrus clones [J]. Hortscience, 1972,7: 118-119
17. Navarro L, Jucurcza J, Ballester JF, et al. Elimination of some citrus pathogens producing psorosis-like leaf symptoms by shoot-tip grafting in vitro [A]. Riverside: In. Proc. 8th Conf. IOCV, 1980: 162-166
18. Mukhoropagation S, Jaishree kai, Sharma BC, et al. Micropropagation of Darjeeling orange by shoot-tip grafting [J]. Journal of Hortscience. 1997,72(3): 493-499
19．赵学源，蒋元晖，李世菱等．我国柑橘栽培品种的裂皮病的鉴定与脱除[J]．园艺学报，1986，13(2)：91-94
20．蒋元晖，邱柱石，苏维芳等．利用茎尖嫁接脱毒培养无裂皮病的脐血橙[J]．植物保护学报，1983，10(8)：166-168
21．宋瑞琳，吴如健，柯冲．茎尖嫁接脱除柑橘主要病原的研究．植物病理学报报，1999，29(3)：275-279
22．郭春慧，马凤桐，经济林木试管茎尖嫁接的研究现状及展望．西北农业大学学报，1997，25(3)：36-39
23．Reuther W, Batchelor LD, Webber HJ 著 (胥洱，孔焱译)．1985：柑橘业(第二卷)：柑橘生产．农业出版社，北京：260-266
24．霍合强，郝玉金，邓秀新．宽皮柑橘品种的胚性愈伤组织诱导．实验生物学报，1999，32(3)：289-295
25. Navarro L. Development of a program for disease-free citrus budwood in Spain [A]. Proc. Int. Soc. Citriculture, 1981: 70-73
26．高峰，陈杰忠，陈善春等．柑橘未受精胚珠离体培养获得无病毒珠心苗．植物学报，1990，32(7)：505-509
27．谭祖国，万蜀渊．朋娜和纽贺尔脐橙胚珠离体培养获得无病毒珠心苗方法研究．湖北农业科学，1998，4：43-45
28. Wallace JM, Drake RJ. Tatter leaf, a previously undescribed virus effect on citrus. Plant Dis. Reptr, 1962,46: 211-212
29. Wallace JM, Drake RJ. New information on symptom effects and host range of the citrus tatter leaf virus. Plant Dis. Reptr, 1963,47: 352-353
30. Garnsey SM. Detection of tatter leaf virus in Florida. Proc. Fla. State Hort, Soc., 1964, 77: 106-109
31. Da Graca JV. Citrus tatter leaf virus in South African Meyer lemon, Citrus and Subtrop. Fruit J, 1977, 529: 18
32. Miyakawa T. A bud-union abnormality of Satsuma mandarm on Poncirus trifoliate in Japan. Proc.7th Conf IOCV., Riverside, 1976: 125-131
33. Fraser LR.Virus and related diseases of citrus in New South Wales. Dept. Agp. New South Wales, Australia, 1979:52
34. Miyakawa T. Occurrence and distribution of tatter leaf-citrange stunt virus and its effects on Japanese citrus. Proc.8th Conf IOCV., Riverside, 1980: 220-224
35. 苏鸿基．Occurrence and distribution of tatter leaf-citrange stunt complex on Taiwanese citrus. NTU Phytopathlogist and Entomologist, 1984, 11: 42-48
36．张天淼，梁仙友．柑橘碎叶病毒在黄岩本地早、槾桔上的发现．中国柑橘，1987，1：27-28
37．赵学源，蒋元晖，李世菱等．柑橘碎叶病的初步鉴定．中国柑橘，1987，1：28
38．张天淼，梁仙友，陈作义等．柑橘碎叶病的发生与初步鉴定．植物病理学报，1988，18(2)：
79-84
39．何新华，蒋元晖，赵学源等．柑橘碎叶病的分布鉴定．植物保护，1991，17(1)：6-7
40．宋顺华，舒广平，刘庚峰等．湖南省柑橘资源病毒类病害鉴定初报．中国柑橘，1995，3：36-37
41. Nishio T, Kawai A, Takahashi T, et al. Purification and properties of citrus tatter leaf virus. Annals of the Phytopathological Sciety of Japan, 1989,55(2): 254-258
42. Yoshikawa N, Imaizumi M, Takahashi T, et al. Striking similarities between the nucleotide sequence and genome organization of citrus tatter leaf and apple stem grooving capillovirus. Journal of general virology, 1993,74(12): 2743-2747
