梨S系矮化砧组培快繁技术体系研究
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摘要
本试验通过对梨S系矮化砧离体再生培养过程的研究，筛选出了直接诱导再生的适宜外殖体和诱导时期，提出了试管苗继代增殖、生根、驯化移栽和微嫁接的主要影响因子。建立了梨S系矮化砧组培快繁的技术体系。主要结果如下：
1．3月份的嫩芽(萌发芽)比休眠芽和4月～6月的茎尖诱导成功率高，1/3MS+6-BA1.0(mg/L，单位下同)+IBA0.2+GA_31.0+蔗糖25g/L+琼脂6.5g/L适于外殖体初代培养。外殖体褐变与多酚氧化酶活力和总酚含量密切相关，PPO活力高峰出现在4月份，总酚含量和褐变率高峰出现在5月份。
2．MS+6-BA1.0+IBA0.2+GA_30.1+蔗糖20g/L+琼脂6.5g/L适宜试管苗继代增殖培养，增殖系数随培养代数的增加而提高，10代以后进入分化高峰期，17代以后开始下降。控制培养基中6-BA浓度低于2.0mg/L和GA_3浓度低于0.2mg/L及使用组培专用封口胶可以有效提高试管苗分化质量，防止玻璃化苗产生。
3．建立了S系矮化砧试管苗生根的技术体系。二步生根法优于一步生根法，首先必需选择充分幼化(12代以后)且生长健壮的试管苗，先在附加IBA2.0mg/L的ASH培养基中暗培养7～9d，以促进根原基的形成，然后转入无激素的ASH+蛭石培养基中光下进行根伸长培养。培养基中添加80～100mg/L间苯三酚可以明显促进IBA2.0mg/L对试管苗生根的诱导效果，单独施用间苯三酚不能诱导不定根发生。试管苗的生根率随继代培养代数的增加而提高。
4．环境条件是试管苗驯化移栽的关键因素。S系矮化砧试管苗移栽前需经闭口强光炼苗10d，然后移栽到蛭石基质中，湿度保持在90％～1006，以后逐渐通风和增加光照，30d后栽入营养钵中。驯化移栽的时期宜选在春季(4月～5月)。
5．经过生根培养基诱导而未生根的无根苗，在适宜的湿度(70％～75％)、温度(25℃)、光照(初期遮光)等环境条件下，嫁接到半木质化杜梨实生砧木上，可以得到较高的成活率。微嫁接技术进一步完善了S系矮化砧组培快繁技术体系。
Through the studies on regeneration of in vitro culture of S series dwarfing rootstock of pear, the proper explant induced directly and the time of induction were chosen, and die main influencing factors of multiplication ,rooting .training, transplanting and niicrografting were evaluated, At the same time, some improving methods were put forward. Technical system of tissue culture and rapid propagation of the S series dwarfing rootstock of pear was established. The main achievements were as follows:
The survival rate of the shoot tip explant in March was higher than that in winter and April桱une, 1/3MS + 6-BA1.0 (mg/L, follows as the same) + IBA0.2 + GA31.0+ 2.5%sugar +0.65% agar was most suitable to initiatory culture of the explant. The browning of explant is relative to the PPO activity and the total phenol contents existed in the explant, The highest PPO activity occured in April, and the total phenol contents and browning rate are highest in May.
MS+6-BA1.0+IBA0.2+GA30.1+2.5%sugar +0.65% agar was suitable to subculture of adventitious bud. The more of the subculture times, the higher of the coefficient of multiplication, The coefficient increased rapidly after the tenth subculture and decreased begun in the seventeenth subculture. 6-BA concentration under 2.0mg/L, GA3 concentration under 0.1 mg/L and use appropriative sealing material could improved the quality of adventitious shoot and overcome vitrification.
Technical system of rooting of the adventitious shoots was established. Two-step-rooting method is better one-step-rooting method, That is, Firstly selecting the juvenile and vigorous shoots , Secondly culturing the shoots in the ASH medium with 2.0mg/L IBA for 7~9d in the darkness in order to promote the formation of primordium of root, then transfering the shoots into the ASH +vermiculite medium without IBA in the light. Adding 80~100mg/L trihydroxybenzene (PG) into the medium promoted on rooting with IBA, but had no effect on rooting without IBA. The more of the subcultural times, the
41
higher of the rooting ratio.
The environmental conditions were main factors of training and transplanting of the rooting shoots. The rooting shoots must be trained for lOd in the light with the sealing material before transplanting , and then transferred into vermiculite medium with 90%~ 100% humidity , winding and lighting gradually after that, then the rooting shoots were transferred into the pots after 30d. Spring (April~May) is most suitable for the in vitro plantlet training and transplanting.
The survival ratio was high when the shoots without rooting after induced in rooting medium were grafted on semi-wooden rootstock ofPyrus betulaefolia Bge. The technical system of tissue culture and rapid propagation of the S series dwarfing rootstock of pear was further perfected through the micrografting.
