摘要
以“二细一粗”银条作为研究试材，对银条茎尖分生组织的培养条件、不同处理对提高茎尖再生植株脱毒率的效果进行了研究，并利用不同外植体探讨了银条再生快繁的适宜途径。目的是获得脱毒种株并进性大量、快速的繁殖，以解决生产中银条由于结籽率低，且难以培养成苗，长期以根状茎进行无性繁殖所造成的病毒累积的问题，并为进一步进行银条的营养利用、生理代谢、生物育种研究奠定基础。试验结果表明：
银条的离体培养以MS作为基本培养基最为适宜，再生苗生长快速，植株健壮。茎尖分生组织培养在MS+6-BA 0.5 mg/L+NAA 0.1mg/L+蔗糖4％+琼脂6.5g/L上，成苗率可达74.1％，最适宜茎尖的培养；经指示植物法、电镜观察检测，再生植株的脱毒率随茎尖切割长度的减小而提高，但过小的茎尖操作、培养困难，培养周期长，成苗率低。0.5mm大小的茎尖成苗率与脱毒率相对较高，分别为71.4％、30.0％，较为适合培养；茎尖的重复切割培养与接种植株进行预热处理均有显著提高脱毒率的作用，但同时也降低了成苗率。植株在42℃高温下，经15d热处理后，0.5mm大小茎尖切割培养成苗率与脱毒率分别达到50.0％、57.1％，重复切割培养3次后则分别为28.6％、87.5％。为减小操作难度，缩短周期，银条植株经42℃，15d热处理后，切取0.5mm大小茎尖进行一次培养较为适宜。
不同生育时期的外植体愈伤组织的诱导能力不同，刚展开的幼嫩叶片、粗壮的幼嫩茎段及花蕾长度在0.5 cm左右时的花药最适宜愈伤的诱导，诱导率最高。幼嫩茎段与花药在MS+6-BA 2.0mg/L+NAA 1.5mg/L+IAA 0.5mg/L培养基上，幼嫩叶片在MS+6-BA 2.5mg/L+NAA 1.5mg/L+IAA 1.0mg/L上，愈伤组织诱导效果最好，愈伤生长快速，颜色鲜艳，继代培养不褐化。愈伤组织不定芽的分化中，花药诱导形成的愈伤难以形成不定芽；叶片与茎段诱导的愈伤可以在MS+6-BA 1.0 mg/L+NAA 0.8mg/L+GA_3 2.0mg/L上分化形成不定芽，诱导分化率分别为71.4％、78.6％。
对幼嫩叶片、幼嫩茎段与茎尖进行不定芽的直接诱导与增殖，发现银条叶片组织未能再生；茎尖、茎段组织直接诱导较大不定芽的再生率较低，平均每个外植体最多可诱导不定芽5～7个。而通过茎段先诱导出不定芽芽团，再进行芽的伸长这一途径，每个外植体诱导的不定芽数平均可提高到14.3个，且芽团经5次继代增殖再生能力没有衰退表现，再生植株生产潜力非常巨大，是一条较优的再生途径。茎段不定芽芽
团诱导培养基为添加6一BA 5.Om留L的MS培养基;增殖培养基为MS+6一BA 4.5m叭
+GA3 1 .Om留L;不定芽伸长培养基以MS+6一BA 1.5 mg几+N AA 0.4m叭+GA30.5
m叭较好。
对不同温度、光照条件、以及蔗糖、6一BA、CCC浓度对离体培养条件下银条根
状茎诱导的影响进行研究表明:全黑暗的培养条件是银条根状茎诱导的必需因子，短
日照(8扮d)与长日照(14扮d)均促使甸旬茎发育成侧枝;高浓度蔗糖有利于根状茎的
诱导，单独使用低浓度的蔗糖(3%)不能诱导根状茎产生而形成黄化的侧枝，再添加
6一BA 5.Om留L十CCC 50Om留L后虽然可以形成根状茎，但单株产量很低。6一BA与CCC
对根状茎形成具有明显的促进作用，能增加根状茎数，提高单株产量。银条根状茎的
诱导温度以20℃为宜，提高温度会降低根状茎数。
再生苗在1/2 Ms+N AA o.sm留L十活性炭0.3%上生根效果最好，将生根的完整植
株移栽到消过毒的1蛙石:1珍珠岩的基质上，成活率高达95.8%。
The culturation of the tip meristem of many flower Betony which was about 0.3-0.5 mm in length with 1 or 2 primordia leaves had been investigated on different culture medium. At the same time, the effect of different techniques on the virus-free rate of regeneration planlets by tip meristem cultured in vitro, the regeneration and rapid propagation system were studied, too. The results showed:
As the basic medium for many flower Betony culturation in vitro, MS was the best in all media include White, MA, B5 and Ne. The optimal medium for the tip meristem culture was MS+6-BA 0.5 mg/L+NAA 0.1 mg/L+sucrose 4%, on which 74.1% plantlet ratio was produced. The different treatment to produce virus-free plantlet by the tip meristem culture was studied. The results showed that the size of excised shoot tip was negative correlated with the virus-free rate of plantlet. Too smaller shoot tip make operation and regeneration difficult. The attempt of using 0.5 mm length shoot tip was successed with 71.4% plantlet rate and 30.0% virus-free rate produced. The virus-free rate could be increased markedly by using the tip meristem culturation technique together with the techniques such as cut of stem tip more than one times, higher temperature pre-treatment of the planr material. However the survival rate might be degraded. The suitable method to obtain virus-free plantlet was to pre-treat the plants at 42℃ for 15 d