43. Ohira K, Namba S, Rozanov M, el al. Complete sequence of an infectious full-length cDNA clone of citrus tatter leaf capillovirus: Comparative sequence analysis of capillovirus genomes. Journal of general virology, 1995,76(9): 2305-2309
44. Magome H, Yoshikawa N, Takahashi T. Molecular variability of the genomes of capilloviruses from apple, Japanese pear, European pear, and citrus trees. Phytopathol, 1996,87: 389-396
45. Inouye N, Maeda T, Mitsuhata K. Citrus tatter left virus isolated from lily. Annals of the Phytopathological Sciety of Japan, 1979,45: 712-720
46. Ohira K, Ito T, Kawai A, Namba S, Kusumi T, Tsuchizaki T. Nucleotide sequence of the 3'-therminal region of citrus tatter leaf virus RNA. Virus genes, 1994, 8(2): 169-172
47. Garnsey SM. Virus in Florida's Meyer lemon trees and their effects on other citrus.. Proc. Florida State Hort. Soc., 1970, 83: 66-71
48. Yarwood CE. Mechanical transmission of alatent lemon virus. Phytopathology, 1963,53:1145
49. Fulton RW. Mechanical transmission of tatter leaf virus from cowpea to citrus. Phytopathology, 1966, 56: 575
50. Roistacher CN. Tatter leaf and infectious variegation viruses on knife blades and its prevention. Proc. 8th Conf. IOCV. Riverside, 1980: 225-229
51. Semancik JS. Partial purification of a mechanical transmissible virus associated with tatter leaf of citrus. Phytopathology, 1965,55: 1354-1358
52．井上成信．Citrus tatter leaf virus．日植病报，1979，45：712-720
53．谢佩华，金显忠，何献武．浙江南部柑橘病毒病和类似病毒病鉴定．中国柑橘，1992，21(4)：13-17
54．谢佩华，金显忠，何献武．浙江南部柑橘碎叶病的调查和鉴定．植物病理学报，1992，22(3)：195-197
55．陈集双，张天淼，周雪平等．柑橘碎叶病毒研究．微生物学报，1995，35(3)：173-180
56. Garnsey SM. Indexing for tatter leaf with cowpea. USDA Agr. Handbook, Washington, D.C., 1968: 80-82
57．家城洋之．热处理对柑橘速衰病毒、矮缩病毒和碎叶病毒的去毒效应[J]．果树试验报告B，1984，11：71-87
58．何新华，蒋元晖，赵学源，周常勇．应用热处理-茎尖嫁接方法培育无碎叶病毒的柑橘．果树科学，1993，10(2)：95-96
59．张天淼．柑橘碎叶病毒、茎陷衰退病毒脱除技术研究．植物保护学报，1997，24(3)：288
60. Roistacher CN, Calvan and Navarro L. Concepts and procedures for importation of citrus budwood [A]. Proc. Int. Soc. Citriculture, 1977,3: 133-136
61. Navarro L, Jiarez J. Obtencion de plants nucelares libres de virus de diversas variedades de agrios del grupo Navel por cultivo de ovules in vitro [A], An. INTA. 1979,12: 95-113
62. Quak F. Meristem culture and virus-free plant. Plant Cell tissue and organ cuture [M], 1977:598-615
63．高原利雄．简易茎尖嫁接法脱除柑橘病毒[J]．果树试验场报告，1986，8：13-24
64．章文才．果树的病毒病及无病毒良种繁育技术[J]．园艺学文摘，1986，6(1)：1-12
65．张秋胜，舒广平，宋顺华．柑橘茎尖嫁接技术的改进[J]．中国柑橘，1991，20(2)：11-13
66．姜玲，万蜀渊，王映红等．柑橘茎尖嫁接操作方法的改进及研究．华中农业大学学报，1995，14(4)：381-385
67．吴力游，刘学端，萧启明．柑橘茎尖脱毒技术的改进．湖南农学院学报，1992，18：161-166
68．赵学源．1993柑橘病毒病和类似病毒病害研讨会记要．中国柑橘，1993，22(3)：23
69. Murashige T, Tucker DPH. Growth factor requirements of citrus tissure culture. Proc 1st Int Citrus Symp, 1969, 3: 1155-1161
70．何水涛，周厚成，刘崇怀等．A蛋白酶联免疫吸附法检测葡萄卷叶病毒的研究．落叶果树，2001 (2)：1-3
71．邓秀新，章文才．柑橘原生质体融合及遗传转化的研究进展．果树科学，1991，8(1)：51-88