1．3月份的嫩芽(萌发芽)比休眠芽和4月～6月的茎尖诱导成功率高，1/3MS+6-BA1.0(mg/L，单位下同)+IBA0.2+GA_31.0+蔗糖25g/L+琼脂6.5g/L适于外殖体初代培养。外殖体褐变与多酚氧化酶活力和总酚含量密切相关，PPO活力高峰出现在4月份，总酚含量和褐变率高峰出现在5月份。
2．MS+6-BA1.0+IBA0.2+GA_30.1+蔗糖20g/L+琼脂6.5g/L适宜试管苗继代增殖培养，增殖系数随培养代数的增加而提高，10代以后进入分化高峰期，17代以后开始下降。控制培养基中6-BA浓度低于2.0mg/L和GA_3浓度低于0.2mg/L及使用组培专用封口胶可以有效提高试管苗分化质量，防止玻璃化苗产生。
3．建立了S系矮化砧试管苗生根的技术体系。二步生根法优于一步生根法，首先必需选择充分幼化(12代以后)且生长健壮的试管苗，先在附加IBA2.0mg/L的ASH培养基中暗培养7～9d，以促进根原基的形成，然后转入无激素的ASH+蛭石培养基中光下进行根伸长培养。培养基中添加80～100mg/L间苯三酚可以明显促进IBA2.0mg/L对试管苗生根的诱导效果，单独施用间苯三酚不能诱导不定根发生。试管苗的生根率随继代培养代数的增加而提高。
4．环境条件是试管苗驯化移栽的关键因素。S系矮化砧试管苗移栽前需经闭口强光炼苗10d，然后移栽到蛭石基质中，湿度保持在90％～1006，以后逐渐通风和增加光照，30d后栽入营养钵中。驯化移栽的时期宜选在春季(4月～5月)。
5．经过生根培养基诱导而未生根的无根苗，在适宜的湿度(70％～75％)、温度(25℃)、光照(初期遮光)等环境条件下，嫁接到半木质化杜梨实生砧木上，可以得到较高的成活率。微嫁接技术进一步完善了S系矮化砧组培快繁技术体系。
Through the studies on regeneration of in vitro culture of S series dwarfing rootstock of pear, the proper explant induced directly and the time of induction were chosen, and die main influencing factors of multiplication ,rooting .training, transplanting and niicrografting were evaluated, At the same time, some improving methods were put forward. Technical system of tissue culture and rapid propagation of the S series dwarfing rootstock of pear was established. The main achievements were as follows:
The survival rate of the shoot tip explant in March was higher than that in winter and April桱une, 1/3MS + 6-BA1.0 (mg/L, follows as the same) + IBA0.2 + GA31.0+ 2.5%sugar +0.65% agar was most suitable to initiatory culture of the explant. The browning of explant is relative to the PPO activity and the total phenol contents existed in the explant, The highest PPO activity occured in April, and the total phenol contents and browning rate are highest in May.
MS+6-BA1.0+IBA0.2+GA30.1+2.5%sugar +0.65% agar was suitable to subculture of adventitious bud. The more of the subculture times, the higher of the coefficient of multiplication, The coefficient increased rapidly after the tenth subculture and decreased begun in the seventeenth subculture. 6-BA concentration under 2.0mg/L, GA3 concentration under 0.1 mg/L and use appropriative sealing material could improved the quality of adventitious shoot and overcome vitrification.
Technical system of rooting of the adventitious shoots was established. Two-step-rooting method is better one-step-rooting method, That is, Firstly selecting the juvenile and vigorous shoots , Secondly culturing the shoots in the ASH medium with 2.0mg/L IBA for 7~9d in the darkness in order to promote the formation of primordium of root, then transfering the shoots into the ASH +vermiculite medium without IBA in the light. Adding 80~100mg/L trihydroxybenzene (PG) into the medium promoted on rooting with IBA, but had no effect on rooting without IBA. The more of the subcultural times, the
41
higher of the rooting ratio.
The environmental conditions were main factors of training and transplanting of the rooting shoots. The rooting shoots must be trained for lOd in the light with the sealing material before transplanting , and then transferred into vermiculite medium with 90%~ 100% humidity , winding and lighting gradually after that, then the rooting shoots were transferred into the pots after 30d. Spring (April~May) is most suitable for the in vitro plantlet training and transplanting.
The survival ratio was high when the shoots without rooting after induced in rooting medium were grafted on semi-wooden rootstock ofPyrus betulaefolia Bge. The technical system of tissue culture and rapid propagation of the S series dwarfing rootstock of pear was further perfected through the micrografting.