ays firstly, and then to cut the shoot tip(0.5 mm long) one times. 50.0% survival rate and 57.1% virus-free rate were gained with this method.
Explants in different development period showed differences in callus induction.The younger and tender the explants were, the better the callus induction would be. Leaf discs, stem segments and anther could all be induced to produce callus, and the induction rate could reach 100%. However, only 61.2% induction rate was got from anther. The callus from variant explants showed differences in buds differentiation. Adventitious buds can be obtained from leaf and stem callus ,but not from anther callus.The best medium for leaf discs to induce callus was MS+6-BA 2.5 mg/L+NAA 1.5 mg/L+IAA 1.0 mg/L. It was
suitable to induce callus from stem segments and anther on MS+6-BA 2.0 mg/L+NAA 1.5 mg/L+IAA 0.5 mg/L. The best adventitious buds induction medium for leaf and stem callus was MS+6-BA 1.0 mg/L+NAA 0.8 mg/L+GA3 2.0mg/L, with 71.4%, 78.6% induction rate, respectively.
The stem apex, leaf disc and stem segments as explants were used to study the effects of different hormones treatments on the adventitious bud induction and propagation.The results showed that stem apex and stem segments had low reproducing rate with only got 5-7 buds per explant in longer buds induction, while leaf disc had not showed ability to reproduce.The best way to get more reproducing plantlet (14.3 per explant)was to lengthen the bud from the bud lump which was gotten from the induced stem segments on MS medium supplemented with 6-BA 5.0 mg/L. The bud lump can be propagated for a long time and the reproducing ability would not decreased on MS medium with 6-BA 4.5 mg/L and GA3 1.0 mg/L. The better medium for bud elongation is MS+6-BA 1.5 mg/L +NAA 0.4 mg/L +GA3 0.5 mg/L.
The effects of the photoperiod, temperature, sucrose, 6-BA and CCC on in vitro stolon induction were studied. The results showed that dark cultural condition is essential to stolon induction. Short photoperiod (8h/d) and long photoperiod (14h/d) would make stolons grow into branches. Lower sucrose concentration (3%) inhibited stolon induction and only got feeble branches were produced. If 6-BA 5mg/l and CCC 500mg/L be supplemented to the medium, smaller stolon could be formed. This procession was apparently promoted by higher sucrose concentration. 6-BA and CCC were not essential to stolon induction, but they could significantly accelerate the development of stolon and enhance the average output per plantlet. The situable temperature was 20℃. Higher temperature may degrade the stolon number.