72. Grosser JW, Gmitter Jr FG. Protoplast fusion and citrus improvement. Plant Breed Rev, 1990,8: 339-374
73．吴金虎，陈吉笙，章文才．柑橘胚珠培养及胚状体发生的研究．果树科学，1990，7(1)：19-24
74．邓秀新，章文才．从山金柑试管苗直接诱导胚性愈伤组织的研究．中国柑橘，1988，17(4)：7-9
75．甘霖，章文才，邓秀新．从败育种子诱导柑橘的胚性愈伤组织．华中农业大学学报，1993，12(5)：490-492
76. Moore GA.Factors affecting in vitro embryogenesis from undeveloped ovules of matrue
citrus fruit. J. Amer.Soc.Hort.Sci, 1985,110(1): 66-70
77. Starrantino A, Russo F.Seedlings from undeveloped ovules of ripe fruits of polyembryonic Citrus cultivars. Hort Science, 1980,15(3): 97
78. Li Dongdong, Shi Wei, Deng Xiuxin. Agrobacterium-mediated transformation of embryogenic calluses of Ponkan mandarin and the regeneration of plants containing the chimeric ribonuclease gene. Plant Cell Report, 2002, 21: 153-156
79. Li Dongdong, Shi Wei, Deng Xiuxin. Agrobacterium-mediated transformation of embryogenic calli of Anliucheng and the regeneration of plants containing the chimeric ribonuclease gene. Agricultural Sciences in China. 2003, 2(2): 127-131
80．霍合强，邓秀新．柑橘胚性愈伤组织的诱导、保存和利用．植物生理学通讯，2000，36(2)：181-187
81. Duran-vila N, Gogorcena Y, Ortega N. Morphogenesis and tissue culture of sweet orange (citrus sinensis (L.) Osb): Effect of temperature and photosythetic radiation. Plant Cell, Tissue and Organ Culture, 1992,29: 11-18
82. Songstad DD, Duncan DR, Widholm JM. Effect of 1-aminocyclopropane-1-carboxylic acid (ACC), silver nitrate and morbornadiene on plant regeneration from maize callus cultures. Plant Cell Report, 1988,7: 262-265
83．唐桂香，周伟军．AgNO3对甘蓝型油菜子叶外植体植株再生的影响．中国油料作物学报，2001，23(3)：9-11
84. Biddington WL, Sutherland RA, Robinson HT. Silver nitrate increanes embryo production in anther culture of Brussel Sproots. Ann Bot, 1988, 62: 181-185
85．吴昌银，叶志彪，李汉霞．AgNO3对不结球白菜子叶离体培养的影响．华中农业大学学报，1999，18(1)：80-82
86. Yang Wenyu, Bai Yongyan, Xu Zhihong. Stimulation of shoot regeneration in leaf tissue culture of solanum tuberosum by silver nitrate. Acta Phytophsiologica Sinica, 1998, 24 (1):86-90
87．叶新荣，章文才，万蜀渊．柑橘胚性细胞系的建立．果树科学，1994，11(2)：81-83
88．谭祖国．红江橙原生质体分离培养以及植株再生的研究．江西农业科技，1998，4：28-31
89．李韬．芦柑、雪柑胚性愈伤组织的诱导．福建果树，1997，101：11-12
90．张鹏，傅爱根，王爱国．AgNO3在植物离体培养中的作用及可能的机制．植物生理学通讯，1997，33(5)：376-379
91．艾鹏飞，罗正荣．甜柿试管苗生根条件的研究．华中农业大学学报，2002，21(2)：154-157
92. Wernicke W, Kohlenbach HW. Investigations on the inhibitory effect of agar and function of active carbon in anther culture. Pflanzenphysiol, 1978, 86: 463-472
93. Martineau B, Hanson MR, Ausubel FM. Effect of charcoal and hormones on anther culture
of Petunica and Nicotiana. Pflanzenphysiol, 1981, 102: 109-116
94．卜学贤，陈维伦．活性炭对培养基中植物生长调节物质的吸附作用．植物生理学报，1988，14(4)：401-405
95．沈德绪，王元裕，陈力耕．柑橘遗传育种学．科学技术出版社，1998
96．邓秀新，肖顺元，邓占鳌，章文才．宜昌橙与伏令夏橙种间体细胞杂种．生物工程学报，1993，9(2)：128-131
97．伊华林．试管嫁接提高柑橘三倍体组培苗成苗率试验．中国果树，2000 (4)：28-29
98．骆学海．柑橘黄龙病对热治疗的研究[J]．植物保护学报，1981，8(1)：47-50