引文
[1] 张克俊等．果树矮化密植和早期丰产[M]．山东科学技术出版社．1993
[2] 贾敬贤等．梨树矮化密植栽培[M]．金盾出版社．1995
[3] 曹孜义等．实用植物组织培养技术教程[M]．甘肃科学技术出版社．1998
[4] Lane W D. Regeneration of pear plants from shoot meristem tips[J]. Plant Sci. lett, 1979,16:337-342
[5] 赵惠祥等．梨种子胚的培养[J]．辽宁农业科学．1981(3)：31—33
[6] 赵惠祥等．梨的试管实生苗茎尖培养再生植株[J]．山西果树．1981(3)：12
[7] Elisabeth Chevreau, Fabienne Mourgues, Martine Neveu et al. Effect of gelling agents and antibiotics on adventitious bud regeneration from in vitro leaves of pear[J]. In Vitro Cellular & Developmental Biology-Plant. 1997, 33(3): 173-179
[8] 8.W.David Lane,Hiroyuki Iketani Tateki Hayashi. Shoot regeneration from cultured leaves of Japanese pear(Pyrus pyrifolia)[J].Plant Cell, Tissue and Organ Culture. 1998,54(1):9-14
[9] 孟庆田．紧凑型矮化梨组织培养获得完整试管苗[J]．天津农林科技．1994(1)：3-5
[10] 王乔春．梨试管苗的生根[J]．果树科学．1994，11(3)：145—148
[11] 王乔春．植物生长调节剂对梨试管苗培育及移栽的影响[J]．果树科学．1995，12(1)：15-20
[12] 杨恩芹，王震星，刘贵仁，等．外源激素对西洋梨快速繁殖的影响[J]．天津农林科技．1996(2)：7-8
[13] 王震星，张磊，刘贵仁．西洋梨茎段离体培养与快速繁殖的探讨[J]．天津农业科学．1998(2)：16-19
[14] 赵惠祥等．梨茎尖离体培养[J]．植物学报．1982，24(4)：392-394
[15] 战淑敏等．莱阳庄梨成龄树茎尖培养的研究[J]．莱阳农学院学报．1992(2)：118—122
[16] 李晓玲，由继红，杨文杰．光对“苹博一号”梨试管苗生长影响[J]．北方园艺．136：56—57
[17]李晓玲等．“苹博一号”梨试管苗的光自养生长和光混合营养生长[J]．特产研究．2000(1)：11-12，20
[18]王定康，王琼芳．玉香梨的组织培养与快速繁殖[J]．植物生理学通讯．1999，35(3)：207
[19]韩文璞，袁明莲．黄金梨的组织培养和快繁研究[J]．落叶果树．2001(2)：7-8
[20]张磊，等．鸭梨三倍体实生苗组织培养快繁试验[J]．中国果树．1996(2)：20—21
[21]孙清荣．梨成熟胚子叶不定梢诱导[J]．河北农业大学学报．1999(2)：50—52
[22]孙清荣，孙洪雁．西洋梨“丰产”叶片不定梢再生[J]．落叶果树．1999(4)：9-10
[23]Wang Q C. Factors affecting rooting of microcuttings of the pear rootstock 8P10030[J]. Scientia Hortic, 1991, 45:209-213
[24]Pious Thomas,M.B.Ravindra. Shoot tip culture in mango: influence of medium, genotype, explant factors, season and decontamination treatments on phenolic exudation, explant survival and axenic culture establishment[J].Journal of Horticultural Science. 1997,72(5): 713-722
[25]晏本菊，李焕秀．梨外殖体褐变与多酚氧化酶及酚类物质的关系[J]．四川农业大学学报．1998，16(3)：310—313
[26]Maneesh Mishra, R.P. Saxena,R.K.Pathak. Effeet of antioxidants, phenol binding agents and explant waxing on in vitro browning of aonla(Emblica officials Gaertn)[J].Progressive Horticulture. 1998,30(3-4): 128-134
[27]赵剑，杨文杰．苹果梨离体快繁过程中产生玻璃化苗时的某些生理生化变化[J]．植物生理学通讯．1998，34(3)：200-201
[28]师校欣，陈四维，马宝琨，等．苹果砧木离体培养中玻璃化问题的研究[J]．河北农业大学学报．1990，13(3)：12-16
[29]程家胜，史永忠，张志云，等．苹果组织培养中的玻璃苗问题[J]．植物生理学通讯．1990(1)：33—35
[30]Masuda Y.,Yoshida Y.,Bessho H. et al. Studies on cell, tissue and shoot-tip culture in apple. I. Relationship between abnormal shoot formation and plant homones[J].Bull Tree Res Stn. 1988,15:21-28
[31]Phan C T and Letouze R.A comparative study of cholorophyll, phenolic and protein contents and of hydroxycinnamate:CoA ligase activity of normal and vitreous plants(Prunus avium L.)obtained in vitro[J]. Plan Sci Letters. 1983,31:323-327
[32]Rugini E, Tarini P and Rossodivita M E. Control of shoot"vitrification" of almond and olive grown in vitro[J]. Acta Hort. 1987,212:177-183
[33]Vieitez A M, Ballester A, San-Jose M C et al. Anatomical and chemical studies of vitrified shoots of chestnut regenerated in vitro[J].Physiol Plant. 1985,65:177-184
[34]赵剑，杨文杰．梨芽离体快繁过程中玻璃化苗的发生与氧自由基胁迫的关系[J]．西北植物学报．1998，18(2)：183—189
[35]陈国菊等．荠菜试管苗玻璃化的控制[J]．西南农业大学学报．1992，14(3)：253
[36]李云等．珠美海棠试管苗玻璃化发生机理的初步研究[J]．北京林业大学学报．1996(1)：53
[37]Marga F., Vebret L., Morvan H..Agar fractions could protect apple shoots cultured in liquid media against hyperhydricity[J]. Plant Cell, Tissue & Organ Culture. 1997,49(1): 1-5
[38]Kevers C. et al. Physiological and biochemical events leading to vitrification of plants cultured in vitro[J]. Physiol. Plant. 1984,61:69-74
[39]Kevers C.et al. Vitrification of carnation in vitro:Changes in ethylene production Acc level and capacity to convert Acc to ethylene[J]. Plant Cell Tissue Culture. 1985,4:215-223