1/2MS medium with 0.5 mg/L NAA and 3% activit
引文
[1] 季宇彬．中药材有效成分药理应用[M]．黑龙江：科技出版社，1994．441～442
[2] 程智慧，孟焕文，周文安，等．地灵高产栽培．西北园艺，2003，(1)：30～32
[3] 胡群宝．脱毒马铃薯繁育体系的研究与应用．马铃薯杂，1999，13(2)：96～99．
[4] 王国平．草莓病毒种类鉴定及培育无病毒种苗的技术研究．中国农业科学，1981，23(4)：43～49
[5] 阎贤伟．甜樱桃茎尖培养和快速繁殖研究．园艺学报，1990，17(4)：275～279
[6] 方中达．植病研究方法．北京：中国农业出版社，1998，250～305
[7] 张仲凯．李毅．云南植物病毒．北京：科学出版社，2001，1～9
[8] 陈君帜，李青．李属植物脱毒技术及病毒检测研究进展．北京林业大学学报，2001，23(5)：71～74．
[9] 郑金城．苹果无病毒苗木繁育技术．北京：中国农业出版社，1995
[10] 洪霓，王国平．苹果腿绿叶斑病毒生理学及生化特性研究．植物病理学报，1999，29(1)：77～81．
[11] 傅润民．果树瓜类生物工程育种，北京：农业出版社．1992
[12] 覃兰英，李青，邓氏秀，等．核果类病毒识别鉴定及脱毒技术．北京农业科学，1997，(10)：23～28
[13] 胡向武，陈士超，张林普．黄瓜花叶病毒的侵染扩散及病变细胞的电镜观察．安徽师范大学学报，1998，(4)：354～357
[14] 李向东，郭兴启，竺晓平，等．西瓜花叶病毒二号侵染西瓜后引起的显微变化．西北农业大学学报，1998，(7)：101．
[15] 粱训生，植物病毒血清学技术．北京：农业出版社．1985
[16] 张建军．一种快速简便的植物病毒检测方法．植物检疫，1998，(1)：222～223
[17] 尚佑芬．国内外植物病毒研究发展概况．山东农业科学，1998，(1)：51～53．
[18] 林清，吕华中．重庆市辣椒TMV和CMV的鉴定．中国蔬菜，2001，(3)：13～15
[19] 王海河，高文臣，魏宁生．渭北烟区黄瓜花叶病毒酶联免疫检测方法．西北农业大学学报，1999，(4)：7～11
[20] 吴雅琴，陈霜莹，王文慧，等．三种ELISA方法检测苹果褪绿叶斑病毒和苹果茎沟病毒的比较．植物保护，1998，25(3)：245～248
[21] 陈剑平．胶体金免疫电镜技术检测和鉴定病汁液中不同形态的植物病毒．植物病理学报，1993，(3)：169～174
[22] 徐平东，李梅．免疫胶体金技术及其在植物病毒研究中的应用．植物检疫，1993，(1)：26～28
[23] 魏梅生．植物无性种质材料中病毒的检测与治疗．世界农业，1995，(10)：30～31
[24] 相宁，周雪荣，孙彤，等．RT-PCR检测烟草环斑病毒的研究．植物检疫，1995，(6)：337～339
[25] 肖荣堂．聚合酶链反应及其在植物病毒鉴定中的应用，植物检疫，1993，(4)：320～322
[26] 蔡伟和，洪益国，黄福新．中国南瓜曲叶双生病毒的生物学、血清学和分子杂交的研究．中国病毒学报，1994，(3)：222～224．
[27] 裘维蕃．植物病毒学．北京：农业出版社．1984，426～442．
[28] 郭明．植物病毒抑制物的研究进展．甘肃农业科技，1992，(7)：28～29
[29] 江山，韩熹莱．植物病毒化学防治的研究进展．中国病毒学，1995，10(1)：1～7
[30] 辛淑英．甘薯分生组织培养．中国甘薯，1989，(3)：16～18
[31] 李昌华，李小川．大蒜茎尖脱毒技术及组织培养研究．华北农学院学报，1995，10(3)：20～25
[32] 杨崇良，尚佑芬，赵九华，等．甘薯脱毒技术及增产效果研究．植物保护学报，1998，25(1)：51～55
[33] 许志刚．茎尖培养脱毒苗防治宝塔菜病毒病的初步研究，南京农业大学学报，1989，12(3)：30
[34] 温孚江．植物抗病毒病基因工程的研究现状．山东农业大学学报，1995，26(1)：126～130