99．杜育映，李伦英，蔡明段等．柑橘病毒病脱毒及无病毒苗木繁育的阶段总结．中国柑橘学会2000年年会论文，2000；1-7
100．张天淼编著．柑橘病毒病．中国农业出版社，2001
101．侯义龙，张开军，胡文玉等．逆转录-聚合酶链式反应检测果树RNA病毒．病毒学报，2002，18(1)：71-73
102. Rowhani A, Maningas MA. Development of a detection system for viruses of woody plants based on PCR analysis of immobilized virions [J]. Phytopathology,1995,85(3):347-352
103. Scott SW. The coat proteins and putative movement proteins of isolates of prunus necrotic ring spot viruses from different host species and geographic origins are extensively conserved [J]. Eur J of Plant Pathol, 1998,104:155-161
104. Spiegel S. Improved detection of prunus necrotic ringspot virus by the polymeras chain reaction [J]. Eur J of Plant Pathol, 1996,102: 681-685
105. Nemchinov L, Hadidi A. Sensitive detection of apple chlorotic leaf spot virus from infected apple or peach tissue using RT-PCR, IC-RT-PCR,or multiplex IC-RT-PCR[J].Acta Hort,1995,386: 51-62
106. Kummer J. Sensitive detection of apple stem grooving and apple stem pitting viruses from infected apple trees by RT-PCR [J]. Acta Hort, 1998,472: 97-104
107. Yoshikawa N, Sasaki K, Kato M, Takahashi T. The nucleotide sequence of apple stem grooving capillovirus genome. Virology, 1993, 191: 98-105
108．刘晓玲，唐伟文．应用PCR技术检测柑橘黄龙病病原的研究．华南农业大学学报，1996，17(3)：119-120
109．田亚男，柯穗，柯冲．应用多聚酶链式反应(PCR)技术检测和定量分析柑橘黄龙病病原．植物病理学报，1996，26(3)：243-250
110．胡勤学，林木兰，张春立等．利用RT-PCR扩增和分析柑橘裂皮病类病毒．植物学报，1997，39(7)：613-617
111. Ayllon1 MA, Lopez C, Navas-Castillo J, Garnsey SM, Guerri J, Flores R, Moreno P.Polymorphism of the 5' terminal region of Citrus tristeza virus (CTV) RNA: Incidence of
three sequence types in isolates of different origin and pathogenicity. Arch Virol, 2001, 146:27-40
112．张开军，侯义龙，胡文玉等．采用RT-PCR技术检测苹果病毒．果树学报，2001，18(6)：370-371
113. James D. Factors affecting the detection of apple stem grooving virus by PCR analysis. Acta Hort. 1998, 472: 119-124
114．何水涛，周厚成，张亚冰等．利用RT-PCR检测葡萄扇叶病毒的研究．果树学报，2001，18(3)：186-187
115．贺国安，肖火根，张曙光等．番木瓜环斑病毒株系的分子生物学方法鉴定．植物病理学报，2002，32(1)：21-25
116．孔宝华，蔡红，刘进元等．香石竹斑驳病毒的鉴定和RT-PCR检测．植物保护学报，2002，28(1)：5-8
117．刘永全．马铃薯病毒检测及其抗病毒基因工程研究进展．生物学通报，2001，36(1)：12-13
118. Lu Xiaoyum, Hirata H, Yamaji Y, et al. Random mutagensis in a plant viral genome using a DNA repair-deficient mutator Escherchia coli strain. Journal of Virological Methods, 2001,94: 37-43
119．张才建，王少成．柑橘无病毒苗木繁育技术及应用推广建议．西南园艺，2000，28(3)：25
2．张映南，李润唐，谷洪源．冰糖橙低产劣质的原因和对策．中国南方果树，2001，30(1)：8-10
3．吴德喜，舒广平，宋顺华．柑橘碎叶病毒生物鉴定研究．中国柑橘，1990，1：47-49
4. Navarro L. The citrus variety improvement program in Spain [A]. In. Proe. 7th conf. IOCV. Riverside. 1976: 198-202
5．何天富．柑橘学．中国农业出版社，1999
6．董高峰，李耿光，张兰英等．柑橘无病原体苗培育研究进展．广西植物，2001，21(1)：53-58
7. Robacker CD, Chang CJ. Shoot-tip culture of Muscadine grape to eleiminate pierce's disease bacteria [J]. Hortscience, 1992, 27 (5): 449-450
8．董雅凤，张尊平，张少瑜等．苹果和梨树茎尖培养结合热处理脱病毒研究．北方果树，2002，2：9-1
