核桃外植体褐化影响因素及增殖培养研究
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摘要
核桃(Juglans regia L．)别名胡桃、羌桃，属核桃科(Juglandacea)核桃属(Juglans)植物，是一种优良的干果树种，具有极高的经济价值。本试验以甘肃山核桃(Juglans regia L．)、美国黑核桃(duglans negro L．)、绵核桃、薄皮核桃、露仁核桃为试材，对外植体褐化的影响因子、初代培养、继代培养各个方面进行了研究，并对生根培养进行了初步的探讨。
核桃外植体的褐化受多种因子的影响：取材时间明显影响外植体的褐化率，在生长季中8月份褐化率最低，7月份褐化率最高；基因型也明显影响褐化率，黑核桃的褐化率在生长季极显著的高于山核桃，露仁核桃的褐化率也明显高于绵核桃；取材树的树龄越小，褐化率越低；随消毒时间的增长，褐化率升高，0.1％的HgCl_2溶液消毒8分钟时褐化率、污染率均较低，8分钟以上则加重褐化；褐化率与PPO活性的相关性不显著；褐化率与酚类物质含量呈极显著的正相关(R=0.791**)；Vc能明显的降低褐化率并减轻褐化发生的程度。无菌外植体建立时，山核桃在5月中旬新梢速长前期、黑核桃在8月中旬新梢二次速长期分别采集当年生新梢，用流水冲洗后先依次在70％酒精中浸泡10秒钟、0.1％HgCl_2中浸泡8分钟，两种核桃分别取得了51.62％和42.68％的成活率。绵核桃、薄皮核桃、露仁核桃实生苗使用0.1％HgCl_2分别消毒6分钟、4.5分钟、4.5分钟，取得了85％、67％、40％的成活率。
将无菌保存的材料接种在DKW+1.0mg／L BA+0.01mg／L IBA培养基上相比于相同激素配比的H培养基，芽萌动早，而且长的快，褐化率低。但不同品种之间的萌动开始期和生长势有明显的差异。绵核桃、山核桃萌动早、生长快，露仁核桃萌动最迟、长势差，黑核桃和薄皮核桃介于两者之间。
继代培养选用DKW培养基，增殖效果好于MS和H培养基；BA在嫩茎的增殖中不可缺少，其浓度在0.5-1.0mg／L时增殖效果较好；低浓度的IBA对BA的增殖有促进作用。在DKW培养基上附加BA1.0mg／L、IBA0.01mg／L取得了最高的增殖倍数。接种材料的大小明显影响褐化率和增殖倍数，以1.8cm左右最为适宜。增殖培养时，前期采用较低温度(22℃)，中期(25-26℃)、后期(28
C）采用较高温度，能明显的促进生长，使增殖倍数上升到4．6。继代周期35
d，适当延长后期的高温阶段，可以显著的增加增殖倍数达到4．8。
用 D邯附加 BAO．25mg／L、IBAO．03。g／L进行壮苗培养，取得了生长整齐健
壮的试管苗。
The micropropagation procedures of walnut (Juglans regia L.) and black walnut (Juglans negro L.) had been researched, including the factors affecting explants browning, the establishment of aseptic cultures, shoot proliferation, selection of suitable medium and hormone. A feasible and practical micropropagation system had been made.
Factors affecting walnut browning are as follows: the time collect shoot affecting browning, in august, two species walnut both have the lowest browning rate; species also affecting browning, black walnut has a higher browning rate than walnut in the growth season; the older the age, the higher the browning rate; explants in 0.1%HgC12 less than 8 mins has a lower rate; PPO and browning rate's correlation is not significant; but phenol concentration and browning rate has a very significant correlation, (R=0.791**); add Vc in the medium can greatly reduce the mortality due to browning. High survival rate of the explants can be achieved when the explants of walnut were collected in May and explants of black walnut were collected in August. Washing the explants under running water and in 0. !%HgC12 for 8 min, rinsed 3-4time with sterile water, the survival rate was 51.62% and 42.86%. Walnut of mianhetao seedling in 0.1%HgC12 for 6min, baopihetao and lourenhetao seedling in 0.1%HgC12 for 4.5min. The survival rate was 85%and 66.7%and 40% respectly.