[40]Kevers C.et al. Vitrification of carnation in vitro:Changes in cell wall mechanical properties,cellulose and ligain cortent[J]. Plant Growth Regulation. 1987,5:59-66
[41]袁丽钗．核桃品种试管苗生根及不定根发生过程中特异蛋白表达的研究[D]．保定：河北农业大学园艺学院，2000
[42]黄学林，李竣菊编著．高等植物组织离体培养的形态建成及其调控[M]．科学出版社．1995
[43]李明军等．多效在玉米组织培养中的作用[J]．作物学报．1997(6)：759
[44]雷逢进，李燕娥，岳建雄，等．苹果试管苗快繁技术体系研究[J]．山西农业科学．2001，29(2)：56—58
[45]李均儒．葡萄试管苗移栽技术研究[J]．中国果树．1994(2)：7—9
[46]曹春英，任术琦，丁世民，等．冬枣试管苗驯化及移植条件研究[J]．落叶果树．2001(2)：10—11
[47]马宝琨，高仪．苹果试管苗生根的研究[J]．河北农业大学学报．1990，13(3)：7—11
[48]吕汰，裴建文，王娟．苹果试管苗生根试验研究[J]．甘肃农业科技．1998(6)：31-32
[49]George E F and Sherrington P D. Plant propagation by tissue culture[J]. Exegetics Ltd. 1984
[50]Maarri K A, Arnaud Y and Miginiac E. In vitro micropropagation of Quince(Cydonia oblonga Mill.)[J]. Sci Hort, 1986,28:315-321
[51]Dunstan D I, Turner K and Lazaroffr W R. Propagation in vitro of the apple rootstock M4: effect of phytohormones on shoot quality[J]. Plant Cell Tissue Organ Culture. 1985,4:55-60
[52]Dornman C H and Vogelmann T C. Effect of rigidity of gel medium on benzyladenine-induced adventitious bud formation and vitrification in vitro in Picea abies[J]. Physiol Plant. 1984,61:505-512
[53]Debergh P C. Effects of agar brand and concentration on the tissue culture medium[J]. Physiol Plant. 1983,59:270-276
[54]Lee C Y, Kagan V, Jaworsk A W. Enezymatic Browning in Relation to phenolic compounds and polyphenoloxidase Activity among various peach cultivnrs[J]. Agril Food chem. 1990, 38:99-101
[55]李焕秀．梨芽和茎尖多酚氧化酶活性和总酚含量的初步研究[J]．四川农业大学学报．1994，2(2)：218—222
[56]鞠志国．莱阳庄梨果实褐变与多酚氧化酶和酚类物质区域化分布的关系[J]．植物生理学报．1988，14(4) 356—361
[57]陈正华．木本植物组织培养及其应用[M]．高等农业出版社．1986
[58]郑均宝等．树木的营养繁殖[M]．北京林业出版社．1989
[59] Rugini E, Jaciboni A, Bazzoffia A. A simple in vitro method to avoid the initial dark period and to increase rooting in fruit tree[J]. Acta Hortic.,1988,227:438-440
[60] Sriskandaraja S, Mullins M G and Nair Y. Induction of adventitious rooting in vitro difficult-to-propagate cultivars of apple[J]. Plant Sci Lett.,1992,24:1-9
[61] Welander M. In vitro rooting of the apple rootstock M26 in adult and rejvenile growth phases and acclimatization of the plantlets[J]. Physiologia Plantarnm. 1988,58:231-238
[62] Zimmerman R H. Micropropagation of woody plants: Post tissue culture aspects[J]. Acta Horticulture. 1988,227:489-496
[63] Van Der Krieken W M, Breteler H, Visser M. The Role of the conversion of IBA into IAA on root regeneration in apple:introduction of test system[J]. Plant cell reports. 1993,12:203-206
[64]王中英等．生物技术在果树上的应用[J]．世界农业．1997(12)：20．
[65]王宇霖．半个世纪以来我国梨果产业与科技发展的回顾[J]．果树科学．1999，16(4)：239—245．
[66]中国农业科学院果树研究所．近几年我国梨树科学研究的进展[J]．中国果树．
[67]覃兰英等．苹果矮化砧木的组织培养[J]．植物生理学通讯．1981(5)：41
[68]汤浩茹，王永清，邓群仙．用8-羟基喹啉硫酸盐防止梨、苹果外殖体褐变[J]．果树科学．1998，15(2)：112—115
[69]程建军，任运宏，杨咏丽，等．果蔬酶褐变控制的研究进展[J]．东北农业大学学报．2000，31(4)：406—410
[70]潘增光，邓秀新．苹果组织培养再生技术研究进展[J]．果树科学．1998，15(3)：261—266
[71]侯修胜，王玉秋，钱茜．美国苹果抗性砧木试管快繁技术研究[J]．河北果树．2001(1)：5—6，8
[72]姜淑荣，谢秀华，何明莉．无病毒苹果试管苗温室嫁接育苗试验[J]．河北果树．1999(2)：7—8
[73] 孙清荣，孙洪雁．诱导樱桃试管苗生根的研究[J]．落叶果树．2000(4)：5-6
[74] 孙清荣，孙洪雁，赵红军．中国樱桃抗病毒选系试管苗生根的研究[J]．园艺学报．2000，27(1)：57—58
[75] 吕汰，裴建文，王娟．苹果试管苗移栽技术[J]．甘肃农业科技．1999(1)：46
[76] 伍克俊，苟永平，贾福明．大樱桃脱毒试管苗移栽条件研究[J]．北方园艺．1997(3)：
[77] 刘用生，李秀菊．6—BA在园艺生产实践中的应用[J]．植物生理学通讯．1994，30(1)：48—51
[78] 辛玉成，秦淑莲，王然，等．IBA处理在苹果组培嫩梢生根中的作用[J]．莱阳农学院学报．1996，13(1)：36—37
[79] Bhojwaniss, Mullins K and Cohen D. In vitro propagation of pyrus pyrifolia[J]. Scientia Horti, 1984, 23:247-254
[80] Shen X-Sh and Mullions M G. Propagation in vitro of pear, Pyrus communis L. Cultivars"william's Bon chretien", "Packhaa's Triumph"and"Beurre Bosc" [J]. Sci Hort, 1984, 23:51-57