9．薛光荣，杨振英，洪霓等．茎尖培养等处理脱除梨病毒的技术研究[J]．中国果树，1996，3：9-11
10．洪霓，王国平，张尊平等．梨病毒脱除技术研究[J]．中国果树，1995，4：5-7
11．陈青瑛，陈景耀．龙眼茎尖离体培养及其脱毒效果[J]．植物生理学通讯，1996，32(2)：126-131
12．谢东，许仁林，魏书．草莓生物技术育种研究进展．果树科学，1996，13(3)：195-198
13．林伯年，胡春根，沈德绪等．甜橙成年树侧芽离体繁殖诱导成功研究．园艺学报，1992，19(3)：209-214
14．宋瑞琳，吴如健，陈青瑛等．脱除柑橘碎叶病毒的初步研究．福建省农科院学报，1994，9(1)：57-59
15．何新华，蒋元晖，赵学源．柑橘碎叶病毒的脱毒．云南农业大学学报，1995，10(2)：100-101
16. Murashinge T, Bitters P, Rangan TS, et al. A technique of shoot apex grafting and its utilization towards recovering virus-free citrus clones [J]. Hortscience, 1972,7: 118-119
17. Navarro L, Jucurcza J, Ballester JF, et al. Elimination of some citrus pathogens producing psorosis-like leaf symptoms by shoot-tip grafting in vitro [A]. Riverside: In. Proc. 8th Conf. IOCV, 1980: 162-166
18. Mukhoropagation S, Jaishree kai, Sharma BC, et al. Micropropagation of Darjeeling orange by shoot-tip grafting [J]. Journal of Hortscience. 1997,72(3): 493-499
19．赵学源，蒋元晖，李世菱等．我国柑橘栽培品种的裂皮病的鉴定与脱除[J]．园艺学报，1986，13(2)：91-94
20．蒋元晖，邱柱石，苏维芳等．利用茎尖嫁接脱毒培养无裂皮病的脐血橙[J]．植物保护学报，1983，10(8)：166-168
21．宋瑞琳，吴如健，柯冲．茎尖嫁接脱除柑橘主要病原的研究．植物病理学报报，1999，29(3)：275-279
22．郭春慧，马凤桐，经济林木试管茎尖嫁接的研究现状及展望．西北农业大学学报，1997，25(3)：36-39
23．Reuther W, Batchelor LD, Webber HJ 著 (胥洱，孔焱译)．1985：柑橘业(第二卷)：柑橘生产．农业出版社，北京：260-266
24．霍合强，郝玉金，邓秀新．宽皮柑橘品种的胚性愈伤组织诱导．实验生物学报，1999，32(3)：289-295
25. Navarro L. Development of a program for disease-free citrus budwood in Spain [A]. Proc. Int. Soc. Citriculture, 1981: 70-73
26．高峰，陈杰忠，陈善春等．柑橘未受精胚珠离体培养获得无病毒珠心苗．植物学报，1990，32(7)：505-509
27．谭祖国，万蜀渊．朋娜和纽贺尔脐橙胚珠离体培养获得无病毒珠心苗方法研究．湖北农业科学，1998，4：43-45
28. Wallace JM, Drake RJ. Tatter leaf, a previously undescribed virus effect on citrus. Plant Dis. Reptr, 1962,46: 211-212
29. Wallace JM, Drake RJ. New information on symptom effects and host range of the citrus tatter leaf virus. Plant Dis. Reptr, 1963,47: 352-353
30. Garnsey SM. Detection of tatter leaf virus in Florida. Proc. Fla. State Hort, Soc., 1964, 77: 106-109
31. Da Graca JV. Citrus tatter leaf virus in South African Meyer lemon, Citrus and Subtrop. Fruit J, 1977, 529: 18
32. Miyakawa T. A bud-union abnormality of Satsuma mandarm on Poncirus trifoliate in Japan. Proc.7th Conf IOCV., Riverside, 1976: 125-131
33. Fraser LR.Virus and related diseases of citrus in New South Wales. Dept. Agp. New South Wales, Australia, 1979:52
34. Miyakawa T. Occurrence and distribution of tatter leaf-citrange stunt virus and its effects on Japanese citrus. Proc.8th Conf IOCV., Riverside, 1980: 220-224
35. 苏鸿基．Occurrence and distribution of tatter leaf-citrange stunt complex on Taiwanese citrus. NTU Phytopathlogist and Entomologist, 1984, 11: 42-48
36．张天淼，梁仙友．柑橘碎叶病毒在黄岩本地早、槾桔上的发现．中国柑橘，1987，1：27-28
37．赵学源，蒋元晖，李世菱等．柑橘碎叶病的初步鉴定．中国柑橘，1987，1：28
38．张天淼，梁仙友，陈作义等．柑橘碎叶病的发生与初步鉴定．植物病理学报，1988，18(2)：
79-84
39．何新华，蒋元晖，赵学源等．柑橘碎叶病的分布鉴定．植物保护，1991，17(1)：6-7