Culture the survival stem on DKW medium with l.Omg/LBA and O.Olmg/LIBA can quickly made the bud germinate and growth well.
During the shoot proliferation, DKW medium was more suitable for growth and subculture of the shoots than on MS and H medium. The exogenous application of cytokinins-BA was very necessary for shoot proliferation, 0.5-l.Omg/LBA have a higher proliferation rate, combination of cytokinin and lower concentration auxin-IB A can stimulate the growth of shoots. Thus the optimal proliferation medium was DKW medium supplement with l.Omg/LBA, O.Olmg/LIBA. It was good to keep the cultures at 22癈,16hr. photoperiod in the first 7 days, then at 25-26癈 in the middle phase, at 28 癈 in the last 7days, the proliferation rate can reached 4.6.Enlonger the last period to 12d,subcultures every 35d, have a proliferation rate 4.8.
DKW medium added BA0.25mg/L, IBA0.03mg/L made the shoot elongation and better growth.
核桃外植体的褐化受多种因子的影响：取材时间明显影响外植体的褐化率，在生长季中8月份褐化率最低，7月份褐化率最高；基因型也明显影响褐化率，黑核桃的褐化率在生长季极显著的高于山核桃，露仁核桃的褐化率也明显高于绵核桃；取材树的树龄越小，褐化率越低；随消毒时间的增长，褐化率升高，0.1％的HgCl_2溶液消毒8分钟时褐化率、污染率均较低，8分钟以上则加重褐化；褐化率与PPO活性的相关性不显著；褐化率与酚类物质含量呈极显著的正相关(R=0.791**)；Vc能明显的降低褐化率并减轻褐化发生的程度。无菌外植体建立时，山核桃在5月中旬新梢速长前期、黑核桃在8月中旬新梢二次速长期分别采集当年生新梢，用流水冲洗后先依次在70％酒精中浸泡10秒钟、0.1％HgCl_2中浸泡8分钟，两种核桃分别取得了51.62％和42.68％的成活率。绵核桃、薄皮核桃、露仁核桃实生苗使用0.1％HgCl_2分别消毒6分钟、4.5分钟、4.5分钟，取得了85％、67％、40％的成活率。
将无菌保存的材料接种在DKW+1.0mg／L BA+0.01mg／L IBA培养基上相比于相同激素配比的H培养基，芽萌动早，而且长的快，褐化率低。但不同品种之间的萌动开始期和生长势有明显的差异。绵核桃、山核桃萌动早、生长快，露仁核桃萌动最迟、长势差，黑核桃和薄皮核桃介于两者之间。
继代培养选用DKW培养基，增殖效果好于MS和H培养基；BA在嫩茎的增殖中不可缺少，其浓度在0.5-1.0mg／L时增殖效果较好；低浓度的IBA对BA的增殖有促进作用。在DKW培养基上附加BA1.0mg／L、IBA0.01mg／L取得了最高的增殖倍数。接种材料的大小明显影响褐化率和增殖倍数，以1.8cm左右最为适宜。增殖培养时，前期采用较低温度(22℃)，中期(25-26℃)、后期(28
C）采用较高温度，能明显的促进生长，使增殖倍数上升到4．6。继代周期35
d，适当延长后期的高温阶段，可以显著的增加增殖倍数达到4．8。
用 D邯附加 BAO．25mg／L、IBAO．03。g／L进行壮苗培养，取得了生长整齐健
壮的试管苗。
The micropropagation procedures of walnut (Juglans regia L.) and black walnut (Juglans negro L.) had been researched, including the factors affecting explants browning, the establishment of aseptic cultures, shoot proliferation, selection of suitable medium and hormone. A feasible and practical micropropagation system had been made.