[35] 冯英，薛庆中．生姜脱菌快繁研究进展．植物学报，2002，19(4)：439～443
[36] 周俊，肖英，程秉铨．辣椒茎尖培养脱毒研究．西北农业学报，1996，5(3)：23～26
[37] 李俊明．植物组织培养教程．北京；中国农业大学出版社，1992
[38] 吴力游，萧启明，刘学端，等．湖南省柑桔良种的脱毒与检测研究．湖南农学院学报，1993，19(3)：241～247
[39] 何水涛，刘沛镇，韩礼星，等．苹果茎顶培养脱毒技术的研究．河南农业科学，1996，11，25～28
[40] 黎定军，张宝玺，赵开军，等，辣椒子叶高效植株再生体系的建立．园艺学报，2002，29(1)：25～29
[41] 李云，冯慧，田砚亭．葡萄遗传转化研究进展．生物工程进展，2000，(3)：49～52．
[42] 谭文澄，戴策刚，等，观赏植物与植物组培技术．北京：中国农业出版社．1991
[43] 王红芳，贾春兰．园艺植物组培苗工厂化生产(三)——优良品种的快速繁殖，农村实用工程技术．1996，(1)：12．
[44] 王玉珍．植物组培快繁技术与产业化研究．林业科技，1997，22(6)：12～13
[45] 张孝棋，魏振承，等，西瓜离体培养技术的研究．中国西瓜甜瓜，1996，(2)：13～16
[46] 韩碧文，刘淑兰，植物离体体细胞胚胎发生．植物生理学通讯，1988，(1)：9～15
[47] 范双喜，植物组培中胚状体诱导的研究．北京农学院学报，1993，8(2)：142～145
[48] 潭文澄，策刚，等．观赏植物与植物组培技术，北京；中国林业出版社．1991
[49] 张英，林贵美，等．芦荟的组织培养和快速繁殖．广西农业科学，1999，(2)：103～105
[50] 周根余，丁洪峰，等．芦荟的无性快繁．园艺学报，1999，26(6)：410～411
[51] 贾春美，忘纪方，杨建荣，等．葡萄试管苗繁殖技术研究．中国果树，1992(1)：25
[52] 牛至清，花药和花粉培养．罗士伟，许智宝主编，经济植物组织培养．北京：北京科学出版社，1988，37
[53] 王敬驹，匡柏健，罾慧．提高甘蔗组织培养效率和研究，植物学通报，1983，(2)：17
[54] 费开韦，薛光荣．国外温带落叶果树组织培养研究综述．中国果树，1980(增刊)：20
[55] 刘丽君等．切花菊组织培养及移栽．园艺作物
[56] E 拉宾诺维奇，高温奇．光合作用，中国科学院植物研究所光合组译．北京：科学出版社，1973，59～60
[57] 杨增海．园艺植物组织培养学．北京：农业出版社，1987，1～3
[58] 张方丕，等．植物组织培养在繁殖上的应用．上海：上海教育出版社，1985，11～13
[59] 桂耀林，徐廷玉，顾淑荣，等．黄连体细胞胚胎发生的研究．植物学报，1989，31(12)：923～927
[60] 肖关丽，杨清辉．植物组织培养过程中内源激素研究进展．云南农业大学学报，2001，16(2)：136～138
[61] 丁永前．组培苗微生态环境中CO_2控制的研究：[硕士论文]．南京：南京农业大学，2000．
[62] 夏铭，吴缃云，张丽梅．红豆杉组织培养中褐变问题的研究生物技术．1996，6(3)18～20
[63] 王秀敏，吴云锋，成巨龙，等．陕西省咸阳市、商州市烟草病毒病毒原鉴定初报．2003，12(1)：139～141．
[64] 赵玖华，等．甘薯脱毒苗的检测研究．山东农业科学，1995，(5)：15～18．
[65] 李卫，董文，赵东力，等．TDZ诱导甘露子茎段高频再生．西北植物学报，2002，22(4)：965～969．
[66] 师素云，练兴明，薛启汗，等．薄荷离体培养愈伤组织诱导与植株分化．江苏农业科学，2000，6：27～28．
[67] 辛淑英．甘薯分生组织培养．中国甘薯．1989，(3)：16～18
[68] 陈玉霞，张朝成，周天虹．甘薯茎尖脱毒培养技术研究．湖北农业科学，1996，2：22～25．
[69] 李昌华，李小川．大蒜茎尖脱毒技术及组织培养研究．华北农学院学报，1995，10(3)：20～25
[70] 许志刚，茎尖培养脱毒苗防治宝塔菜病毒病的初步研究，南京农业大学学报，1989，12(3)：30．