40．宋顺华，舒广平，刘庚峰等．湖南省柑橘资源病毒类病害鉴定初报．中国柑橘，1995，3：36-37
41. Nishio T, Kawai A, Takahashi T, et al. Purification and properties of citrus tatter leaf virus. Annals of the Phytopathological Sciety of Japan, 1989,55(2): 254-258
42. Yoshikawa N, Imaizumi M, Takahashi T, et al. Striking similarities between the nucleotide sequence and genome organization of citrus tatter leaf and apple stem grooving capillovirus. Journal of general virology, 1993,74(12): 2743-2747
43. Ohira K, Namba S, Rozanov M, el al. Complete sequence of an infectious full-length cDNA clone of citrus tatter leaf capillovirus: Comparative sequence analysis of capillovirus genomes. Journal of general virology, 1995,76(9): 2305-2309
44. Magome H, Yoshikawa N, Takahashi T. Molecular variability of the genomes of capilloviruses from apple, Japanese pear, European pear, and citrus trees. Phytopathol, 1996,87: 389-396
45. Inouye N, Maeda T, Mitsuhata K. Citrus tatter left virus isolated from lily. Annals of the Phytopathological Sciety of Japan, 1979,45: 712-720
46. Ohira K, Ito T, Kawai A, Namba S, Kusumi T, Tsuchizaki T. Nucleotide sequence of the 3'-therminal region of citrus tatter leaf virus RNA. Virus genes, 1994, 8(2): 169-172
47. Garnsey SM. Virus in Florida's Meyer lemon trees and their effects on other citrus.. Proc. Florida State Hort. Soc., 1970, 83: 66-71
48. Yarwood CE. Mechanical transmission of alatent lemon virus. Phytopathology, 1963,53:1145
49. Fulton RW. Mechanical transmission of tatter leaf virus from cowpea to citrus. Phytopathology, 1966, 56: 575
50. Roistacher CN. Tatter leaf and infectious variegation viruses on knife blades and its prevention. Proc. 8th Conf. IOCV. Riverside, 1980: 225-229
51. Semancik JS. Partial purification of a mechanical transmissible virus associated with tatter leaf of citrus. Phytopathology, 1965,55: 1354-1358
52．井上成信．Citrus tatter leaf virus．日植病报，1979，45：712-720
53．谢佩华，金显忠，何献武．浙江南部柑橘病毒病和类似病毒病鉴定．中国柑橘，1992，21(4)：13-17
54．谢佩华，金显忠，何献武．浙江南部柑橘碎叶病的调查和鉴定．植物病理学报，1992，22(3)：195-197
55．陈集双，张天淼，周雪平等．柑橘碎叶病毒研究．微生物学报，1995，35(3)：173-180
56. Garnsey SM. Indexing for tatter leaf with cowpea. USDA Agr. Handbook, Washington, D.C., 1968: 80-82
57．家城洋之．热处理对柑橘速衰病毒、矮缩病毒和碎叶病毒的去毒效应[J]．果树试验报告B，1984，11：71-87
58．何新华，蒋元晖，赵学源，周常勇．应用热处理-茎尖嫁接方法培育无碎叶病毒的柑橘．果树科学，1993，10(2)：95-96
59．张天淼．柑橘碎叶病毒、茎陷衰退病毒脱除技术研究．植物保护学报，1997，24(3)：288
60. Roistacher CN, Calvan and Navarro L. Concepts and procedures for importation of citrus budwood [A]. Proc. Int. Soc. Citriculture, 1977,3: 133-136
61. Navarro L, Jiarez J. Obtencion de plants nucelares libres de virus de diversas variedades de agrios del grupo Navel por cultivo de ovules in vitro [A], An. INTA. 1979,12: 95-113
62. Quak F. Meristem culture and virus-free plant. Plant Cell tissue and organ cuture [M], 1977:598-615
63．高原利雄．简易茎尖嫁接法脱除柑橘病毒[J]．果树试验场报告，1986，8：13-24