Factors affecting walnut browning are as follows: the time collect shoot affecting browning, in august, two species walnut both have the lowest browning rate; species also affecting browning, black walnut has a higher browning rate than walnut in the growth season; the older the age, the higher the browning rate; explants in 0.1%HgC12 less than 8 mins has a lower rate; PPO and browning rate's correlation is not significant; but phenol concentration and browning rate has a very significant correlation, (R=0.791**); add Vc in the medium can greatly reduce the mortality due to browning. High survival rate of the explants can be achieved when the explants of walnut were collected in May and explants of black walnut were collected in August. Washing the explants under running water and in 0. !%HgC12 for 8 min, rinsed 3-4time with sterile water, the survival rate was 51.62% and 42.86%. Walnut of mianhetao seedling in 0.1%HgC12 for 6min, baopihetao and lourenhetao seedling in 0.1%HgC12 for 4.5min. The survival rate was 85%and 66.7%and 40% respectly.
Culture the survival stem on DKW medium with l.Omg/LBA and O.Olmg/LIBA can quickly made the bud germinate and growth well.
During the shoot proliferation, DKW medium was more suitable for growth and subculture of the shoots than on MS and H medium. The exogenous application of cytokinins-BA was very necessary for shoot proliferation, 0.5-l.Omg/LBA have a higher proliferation rate, combination of cytokinin and lower concentration auxin-IB A can stimulate the growth of shoots. Thus the optimal proliferation medium was DKW medium supplement with l.Omg/LBA, O.Olmg/LIBA. It was good to keep the cultures at 22癈,16hr. photoperiod in the first 7 days, then at 25-26癈 in the middle phase, at 28 癈 in the last 7days, the proliferation rate can reached 4.6.Enlonger the last period to 12d,subcultures every 35d, have a proliferation rate 4.8.
DKW medium added BA0.25mg/L, IBA0.03mg/L made the shoot elongation and better growth.
引文
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62． Chenevard D, Jay-Allemand C;Gendraud M,Frossard J.S The effect of sucrose on the development of hybrid walnut microcuttings consequences on their survival during acclimation. Annales des sciences Forestieres,1995,52(2) :147-156
63． Drew R.A., The effects of medium composition and cultural conditions on in vitro root initiation and growth of papaya. Journal Hort sci, 1987, 62:551-556
64． Druart, Kevers. C, Boxus, In vitro promotion of root formation by apple shoots through darkness: effect on endogenous phenols peroxidases. physiol,1982,5:429-436
65． Gebhardt K, Activation of indole-3-acetic acid oxidase from horseradish and prunus by phenols and hydrogen peroxidase. Plant Growth Regul, 1982 1(2) :73-84
66． Gonzalez A,Tames SR, Ethylene in relation to protein, peroxidase and polyphenol oxidase activities during rooting inhazelnut cotyledons. Physiol Plant. 1991． 83． 611-620
67． Gruselle,,R,Badia,N. And Boxus,P., Walnut micropropagation: first results. Acta Hortic.,1987 (212) :511-515
68． H.J.Scholten, RL.M.Pierik, Agar as a gelling: differential biological effects in vitro. Scientia Horticulturae 77(1998) :109-116