[81] Sarwar M; Skirvin RM; Kushad M; Norton MA. Selecting dwarf apple (Malus X domestica Borkh.) trees in vitro: multiple cytokinin tolerance expressed among three strains of'McIntosh' that differ in their growth habit under field conditions[J].Plant Cell, Tissue and Organ Culture. 1998, 54(2):71-76
[82] Zhu LH; Holefors A; Ahlman A. Transformation of the apple rootstock M.9/29 with the rolB gene and itsinfluence on rooting and growth. [J].Plant Science.2001,160(3):433-439
[83] Possum MWPC van; Klerk GJM de; Plas LHW van der. Adventitious regeneration from tulip, lily and apple explants at different oxygen levels[J].Journal of Plant Physiology. 1998,153(1-2):141-145
[84] L.H.Zhu; M.Welander. Adventitious shoot regeneration of two dwarfing pear rootstocks and the development of a transformation protocol[J].The Journal of Horticultural Science & Biotechnology. 2000,75(6):745-752
[85] Tony Webster; Ken Tobutt; Kate Evans. Breeding and evaluation of new rootstocks for apple, pear and sweet cherry[J].Compact Fruit Tree. 2000,33(4): 100-104
[86] Sridevy Sriskandarajah; Goodwin P; Sriskandarajah S.Conditioning promotes regeneration and transformation in apple leaf explants[J].Plant Cell, Tissue &
Culture. Vol.53,No. 1,1998,53(1) : 1-11
[87] Lambardi M; Benelli C; Fabbri A.In vitro axillary shoot proliferation of apple rootstocks under different ethylene conditions[J].In Vitro Cellular & Developmental Biology-PlanLl997,33(1) : 70-74
[88] Faggioli F; Martino L; Barba M.In vitro micrografting of Pyrus communis shoot tips[J].Advances in Horticultural Science. 1997,1 l(1) :25-29
[89] Caboni E; Tonelli MG; Lauri P.In vitro shoot regeneration from leaves of wild pear[J].Plant Cell, Tissue and Organ Culture. 1999,59(1) : 1-7
[90] Shibli RA; Ajlouni MM; Jaradat A.Micropropagation in wild pear (Pyrus syrica) [J].Scientia Horriculturae. 1997,68(l-4) :237-242
[91] Jacob HB.Pyrodwarf, a new rootstock for high density pears[J]. Good Fruit Grower. 2000,51(4) :47-50
[92] Caboni E; Lauri P; Damiano C. Somaclonal variation induced by adventitious shoot regeneration in pear and apple[J]. Acta Horticulturae. 2000,530:195-201
[93] Shibli RA; Mohammad MJ; Ajlouni MM. Stability of chemical parameters of tissue culture medium (pH, osmolarity, electrical conductivity) as a function of time of growth[J]..Journal of Plant Nutrition. 1999,22(3) :501-510
[94] Karhu ST. Sugar use in relation to shoot induction by sorbitol and cytokinin in apple[J].Journal of the American Society for Horticultural Science, 1997 ,122(4) :476-480
[95] Llamoca-Zarate RM.; Aguiar LF.; Landsmann J. Whole plant regeneration from the shootapical meristem of Opuntiaficus-indica Mill. (Cactaceae) [J].Journal of Applied Botany Angewandte Botaiuk, 1999,73(3-4) :.83-85
[96] Perez-Tornero 0． ; Vanoostende A.; Burgos L. Assessment of factors affecting adventitious shoot regeneration from in vitro cultured leaves of apricot[J].Plant Science, 2000,158(1-2) : 61-70
[97] E. Caboni; P.Lauri; S.D'Angeli. In vitro plant regeneration from callus of shoot apices in apple shoot culture[J]. Plant Cell Reports, 2000,19(8) :755-760
[98] Astrid Lux-Endrich; Dieter Treutter; Walter Feucht. Influence of nutrients and carbohydrate supply on the phenol composition of apple shoot cultures[J].Plant Cell, Tissue and Organ Culture, 2000,60(1) :15-21
[99] Saito A; Suzuki M. Plant regeneration from meristem-derived callus protoplasts of apple (Malus X domestica cv. Tuji') [J].Plant Cell Reports,1999,18(7-8) :549-553
[100] Sansavini S: Musacchi S. New pear orchards: densities, rootstocks, training systems [J]. Rivista di Frutticoltura e di Ortofloricoltura ,
2000 ,62(9) :84-94,
[101] Klerk GJ de: de Klerk GJ: Cassells AC (ed.). Rooting treatment and the ex vitrum performance of micropropagated plants[J]. ActaHorticulturae, 2000 ,530: 277-288