64．章文才．果树的病毒病及无病毒良种繁育技术[J]．园艺学文摘，1986，6(1)：1-12
65．张秋胜，舒广平，宋顺华．柑橘茎尖嫁接技术的改进[J]．中国柑橘，1991，20(2)：11-13
66．姜玲，万蜀渊，王映红等．柑橘茎尖嫁接操作方法的改进及研究．华中农业大学学报，1995，14(4)：381-385
67．吴力游，刘学端，萧启明．柑橘茎尖脱毒技术的改进．湖南农学院学报，1992，18：161-166
68．赵学源．1993柑橘病毒病和类似病毒病害研讨会记要．中国柑橘，1993，22(3)：23
69. Murashige T, Tucker DPH. Growth factor requirements of citrus tissure culture. Proc 1st Int Citrus Symp, 1969, 3: 1155-1161
70．何水涛，周厚成，刘崇怀等．A蛋白酶联免疫吸附法检测葡萄卷叶病毒的研究．落叶果树，2001 (2)：1-3
71．邓秀新，章文才．柑橘原生质体融合及遗传转化的研究进展．果树科学，1991，8(1)：51-88
72. Grosser JW, Gmitter Jr FG. Protoplast fusion and citrus improvement. Plant Breed Rev, 1990,8: 339-374
73．吴金虎，陈吉笙，章文才．柑橘胚珠培养及胚状体发生的研究．果树科学，1990，7(1)：19-24
74．邓秀新，章文才．从山金柑试管苗直接诱导胚性愈伤组织的研究．中国柑橘，1988，17(4)：7-9
75．甘霖，章文才，邓秀新．从败育种子诱导柑橘的胚性愈伤组织．华中农业大学学报，1993，12(5)：490-492
76. Moore GA.Factors affecting in vitro embryogenesis from undeveloped ovules of matrue
citrus fruit. J. Amer.Soc.Hort.Sci, 1985,110(1): 66-70
77. Starrantino A, Russo F.Seedlings from undeveloped ovules of ripe fruits of polyembryonic Citrus cultivars. Hort Science, 1980,15(3): 97
78. Li Dongdong, Shi Wei, Deng Xiuxin. Agrobacterium-mediated transformation of embryogenic calluses of Ponkan mandarin and the regeneration of plants containing the chimeric ribonuclease gene. Plant Cell Report, 2002, 21: 153-156
79. Li Dongdong, Shi Wei, Deng Xiuxin. Agrobacterium-mediated transformation of embryogenic calli of Anliucheng and the regeneration of plants containing the chimeric ribonuclease gene. Agricultural Sciences in China. 2003, 2(2): 127-131
80．霍合强，邓秀新．柑橘胚性愈伤组织的诱导、保存和利用．植物生理学通讯，2000，36(2)：181-187
81. Duran-vila N, Gogorcena Y, Ortega N. Morphogenesis and tissue culture of sweet orange (citrus sinensis (L.) Osb): Effect of temperature and photosythetic radiation. Plant Cell, Tissue and Organ Culture, 1992,29: 11-18
82. Songstad DD, Duncan DR, Widholm JM. Effect of 1-aminocyclopropane-1-carboxylic acid (ACC), silver nitrate and morbornadiene on plant regeneration from maize callus cultures. Plant Cell Report, 1988,7: 262-265
83．唐桂香，周伟军．AgNO3对甘蓝型油菜子叶外植体植株再生的影响．中国油料作物学报，2001，23(3)：9-11
84. Biddington WL, Sutherland RA, Robinson HT. Silver nitrate increanes embryo production in anther culture of Brussel Sproots. Ann Bot, 1988, 62: 181-185
85．吴昌银，叶志彪，李汉霞．AgNO3对不结球白菜子叶离体培养的影响．华中农业大学学报，1999，18(1)：80-82
86. Yang Wenyu, Bai Yongyan, Xu Zhihong. Stimulation of shoot regeneration in leaf tissue culture of solanum tuberosum by silver nitrate. Acta Phytophsiologica Sinica, 1998, 24 (1):86-90
87．叶新荣，章文才，万蜀渊．柑橘胚性细胞系的建立．果树科学，1994，11(2)：81-83
88．谭祖国．红江橙原生质体分离培养以及植株再生的研究．江西农业科技，1998，4：28-31
89．李韬．芦柑、雪柑胚性愈伤组织的诱导．福建果树，1997，101：11-12
90．张鹏，傅爱根，王爱国．AgNO3在植物离体培养中的作用及可能的机制．植物生理学通讯，1997，33(5)：376-379
91．艾鹏飞，罗正荣．甜柿试管苗生根条件的研究．华中农业大学学报，2002，21(2)：154-157
92. Wernicke W, Kohlenbach HW. Investigations on the inhibitory effect of agar and function of active carbon in anther culture. Pflanzenphysiol, 1978, 86: 463-472