69． Haissig B.E., Influence of auxins and synergists on adventitious root primordium in initiation and development. New Zealand J For sci, 1974,4:311-323
70． Haissig BE.,Metabolic processes in adventitious rooting of cutting. New root Formation of Plant and Cutting.l986,141-189
71． Hammerschlag F.A., Bancham G.R, Scoraz R, Factors influencing in vitro multiplication and rooting of peach cultivars. Plant Cell Tissue Organ Cult. 1987． 8:235-242
72． Hejie-Sudholt.C, Huetteman.C.A; In vitro embryonic axis and seedling shoot tip culture of Juglans nigro L.. Plant cell. Tissue and organ culture. 1986,16(2) :189-197
73． Hoza D; STANDARDI A;STANICA F,TUDOR T.A,Preliminary data on in vitro culture of walnut (Juglans regia). 7 (3) :319-324
74． Jay-allmend. C.Peng.S, Capelli, Micropropagation of hybrid walnut trees: some factors involved in rooting. Acta Hortculturae 1993,311:117-124
75． John A. Driver, Andrew H.Kuniyuki, In vitro propagation of paradox walnut rootstock. Hortscience, 1984,19(4) :507-509
76． Jose Carlos Goncalves,Graca Diogo, sara Amancio, In vitro propagation of chestnut (Castanea sativa × C.crenata): Effects of rooting trements on plant survival, peroxidase activity and anatomocal changes during adventitious root formation. Scientia Horticulturae 72(1998) :265-275
77． K.AL-Maarri, Y.Amaud, E.Miginiac; Micropropagation of Pyrus communis cultivar 'passe Crassane' seedlings and cultivar 'Williams': factors affecting root formation in vitro and ex vitro. Scientia Horticulturae 58(1994) :207-214
78． Kornova. K, Stephanova. A, Teriusky.D, In vitro culture of immature embryos and cotyledons of Juglans regia L,Morphological analyses of some regenerants. Acta Hortculturae 1993,311:129-133
79． Lydia Bouza, Monique Jacques,Emile Miginiac, Requirements for in vitro rooting of paeonia suffruticosa Andr. Cv.'Mme de Vatry'. Scientia Horticulture 58(1994) :223-233
80． Opatrny Z; Klerk G, J.de, Quantitative Variations of indoly compounds including IAA, IAA-asparate and serotonin in walnut microcuttings during root induction, Biologia Plantarum, 1997, 39 (1) : 131-137
81． Paolo Curir. Salvatore Sulis, etal, Influence of endogenous phenols on rootability of chamaelaucium uncinatum Schauer stem cuttings. Scientia Horticulturae, 55 (1993) 303-314
82． Poapst P A, Durkee A B, Root differentiating properties of some simple aromatic substances of the apple and pear fruit. Hort sci;1976,42: 429-438
83． Ponchia G;TONON,G, Preliminary results of research onmicropropagation of walnut .Rivista di frutticolotura edi ortofloricolture,1993,55(1) :91-94
84． R.L.M. Pierik. J.Oosterkamp, M.A.C.Ebbing, Factors controlling adventitious root formation of explants from juvenile and adult Ouercus robur ''astigiata'.
85． Ra'anan Friedman, Arie Altman, Uriel Bachrach, Polyamines and root formation in mung bean hypocotyl cuttings. Plant Physiol 1982(70) : 844-848
86． Rodriguez 刺激核桃种子幼芽丛形成. Hortiscience 1982 17(4) : 592
87． Rodriguez. R 等 促使胡桃种胚形成芽丛. Hort science 1982 17(4)
88． Ruipetti V,Kevers C;Gaspar T, Two successive media for the rooting of walnut shoot in vitro,changes in peroxidase activity and in the ethylene production. Advance in Horticultural Science,1994(8) :29-32
89． Sambeek, J.W.VAN, Lambus L.J, Khan, S.B, In vitro establishment of tissue from adult black walnut. Preece, 2000 JE.