[2] 贾敬贤等．梨树矮化密植栽培[M]．金盾出版社．1995
[3] 曹孜义等．实用植物组织培养技术教程[M]．甘肃科学技术出版社．1998
[4] Lane W D. Regeneration of pear plants from shoot meristem tips[J]. Plant Sci. lett, 1979,16:337-342
[5] 赵惠祥等．梨种子胚的培养[J]．辽宁农业科学．1981(3)：31—33
[6] 赵惠祥等．梨的试管实生苗茎尖培养再生植株[J]．山西果树．1981(3)：12
[7] Elisabeth Chevreau, Fabienne Mourgues, Martine Neveu et al. Effect of gelling agents and antibiotics on adventitious bud regeneration from in vitro leaves of pear[J]. In Vitro Cellular & Developmental Biology-Plant. 1997, 33(3): 173-179
[8] 8.W.David Lane,Hiroyuki Iketani Tateki Hayashi. Shoot regeneration from cultured leaves of Japanese pear(Pyrus pyrifolia)[J].Plant Cell, Tissue and Organ Culture. 1998,54(1):9-14
[9] 孟庆田．紧凑型矮化梨组织培养获得完整试管苗[J]．天津农林科技．1994(1)：3-5
[10] 王乔春．梨试管苗的生根[J]．果树科学．1994，11(3)：145—148
[11] 王乔春．植物生长调节剂对梨试管苗培育及移栽的影响[J]．果树科学．1995，12(1)：15-20
[12] 杨恩芹，王震星，刘贵仁，等．外源激素对西洋梨快速繁殖的影响[J]．天津农林科技．1996(2)：7-8
[13] 王震星，张磊，刘贵仁．西洋梨茎段离体培养与快速繁殖的探讨[J]．天津农业科学．1998(2)：16-19
[14] 赵惠祥等．梨茎尖离体培养[J]．植物学报．1982，24(4)：392-394
[15] 战淑敏等．莱阳庄梨成龄树茎尖培养的研究[J]．莱阳农学院学报．1992(2)：118—122
[16] 李晓玲，由继红，杨文杰．光对“苹博一号”梨试管苗生长影响[J]．北方园艺．136：56—57
[17]李晓玲等．“苹博一号”梨试管苗的光自养生长和光混合营养生长[J]．特产研究．2000(1)：11-12，20
[18]王定康，王琼芳．玉香梨的组织培养与快速繁殖[J]．植物生理学通讯．1999，35(3)：207
[19]韩文璞，袁明莲．黄金梨的组织培养和快繁研究[J]．落叶果树．2001(2)：7-8
[20]张磊，等．鸭梨三倍体实生苗组织培养快繁试验[J]．中国果树．1996(2)：20—21
[21]孙清荣．梨成熟胚子叶不定梢诱导[J]．河北农业大学学报．1999(2)：50—52
[22]孙清荣，孙洪雁．西洋梨“丰产”叶片不定梢再生[J]．落叶果树．1999(4)：9-10
[23]Wang Q C. Factors affecting rooting of microcuttings of the pear rootstock 8P10030[J]. Scientia Hortic, 1991, 45:209-213
[24]Pious Thomas,M.B.Ravindra. Shoot tip culture in mango: influence of medium, genotype, explant factors, season and decontamination treatments on phenolic exudation, explant survival and axenic culture establishment[J].Journal of Horticultural Science. 1997,72(5): 713-722
[25]晏本菊，李焕秀．梨外殖体褐变与多酚氧化酶及酚类物质的关系[J]．四川农业大学学报．1998，16(3)：310—313
[26]Maneesh Mishra, R.P. Saxena,R.K.Pathak. Effeet of antioxidants, phenol binding agents and explant waxing on in vitro browning of aonla(Emblica officials Gaertn)[J].Progressive Horticulture. 1998,30(3-4): 128-134
[27]赵剑，杨文杰．苹果梨离体快繁过程中产生玻璃化苗时的某些生理生化变化[J]．植物生理学通讯．1998，34(3)：200-201
[28]师校欣，陈四维，马宝琨，等．苹果砧木离体培养中玻璃化问题的研究[J]．河北农业大学学报．1990，13(3)：12-16
[29]程家胜，史永忠，张志云，等．苹果组织培养中的玻璃苗问题[J]．植物生理学通讯．1990(1)：33—35
[30]Masuda Y.,Yoshida Y.,Bessho H. et al. Studies on cell, tissue and shoot-tip culture in apple. I. Relationship between abnormal shoot formation and plant homones[J].Bull Tree Res Stn. 1988,15:21-28
[31]Phan C T and Letouze R.A comparative study of cholorophyll, phenolic and protein contents and of hydroxycinnamate:CoA ligase activity of normal and vitreous plants(Prunus avium L.)obtained in vitro[J]. Plan Sci Letters. 1983,31:323-327
[32]Rugini E, Tarini P and Rossodivita M E. Control of shoot"vitrification" of almond and olive grown in vitro[J]. Acta Hort. 1987,212:177-183
[33]Vieitez A M, Ballester A, San-Jose M C et al. Anatomical and chemical studies of vitrified shoots of chestnut regenerated in vitro[J].Physiol Plant. 1985,65:177-184
[34]赵剑，杨文杰．梨芽离体快繁过程中玻璃化苗的发生与氧自由基胁迫的关系[J]．西北植物学报．1998，18(2)：183—189
[35]陈国菊等．荠菜试管苗玻璃化的控制[J]．西南农业大学学报．1992，14(3)：253
[36]李云等．珠美海棠试管苗玻璃化发生机理的初步研究[J]．北京林业大学学报．1996(1)：53
[37]Marga F., Vebret L., Morvan H..Agar fractions could protect apple shoots cultured in liquid media against hyperhydricity[J]. Plant Cell, Tissue & Organ Culture. 1997,49(1): 1-5
[38]Kevers C. et al. Physiological and biochemical events leading to vitrification of plants cultured in vitro[J]. Physiol. Plant. 1984,61:69-74
[39]Kevers C.et al. Vitrification of carnation in vitro:Changes in ethylene production Acc level and capacity to convert Acc to ethylene[J]. Plant Cell Tissue Culture. 1985,4:215-223