93. Martineau B, Hanson MR, Ausubel FM. Effect of charcoal and hormones on anther culture
of Petunica and Nicotiana. Pflanzenphysiol, 1981, 102: 109-116
94．卜学贤，陈维伦．活性炭对培养基中植物生长调节物质的吸附作用．植物生理学报，1988，14(4)：401-405
95．沈德绪，王元裕，陈力耕．柑橘遗传育种学．科学技术出版社，1998
96．邓秀新，肖顺元，邓占鳌，章文才．宜昌橙与伏令夏橙种间体细胞杂种．生物工程学报，1993，9(2)：128-131
97．伊华林．试管嫁接提高柑橘三倍体组培苗成苗率试验．中国果树，2000 (4)：28-29
98．骆学海．柑橘黄龙病对热治疗的研究[J]．植物保护学报，1981，8(1)：47-50
99．杜育映，李伦英，蔡明段等．柑橘病毒病脱毒及无病毒苗木繁育的阶段总结．中国柑橘学会2000年年会论文，2000；1-7
100．张天淼编著．柑橘病毒病．中国农业出版社，2001
101．侯义龙，张开军，胡文玉等．逆转录-聚合酶链式反应检测果树RNA病毒．病毒学报，2002，18(1)：71-73
102. Rowhani A, Maningas MA. Development of a detection system for viruses of woody plants based on PCR analysis of immobilized virions [J]. Phytopathology,1995,85(3):347-352
103. Scott SW. The coat proteins and putative movement proteins of isolates of prunus necrotic ring spot viruses from different host species and geographic origins are extensively conserved [J]. Eur J of Plant Pathol, 1998,104:155-161
104. Spiegel S. Improved detection of prunus necrotic ringspot virus by the polymeras chain reaction [J]. Eur J of Plant Pathol, 1996,102: 681-685
105. Nemchinov L, Hadidi A. Sensitive detection of apple chlorotic leaf spot virus from infected apple or peach tissue using RT-PCR, IC-RT-PCR,or multiplex IC-RT-PCR[J].Acta Hort,1995,386: 51-62
106. Kummer J. Sensitive detection of apple stem grooving and apple stem pitting viruses from infected apple trees by RT-PCR [J]. Acta Hort, 1998,472: 97-104
107. Yoshikawa N, Sasaki K, Kato M, Takahashi T. The nucleotide sequence of apple stem grooving capillovirus genome. Virology, 1993, 191: 98-105
108．刘晓玲，唐伟文．应用PCR技术检测柑橘黄龙病病原的研究．华南农业大学学报，1996，17(3)：119-120
109．田亚男，柯穗，柯冲．应用多聚酶链式反应(PCR)技术检测和定量分析柑橘黄龙病病原．植物病理学报，1996，26(3)：243-250
110．胡勤学，林木兰，张春立等．利用RT-PCR扩增和分析柑橘裂皮病类病毒．植物学报，1997，39(7)：613-617
111. Ayllon1 MA, Lopez C, Navas-Castillo J, Garnsey SM, Guerri J, Flores R, Moreno P.Polymorphism of the 5' terminal region of Citrus tristeza virus (CTV) RNA: Incidence of
three sequence types in isolates of different origin and pathogenicity. Arch Virol, 2001, 146:27-40
112．张开军，侯义龙，胡文玉等．采用RT-PCR技术检测苹果病毒．果树学报，2001，18(6)：370-371
113. James D. Factors affecting the detection of apple stem grooving virus by PCR analysis. Acta Hort. 1998, 472: 119-124
114．何水涛，周厚成，张亚冰等．利用RT-PCR检测葡萄扇叶病毒的研究．果树学报，2001，18(3)：186-187
115．贺国安，肖火根，张曙光等．番木瓜环斑病毒株系的分子生物学方法鉴定．植物病理学报，2002，32(1)：21-25
116．孔宝华，蔡红，刘进元等．香石竹斑驳病毒的鉴定和RT-PCR检测．植物保护学报，2002，28(1)：5-8
117．刘永全．马铃薯病毒检测及其抗病毒基因工程研究进展．生物学通报，2001，36(1)：12-13
118. Lu Xiaoyum, Hirata H, Yamaji Y, et al. Random mutagensis in a plant viral genome using a DNA repair-deficient mutator Escherchia coli strain. Journal of Virological Methods, 2001,94: 37-43
119．张才建，王少成．柑橘无病毒苗木繁育技术及应用推广建议．西南园艺，2000，28(3)：25