90． Sriskandarajah. S., Mullins M. G.,Nair Y., Induction of adventitious rooting in vitro diffcult to propagate cultivars of apple. Plant sci. Lett, 1982,24:1-9
91． Srivastava OP, Van Huystee R B,An interrelationship among peroxidase, IAA oxidase and polyphenol oxidase from peanut cell. Can,J Bot, 1977． 55:2630-2635
92． Stephens.L.C, Krell.S.L; Domoto. P.A, In vitro propagation of Juglans regia In 81st annual report of the northern nut grower's association. HAMDEN. CONNECTICUT USA 1990 publish
93． Turems, N; comlekcloglun , determination of the most suitable explants for walnut micropropagation. Acta Horticulturae 1994,no.441:379-381
94． Vahdati K, Kalighi A, Naderi R, Majda, Effects of some growth regulations and complementary nitrogen compounds on in vitro shoot proliferation of persian walnut. Iranian Journal of Agricultural Sciences, 1998,29(3) : 599-606
95． Wood B.W. 美国山核桃茎尖离体繁殖. Hortiscience 1982 17(6) :860-891
96． Z.AL BARAZL , W.W. SCHWABE, The possible involvement of polyphenol-oxidase and the auxin-oxidase system in root formation and development in cuttings of Pistacia vera. Journal of Horticultural Science (1985) 59:453-461
2．曹孜义，刘国民，实用植物组织培养技术教程．兰州．甘肃科学技术出版社，1996
3．常永健，陈四维，马宝锟，苹果茎尖培养中植物激素与不定根形成关系的研究．河北农业大学学报，1991，14(4)：1-6
4．陈龙清，鞠庆坤，活性炭对几种植物试管苗生根的影响．华中农大学报 1996，14(6)：600-602
5．陈伟，生长调节剂、酚类化合物和多胺对核桃组织培养生根的影响．福建农业大学学报，1994，23(4)：490-494
6．陈伟，影响胡桃体细胞胚萌发率若干因素的研究．福建林学院学报，1993，13(4)：381-385
7．陈正华，《木本植物组织培养及其应用》．北京：高等教育出版社 1986
8．方宏筠，王关林，黑核桃体细胞胚状体发生及其基因转化系统的建立．园艺学报，2000，27(6)：406-411
9．肖关丽 甘蔗组培苗继代培养中内源激素与绿苗生根率关系研究．云南农业大学学报，2001，16(4) 271-273
10．高清华，段可，甘霖，张大福，我国核桃繁殖技术研究进展．果树科学 2000，17(3) 220-224
11．郭军战，舒庆艳，王丽玲，刘志勤，四倍体刺槐组织培养中的外植体选择和消毒的研究．2002，17(1)：15-18
12．韩文璞，袁明莲，活性炭在甜樱桃组织培养中的应用．落叶果树 2001，(3)：7-8
13．郝刚平，杜希华，尤勇，等，不同因素对离体培养的银杏幼茎腋芽生长发育的影响．林业科学研究，2000，13(2)：217-221
14．黄卓烈、林韶湘，谭绍满，桉树体内的生根抑制物质研究综述．林业科学研究，1994，
15．康冰，张小红，陈彦生，等，PP333及CCC对香椿试管苗增殖及生根移栽的影响．西北植物学报，2002，22(2)：411-415
16．李焕秀，王乔春，李春秀，梨芽和茎尖多酚氧化酶活性和总酚含量的初步研究．四川农业大学学报，1994，2(2)：218-222
17．李焕秀，晏本菊，梨外植体组培褐变与氧化酶活性和同工酶的关系．西南农业大学学报，2001，23(5)：432-434
18．李明，黄卓烈，谭绍满，等，萘乙酸处理桉树插条后多酚氧化酶活性及同工酶变化与插条生根的关系．华南农业大学学报，2002，23(1)：45-48
19．李明，黄卓烈，谭绍满，等，难易生根桉树多酚氧化酶、吲哚乙酸氧化酶活性及其同工酶的比较研究．林业科学研究，2000，13(5)：493-500
20．林海球，龙腾，黄晓勇，等，抗酚剂对桉树扦插生根的影响研究．林业科学研究，2000，13(1)：86-92
21．刘兰英，核桃的组织培养和快速繁殖．植物生理学通讯，2000，36(5)，434-435
22．刘兰英，薄壳香核桃组培中的褐化及防止措施的研究．园艺学报，2002，29(2)：171-172
23．刘淑兰，韩碧文，陈正华，核桃叶柄体细胞胚胎发生及其细胞学观察．北京农业大学学报，1992，18(1)：29-32
24．刘淑兰，韩碧文，核桃离体胚的植株再生．植物生理学报 1989，15(1)：98-100
25．刘淑兰，韩碧文，核桃愈伤组织的诱导
26．陆振鑫，夏振澳，戴维逊棉的组织培养与原生质体培养研究．植物学报，1991，33(2)：98-103
27．罗晓芳，田砚亭，姚红军，组织培养过程中PPO活性和总酚含量的研究．北京林业大学报，1999，21(1)：92-95
28．马锋旺，王居仓，雍文，植物生长调节剂对‘长富—2’苹果离体繁殖的效应．果树科学，1990，7(4)：201-206
29．裴东，奚声珂，董凤祥，核桃砧穗生理状态对微枝嫁接成活的影响．林业科学研究，1998，11(2)：119-123
30．裴东，奚声珂，核桃早实品种微枝接穗试管快繁技术的研究．林业科学研究 1998，11(4)：350-354