[40]Kevers C.et al. Vitrification of carnation in vitro:Changes in cell wall mechanical properties,cellulose and ligain cortent[J]. Plant Growth Regulation. 1987,5:59-66
[41]袁丽钗．核桃品种试管苗生根及不定根发生过程中特异蛋白表达的研究[D]．保定：河北农业大学园艺学院，2000
[42]黄学林，李竣菊编著．高等植物组织离体培养的形态建成及其调控[M]．科学出版社．1995
[43]李明军等．多效在玉米组织培养中的作用[J]．作物学报．1997(6)：759
[44]雷逢进，李燕娥，岳建雄，等．苹果试管苗快繁技术体系研究[J]．山西农业科学．2001，29(2)：56—58
[45]李均儒．葡萄试管苗移栽技术研究[J]．中国果树．1994(2)：7—9
[46]曹春英，任术琦，丁世民，等．冬枣试管苗驯化及移植条件研究[J]．落叶果树．2001(2)：10—11
[47]马宝琨，高仪．苹果试管苗生根的研究[J]．河北农业大学学报．1990，13(3)：7—11
[48]吕汰，裴建文，王娟．苹果试管苗生根试验研究[J]．甘肃农业科技．1998(6)：31-32
[49]George E F and Sherrington P D. Plant propagation by tissue culture[J]. Exegetics Ltd. 1984
[50]Maarri K A, Arnaud Y and Miginiac E. In vitro micropropagation of Quince(Cydonia oblonga Mill.)[J]. Sci Hort, 1986,28:315-321
[51]Dunstan D I, Turner K and Lazaroffr W R. Propagation in vitro of the apple rootstock M4: effect of phytohormones on shoot quality[J]. Plant Cell Tissue Organ Culture. 1985,4:55-60
[52]Dornman C H and Vogelmann T C. Effect of rigidity of gel medium on benzyladenine-induced adventitious bud formation and vitrification in vitro in Picea abies[J]. Physiol Plant. 1984,61:505-512
[53]Debergh P C. Effects of agar brand and concentration on the tissue culture medium[J]. Physiol Plant. 1983,59:270-276
[54]Lee C Y, Kagan V, Jaworsk A W. Enezymatic Browning in Relation to phenolic compounds and polyphenoloxidase Activity among various peach cultivnrs[J]. Agril Food chem. 1990, 38:99-101
[55]李焕秀．梨芽和茎尖多酚氧化酶活性和总酚含量的初步研究[J]．四川农业大学学报．1994，2(2)：218—222
[56]鞠志国．莱阳庄梨果实褐变与多酚氧化酶和酚类物质区域化分布的关系[J]．植物生理学报．1988，14(4) 356—361
[57]陈正华．木本植物组织培养及其应用[M]．高等农业出版社．1986
[58]郑均宝等．树木的营养繁殖[M]．北京林业出版社．1989
[59] Rugini E, Jaciboni A, Bazzoffia A. A simple in vitro method to avoid the initial dark period and to increase rooting in fruit tree[J]. Acta Hortic.,1988,227:438-440
[60] Sriskandaraja S, Mullins M G and Nair Y. Induction of adventitious rooting in vitro difficult-to-propagate cultivars of apple[J]. Plant Sci Lett.,1992,24:1-9
[61] Welander M. In vitro rooting of the apple rootstock M26 in adult and rejvenile growth phases and acclimatization of the plantlets[J]. Physiologia Plantarnm. 1988,58:231-238
[62] Zimmerman R H. Micropropagation of woody plants: Post tissue culture aspects[J]. Acta Horticulture. 1988,227:489-496
[63] Van Der Krieken W M, Breteler H, Visser M. The Role of the conversion of IBA into IAA on root regeneration in apple:introduction of test system[J]. Plant cell reports. 1993,12:203-206
[64]王中英等．生物技术在果树上的应用[J]．世界农业．1997(12)：20．
[65]王宇霖．半个世纪以来我国梨果产业与科技发展的回顾[J]．果树科学．1999，16(4)：239—245．
[66]中国农业科学院果树研究所．近几年我国梨树科学研究的进展[J]．中国果树．
[67]覃兰英等．苹果矮化砧木的组织培养[J]．植物生理学通讯．1981(5)：41
[68]汤浩茹，王永清，邓群仙．用8-羟基喹啉硫酸盐防止梨、苹果外殖体褐变[J]．果树科学．1998，15(2)：112—115
[69]程建军，任运宏，杨咏丽，等．果蔬酶褐变控制的研究进展[J]．东北农业大学学报．2000，31(4)：406—410
[70]潘增光，邓秀新．苹果组织培养再生技术研究进展[J]．果树科学．1998，15(3)：261—266
[71]侯修胜，王玉秋，钱茜．美国苹果抗性砧木试管快繁技术研究[J]．河北果树．2001(1)：5—6，8
[72]姜淑荣，谢秀华，何明莉．无病毒苹果试管苗温室嫁接育苗试验[J]．河北果树．1999(2)：7—8
[73] 孙清荣，孙洪雁．诱导樱桃试管苗生根的研究[J]．落叶果树．2000(4)：5-6
[74] 孙清荣，孙洪雁，赵红军．中国樱桃抗病毒选系试管苗生根的研究[J]．园艺学报．2000，27(1)：57—58
[75] 吕汰，裴建文，王娟．苹果试管苗移栽技术[J]．甘肃农业科技．1999(1)：46
[76] 伍克俊，苟永平，贾福明．大樱桃脱毒试管苗移栽条件研究[J]．北方园艺．1997(3)：
[77] 刘用生，李秀菊．6—BA在园艺生产实践中的应用[J]．植物生理学通讯．1994，30(1)：48—51
[78] 辛玉成，秦淑莲，王然，等．IBA处理在苹果组培嫩梢生根中的作用[J]．莱阳农学院学报．1996，13(1)：36—37
[79] Bhojwaniss, Mullins K and Cohen D. In vitro propagation of pyrus pyrifolia[J]. Scientia Horti, 1984, 23:247-254
[80] Shen X-Sh and Mullions M G. Propagation in vitro of pear, Pyrus communis L. Cultivars"william's Bon chretien", "Packhaa's Triumph"and"Beurre Bosc" [J]. Sci Hort, 1984, 23:51-57