[71] 王纪方，金波，高秀云，等．蔬菜组织培养．上海：上海科技出版社．1983．
[72] 陈火英，张建华，俞俊棠，等．番茄离体培养过程中器官发生的细胞组织学观察．武汉植物学研究，2001，19(2)：91～95．
[73] 李灿辉，王军，管朝旭．等．离体培养条件下植物生长物质对马铃薯块茎形成的影响．马铃薯杂志，1998，12(2)：67～73．
[74] Abou-mandour A A, W Hartung. The effect of abscisic acid and increased osmotic potential of the media on growth and root regeeration of zea mays callus. Plant Physioll, 1986,22:139～145.
[75] Baranwal V K, Vermb H N. Characteristics of a virus inhibitor from the leaf extract of Celosia cristata. Plant Pathology, 1997,46(4):523～529.
[76] Boggetti. B., Jasik. J. and Mantell. S. In vitro multiplicastion of cashew (Anacardium Oceidentale. L.) Using shoot node explants of glasshouse raised plants. Plant cell Report. 1999. 18, 456～461
[77] Cenger, B. V. Cloning agriculture Plants via in vitro technigues, CRC Press Inc. USA. 1981.
[78] Cronauerss. Reinition of vegetable growth from aseptically. Cultured terminal flonal apex of banana. Amer J. Bot. 1985, 72(1):1598
[79] Deogratias J M, Disba F, Lutz A. Elimination of PDV, NRSV and CLSV from sweet cherries by mean of in vitro culture. Acta Horticulturae, 1988,235:189
[80] Deogratias J M, Lutz A, Dosba F. In vitro micrografting of shoot tips from juvenile and adult Prunus avium and Prunus persica(L.) Batsch to produce vires-free plants. Acta Horticulturae, 1986,193:139～145
[81] Deogratias J M, Disba F, Lutz A. Eradication of prune dwarf virus, Prunus necrotic ringspot virus and apple chlorotic leaf spot virus in sweet cherries by a combination of chemotherapy, thermotherapoy, and in vitro culture. Canadian Journal of Plant Pathology, 1989,11:337～342.
[82] Desjardins Y. Factors affecting CO_2 fixation in strving to optimize photoautotropby in micropropagated plantlets. Plant Tissue Culture and Biotechnology, 1995,1(1):13～25.