31．裘文达，《园艺植物组织培养》．上海科学技术出版社 1984
32．曲泽州，《果树栽培学各论》．北京：中国农业出版社．1987，
33．宋金耀，何文林，李松波，等，毛白杨嵌合体扦插生根相关理化特性得分析．林业科学，2001，37(5)：64-67
34．宋平，沈丙辉，活性炭培养基对棉花愈伤组织诱导的效果．植物生理学通讯，1988 (5)：47-48
35．汤浩如，王永清，任正隆，果树的体细胞胚发生．四川农业大学学报 1999，17(1)：69-73
36．汤浩茹，王永清，核桃体细胞胚发生与转基因研究进展．林业科学，2000，36(3) 102-110
37．汤浩茹，王永清，任正隆，G.Kreal，德国核桃‘No.120’幼胚胚轴与子叶体细胞胚胎发生及其植株再生．园艺学报，2000，27(1)：59-61
38．王海光，张国良，张淑云，刘四围，柿组织培养研究进展．河北农业大学学报，2001，24(4)：93-97
39．郗荣庭，张毅萍，《中国核桃》．北京：中国林业出版社 1992
40．郗荣庭，张毅萍《中国果树志》核桃卷．北京：中国林业出版社 1996
41．奚声柯，杨彬，核桃微枝嫁接技术的研究．林业科学研究，1992，5(5)：531-535
42．杨增海，胡霓云，路广明，桃试管实生苗茎尖培养的研究．园艺学报，19884，11(1)：7-13
43．姚红军，罗晓芳，田砚亭，植物组织培养外植体褐变的研究进展．北京林业大学学报 1999，21(3) 78-84
44．袁巧平，董茂山，黄钦才，刘颂，核桃体细胞胚诱导的初步研究．植物生理学报，1994，20(3)
45．袁巧平，董茂山，黄请才，刘颂，核桃的组织培养．植物生理学报 1994，20(2)：118-119
46 曾炳山，许煌灿，刘英，棕榈藤组培苗根的诱导研究．林业科学研究，1998，11(3)：260-264
47．张继祥，魏钦平，毛志泉，美国黑核桃种质资源及其开发利用．山东农业大学学报 2001，32(4)：555-560
48．张妙霞，孔祥生，郭秀璞，张益民，柿树组织培养防止外植体褐变的研究．河南农业大学学报，1999，33(1)：87-90
49．郑红军，张力思，李勃，黑色培养基促进中国樱桃试管苗生根的研究．落叶果树 2001 (4)：6-7
50．郑均宝，刘红军等，几种木本植物接穗生根与内源 IAA、ABA的关系．植物生理学报，1991，17(3)：313-316
51．周清桂，杨克钦，译，苹果幼茎离体繁殖和生长调节．国外农学——果树，1981，(2)，8-11
52．朱宁华，李芳东，张昭炜，赤桉组培苗生根与多酚氧化酶、过氧化物酶活性的关系中南林学院学报，2000，20(3)：67-69
53． 朱玉球,廖望仪,黄坚看,孙小萍,山核桃愈伤组织诱导的初步研究.浙江林学院学报,2001,18(2) :115-118
54． Alain Rival, Florence Bernand, Yvan Mathieu, Changes in peroxidase activity during in vitro rooting of oil palm (Elaeis gunsis Jacq.). Scientia Horticulturae 71(1997) :103-112
55． B.C.Jarvis, P.RM.Shannon and yasmin Involvement of polyamines with adventitious root development in stem cuttings of Mung Bean. Scientia Horticulturae Plant Physiol 1983: 677-683
56． Balakrish namurthy G., Changes in phenols during rhizogenesis in rose. Curr sci, 1988,57(17) :960-962
57． BARBAS,E;JAY-ALLMAND C;DOUMAS,P;et al, Effects of gelling agents on growth mineral of composition and naphthoquinone content of in vitro explants of hybrid walnut tree(Juglans regia x Juglans nigra). Annales des sciences Forestieres 1993,50(2) : 172-181
58． Bassuk NL, Hunter LD, The apparent of polyphenol oxidase and phloridzin in the production of apple rooting cofactors. Hort sci, 1981,56(4) :313-322
59． Bhattacharya N.C,Kumar A., physiological and biological studies associated with adventitious root formation in phaseolus mungo L.in relation to auxin-phenol synergism. Biochem physiol pflanz, 1980,175:421-435
60． C.Brutti, N.M.Apostolo, S.A.Ferrarotti, B.E. Llorente, N. Krymkiewicz, Micropropagation of Cynara scolymus L. employing cyclodextrins to promote rhizogenesis. Scientia Horticulturae 83(2000) 1-10
61． C.Jay-Allemand, P.Capelli and D.Cornu, Root development of in vitro hybrid walnut microcuttings in a venniculite-containing gelrite medium. Scientia Horticulturae 51(1992) :335-342
62． Chenevard D, Jay-Allemand C;Gendraud M,Frossard J.S The effect of sucrose on the development of hybrid walnut microcuttings consequences on their survival during acclimation. Annales des sciences Forestieres,1995,52(2) :147-156
63． Drew R.A., The effects of medium composition and cultural conditions on in vitro root initiation and growth of papaya. Journal Hort sci, 1987, 62:551-556
64． Druart, Kevers. C, Boxus, In vitro promotion of root formation by apple shoots through darkness: effect on endogenous phenols peroxidases. physiol,1982,5:429-436
65． Gebhardt K, Activation of indole-3-acetic acid oxidase from horseradish and prunus by phenols and hydrogen peroxidase. Plant Growth Regul, 1982 1(2) :73-84
66． Gonzalez A,Tames SR, Ethylene in relation to protein, peroxidase and polyphenol oxidase activities during rooting inhazelnut cotyledons. Physiol Plant. 1991． 83． 611-620
67． Gruselle,,R,Badia,N. And Boxus,P., Walnut micropropagation: first results. Acta Hortic.,1987 (212) :511-515
68． H.J.Scholten, RL.M.Pierik, Agar as a gelling: differential biological effects in vitro. Scientia Horticulturae 77(1998) :109-116
69． Haissig B.E., Influence of auxins and synergists on adventitious root primordium in initiation and development. New Zealand J For sci, 1974,4:311-323
70． Haissig BE.,Metabolic processes in adventitious rooting of cutting. New root Formation of Plant and Cutting.l986,141-189
71． Hammerschlag F.A., Bancham G.R, Scoraz R, Factors influencing in vitro multiplication and rooting of peach cultivars. Plant Cell Tissue Organ Cult. 1987． 8:235-242
72． Hejie-Sudholt.C, Huetteman.C.A; In vitro embryonic axis and seedling shoot tip culture of Juglans nigro L.. Plant cell. Tissue and organ culture. 1986,16(2) :189-197
73． Hoza D; STANDARDI A;STANICA F,TUDOR T.A,Preliminary data on in vitro culture of walnut (Juglans regia). 7 (3) :319-324
74． Jay-allmend. C.Peng.S, Capelli, Micropropagation of hybrid walnut trees: some factors involved in rooting. Acta Hortculturae 1993,311:117-124
75． John A. Driver, Andrew H.Kuniyuki, In vitro propagation of paradox walnut rootstock. Hortscience, 1984,19(4) :507-509
76． Jose Carlos Goncalves,Graca Diogo, sara Amancio, In vitro propagation of chestnut (Castanea sativa × C.crenata): Effects of rooting trements on plant survival, peroxidase activity and anatomocal changes during adventitious root formation. Scientia Horticulturae 72(1998) :265-275
77． K.AL-Maarri, Y.Amaud, E.Miginiac; Micropropagation of Pyrus communis cultivar 'passe Crassane' seedlings and cultivar 'Williams': factors affecting root formation in vitro and ex vitro. Scientia Horticulturae 58(1994) :207-214
78． Kornova. K, Stephanova. A, Teriusky.D, In vitro culture of immature embryos and cotyledons of Juglans regia L,Morphological analyses of some regenerants. Acta Hortculturae 1993,311:129-133