[83] Devi, Borthakur B, Deka P C. Clonal propagation of denelrobium moschatum and cymbidium aloifolium through
shoot-tip culture. Journal of the Orchid Society of India, 1997,11 (1-2) : 19．
[84] Ewing E E. Induction of tuberization in potato. In: Vayda M E and Park W D eds. The Molecular and cellular biology of the potato. 1990,25-41．
[85] Gibson RW.Pickett JA.Wild potato repels aphids by release of aphid alarm pheromone.Nature, 1983,302:608．
[86] Gclla R, Errla P. Application of in vitro therapy for ilarvirus elimination in three Prunus species. Journal of Phytopathology, 1998,146(8/9) :445-449．
[87] Gabryszewska-E et al The influence of light quality on the shoot proliferation and rooting of Gerbera gamesonii in vitro Acta Agrobotanica . 1995,48(2) : 105-111
[88] Hayashik, Nagmatsut, ItoM, et al. Acteoside:a component of Stachys sieboldii Miq. , may be a promising antinephritic agent(3) : effect of acteoside on expression of intercellular adhesion molecule-1 in experimental nephritic glomeruli in rats and cultured endothelial cells[J]. PhJPharmacol, 1996, 70 (2) : 157-168
[89] Hosoki-T,Yasufuku-T,Hayashi-M,et al. In vitro mass-propagation of Chinese artichoke(stachys sieboldii Miq.). Acta-Horticulturae, 1992,No.319, 149-152．
[90] Hollins M, Stone O.M.,1964, Ann. Appl. Biool. 53: 103-108．
[91] Hakkaart F.A. Quak F. 1964,Neth. J. Plant Path. 70:154-157
[92] Hazel Y.W.,Choongsir K,Harry E.S.Vessel volume.Gelling Ageent ans Basal Salts effect pH and gel stnength of autoclaved tissue culture media.Hortscience,1994,29(6) :683-685
[93] Jackson L D and L Willmitzer. Jasomonic acid spraying does not induce tuberization in short-day-requiring potato species kept in non-inducing conditions. Planta, 1994, 194:155-159．
[94] Kohler G.Milstein C. Continuous culoures of fused cells secreting antibody of predefined specificity. Nature, 1 975,256;490-496．
[95] Kaozai T, Kushihashi S. Effects of the difference betweenphotoperiod and dark period temperatures and photosynthetic photo flux density on the shoot length and growth of potato plantlets in vitro. J. Jpn.Soc.Hort.Sci., 1992,61 (1) :93-98．
[96] Lawrence F J et al. Effect of virus infection on the strawberry cultivar. Northwest Plant Disease Rep52
[97] Ladyman.T.A.R. and B.Girard.Cucumber Somatic embrgo development on various gelling agents and carbohydrate sources.Hortscience 27:164-165
[98] Lee.N,H.Y.Wetzstein,and H.E.sommer.1986:The effect of ager vs.Liquid medium on rooting in tissue-cultured Sweetgum-Hortscience 21:317-320
[99] Murashige T. et al. 1972, Hortscience 7: 118-119．
[100] Msikita W, Skirvin RM, Juvick JA and Splittstoesser WE. 1988． In vitro regeneration and flowering of cucumber cultivars and lines cultured from excised seed. Cucurbit Genet. Coop. Rep. 11:5-7
[101] Mudge KW. Micropropagation of muge pine from embrgonic and seedling explants. Hortsci, 1986, 21(2) :298
[102] Mohsen K.H Ebrahim.Ibrahim A, Influence of medium solidification and PH value on in vitro propagation of marauta.leuconeura CV.Kerchoviana Science Horticulture 2000,86:211-221
[103] Navarro L, Llacer G, Cambra M, et al. Shoot-tip grafting in vitro for elimination of viruses in peach plants(Prunus persica L. Batsch). Acta Horticulturae, 1982, 130: 185-192
[104] Nemchinov L.Hadidi A,candtesse A,et al. Sensitive detection of apple chlorotic leafspot virus from infected apple or peach tissue using RT-PCR,IC-RT-PCR,or multiplex IC-RT-PCR.Acta Horticulturae,1995,386:51-62．
[105] Owens.L.D. and C.A. wozniar.Measurement and effects of gel.matrix potential and expressibility on production of morpHogenic callus by cultures sugar beet leaf discs.Plant Cell Tissue Organ Cult.26: 127-133
[106] Pankai sharma et al. Genotype, explant and position effects on organogenesis and somatic embryogenesis in eggplant. Journal of Experimetal Botany. 1995,46(282) : 135-141．
[107] Punja, Z.K. Abbas N. Sarmento G.G. and Tang, F.A. Influence of explant source, growth regulator regime and genotype. Plant Cell, Tissue and Organ Culture.2 1:93-102
[108] Pvrohit, S.D. singhvi, A. Microprapogation of Achras sapota throgh enhence axillary branching. Scientia Horticulturae. 1998,76:219-229．
[109] Proft,M.P.P.;Maene,L.J.Debcrgh,P.C.,Carbon dioxide and cthylene evolution in the culture atmospHere of Magnolia Cultures in vitro.Physiol.Plantarum .1985,65:375-379
[110] Pelacho A M and A M Mingo-Castel. Jasmonic acid induces tuberization of potato stolons cuturee in vitro. Plant
Physiology, 1991,97;1253-1255．
[111] Rowhani A.Maningas M A,Lile L S,et al. Development of a detection system for viruses of woody plants based on PCR analysis of a immobilized virions.Hytopathology,1995,85(3) :347-352．
[112] Ross H. Potato breeding-problems and perspectives. In Horn W. et al(ed)Advances in plant breeding(supplement 13 to journal of plant Breeding). Verlag Paul Parey Berlin and Hamburg, 1986,31-81
[113] Rao PVL, Singh B. Plantlet regeneration from encapsulated somatic embryos of hybrid solanum melongena L. Plant Cell Reports, 1991,10(1) :7-11．
[114] Richard M.S. Mulua and Prem L.Bhalla.Invitro shoot proliferation of Macadamia tetrapHylla L.Johnson.J.OURNAL OF horticultural Science and Biotechnology 75(1) : 1-5
[115] Richard M.S. Mulua and Prem L. Bhalla.Invitro shoot multiplication of Macadamia tetraphylla L. Johnson. J. Oumal of horticultural Science and Biotechnology 75(1) :1-5
[116] Rathore TS.Micropropogation of jujube by in vitro culture.Sci.Horticult Amsterdam. 1992, 51(1-2) : 165
[117] Roche T D, Long R D, Sayegh A J. Commercial-scale photoautotrophic micropropagations in Irish agriculture.horticulture and forestry. Acta Hort, 1996,440:515-520
[118] Simons P. The potato bites back, New Scientist, 1981,1276:470．
[119] Stace-Smith R. Mellor F.C. 1968,Phytopathology 58:199-203
[120] Stein A. Spiegel S, Faingersh G, et al. Responses of micropropagated peach cultivars to thermotherapy for the elimination of Prunus necrotic ringspot virus. Ann Appl Bilo, 1991,119:265-271．
[121] Smith.D.L. ans A.D.Krirorian Somatic embryogenesis of carrot in hormone-free medium:External PH control over morphogenesis. Amer.J.Bot. 1990,77:1634-1647
[122] Serby ,C.,R.Lee. and BM.R. Havey. 1989． The effects of culture medium rigidity on advenfitious bud production andtissue vitrification in needle cultures of sitra spruce (picea sitchensis carr).New Phytal.113:203-210
[123] Seko Y,Nishimura M. Effects of CO2 and light on survival and growth of rice regenerants in vitro on sugar-free medium. Plant Cell, Tissue and Organ Culture,1997, 46(3) :257-264．
[124] Stamp J A, Colby S M. Improved shoot orgnogenesis from leaves of grape. J. Amer. Soc. Hort. Sci. 1990,115(6) :1038-1042．
[125] Tarayama,S.,Misawa,M.,Differentiation in Lilium auratuno bulb scales grown in vitro.Effects of various cultural conditions Physiol.Plant. 1979,46,184-190
[126] Venner c.,The formation of adventitious organ.Acomparison of root and shoot formation on Nautilocalyx explants.pflanzen Physiol 1976,80,310-322
[127] Wilson TMA,Davies JW. New reads to crop protection against viruses. Outlook on Agriculture,1994,23(1) :33-39．
[128] Wilson TMA.Structual intreractions between plant RNA viruses and cells. Oxford Survey of plant Molecular and Cell Biology, 1988,5:89．
[129] Walkey D. G. A. Cooper V. C. 1975, Ann. Appl. Biol. 80:185-190．
[130] Warren K. Coleman, Danielle J. Donnelly, Shirlyn E. Coleman. Potato Microtubers as Research Tools: A Review. Amer J of Potato Res(2001) 78:47-55．
[131] Zilkah S, Evgenia Faingersh,Rotbaum A, et al. In vitro micropropogation of indicator plants for indexing Prunus necrotic ringspect virus. Acta Horticulturae,1993,336:121-125．