79． Lydia Bouza, Monique Jacques,Emile Miginiac, Requirements for in vitro rooting of paeonia suffruticosa Andr. Cv.'Mme de Vatry'. Scientia Horticulture 58(1994) :223-233
80． Opatrny Z; Klerk G, J.de, Quantitative Variations of indoly compounds including IAA, IAA-asparate and serotonin in walnut microcuttings during root induction, Biologia Plantarum, 1997, 39 (1) : 131-137
81． Paolo Curir. Salvatore Sulis, etal, Influence of endogenous phenols on rootability of chamaelaucium uncinatum Schauer stem cuttings. Scientia Horticulturae, 55 (1993) 303-314
82． Poapst P A, Durkee A B, Root differentiating properties of some simple aromatic substances of the apple and pear fruit. Hort sci;1976,42: 429-438
83． Ponchia G;TONON,G, Preliminary results of research onmicropropagation of walnut .Rivista di frutticolotura edi ortofloricolture,1993,55(1) :91-94
84． R.L.M. Pierik. J.Oosterkamp, M.A.C.Ebbing, Factors controlling adventitious root formation of explants from juvenile and adult Ouercus robur ''astigiata'.
85． Ra'anan Friedman, Arie Altman, Uriel Bachrach, Polyamines and root formation in mung bean hypocotyl cuttings. Plant Physiol 1982(70) : 844-848
86． Rodriguez 刺激核桃种子幼芽丛形成. Hortiscience 1982 17(4) : 592
87． Rodriguez. R 等 促使胡桃种胚形成芽丛. Hort science 1982 17(4)
88． Ruipetti V,Kevers C;Gaspar T, Two successive media for the rooting of walnut shoot in vitro,changes in peroxidase activity and in the ethylene production. Advance in Horticultural Science,1994(8) :29-32
89． Sambeek, J.W.VAN, Lambus L.J, Khan, S.B, In vitro establishment of tissue from adult black walnut. Preece, 2000 JE.
90． Sriskandarajah. S., Mullins M. G.,Nair Y., Induction of adventitious rooting in vitro diffcult to propagate cultivars of apple. Plant sci. Lett, 1982,24:1-9
91． Srivastava OP, Van Huystee R B,An interrelationship among peroxidase, IAA oxidase and polyphenol oxidase from peanut cell. Can,J Bot, 1977． 55:2630-2635
92． Stephens.L.C, Krell.S.L; Domoto. P.A, In vitro propagation of Juglans regia In 81st annual report of the northern nut grower's association. HAMDEN. CONNECTICUT USA 1990 publish
93． Turems, N; comlekcloglun , determination of the most suitable explants for walnut micropropagation. Acta Horticulturae 1994,no.441:379-381
94． Vahdati K, Kalighi A, Naderi R, Majda, Effects of some growth regulations and complementary nitrogen compounds on in vitro shoot proliferation of persian walnut. Iranian Journal of Agricultural Sciences, 1998,29(3) : 599-606
95． Wood B.W. 美国山核桃茎尖离体繁殖. Hortiscience 1982 17(6) :860-891
96． Z.AL BARAZL , W.W. SCHWABE, The possible involvement of polyphenol-oxidase and the auxin-oxidase system in root formation and development in cuttings of Pistacia vera. Journal of Horticultural Science (1985) 59:453-461