[81] Sarwar M; Skirvin RM; Kushad M; Norton MA. Selecting dwarf apple (Malus X domestica Borkh.) trees in vitro: multiple cytokinin tolerance expressed among three strains of'McIntosh' that differ in their growth habit under field conditions[J].Plant Cell, Tissue and Organ Culture. 1998, 54(2):71-76
[82] Zhu LH; Holefors A; Ahlman A. Transformation of the apple rootstock M.9/29 with the rolB gene and itsinfluence on rooting and growth. [J].Plant Science.2001,160(3):433-439
[83] Possum MWPC van; Klerk GJM de; Plas LHW van der. Adventitious regeneration from tulip, lily and apple explants at different oxygen levels[J].Journal of Plant Physiology. 1998,153(1-2):141-145
[84] L.H.Zhu; M.Welander. Adventitious shoot regeneration of two dwarfing pear rootstocks and the development of a transformation protocol[J].The Journal of Horticultural Science & Biotechnology. 2000,75(6):745-752
[85] Tony Webster; Ken Tobutt; Kate Evans. Breeding and evaluation of new rootstocks for apple, pear and sweet cherry[J].Compact Fruit Tree. 2000,33(4): 100-104
[86] Sridevy Sriskandarajah; Goodwin P; Sriskandarajah S.Conditioning promotes regeneration and transformation in apple leaf explants[J].Plant Cell, Tissue &
Culture. Vol.53,No. 1,1998,53(1) : 1-11
[87] Lambardi M; Benelli C; Fabbri A.In vitro axillary shoot proliferation of apple rootstocks under different ethylene conditions[J].In Vitro Cellular & Developmental Biology-PlanLl997,33(1) : 70-74
[88] Faggioli F; Martino L; Barba M.In vitro micrografting of Pyrus communis shoot tips[J].Advances in Horticultural Science. 1997,1 l(1) :25-29
[89] Caboni E; Tonelli MG; Lauri P.In vitro shoot regeneration from leaves of wild pear[J].Plant Cell, Tissue and Organ Culture. 1999,59(1) : 1-7
[90] Shibli RA; Ajlouni MM; Jaradat A.Micropropagation in wild pear (Pyrus syrica) [J].Scientia Horriculturae. 1997,68(l-4) :237-242
[91] Jacob HB.Pyrodwarf, a new rootstock for high density pears[J]. Good Fruit Grower. 2000,51(4) :47-50
[92] Caboni E; Lauri P; Damiano C. Somaclonal variation induced by adventitious shoot regeneration in pear and apple[J]. Acta Horticulturae. 2000,530:195-201
[93] Shibli RA; Mohammad MJ; Ajlouni MM. Stability of chemical parameters of tissue culture medium (pH, osmolarity, electrical conductivity) as a function of time of growth[J]..Journal of Plant Nutrition. 1999,22(3) :501-510
[94] Karhu ST. Sugar use in relation to shoot induction by sorbitol and cytokinin in apple[J].Journal of the American Society for Horticultural Science, 1997 ,122(4) :476-480
[95] Llamoca-Zarate RM.; Aguiar LF.; Landsmann J. Whole plant regeneration from the shootapical meristem of Opuntiaficus-indica Mill. (Cactaceae) [J].Journal of Applied Botany Angewandte Botaiuk, 1999,73(3-4) :.83-85
[96] Perez-Tornero 0． ; Vanoostende A.; Burgos L. Assessment of factors affecting adventitious shoot regeneration from in vitro cultured leaves of apricot[J].Plant Science, 2000,158(1-2) : 61-70
[97] E. Caboni; P.Lauri; S.D'Angeli. In vitro plant regeneration from callus of shoot apices in apple shoot culture[J]. Plant Cell Reports, 2000,19(8) :755-760
[98] Astrid Lux-Endrich; Dieter Treutter; Walter Feucht. Influence of nutrients and carbohydrate supply on the phenol composition of apple shoot cultures[J].Plant Cell, Tissue and Organ Culture, 2000,60(1) :15-21
[99] Saito A; Suzuki M. Plant regeneration from meristem-derived callus protoplasts of apple (Malus X domestica cv. Tuji') [J].Plant Cell Reports,1999,18(7-8) :549-553
[100] Sansavini S: Musacchi S. New pear orchards: densities, rootstocks, training systems [J]. Rivista di Frutticoltura e di Ortofloricoltura ,
2000 ,62(9) :84-94,
[101] Klerk GJ de: de Klerk GJ: Cassells AC (ed.). Rooting treatment and the ex vitrum performance of micropropagated plants[J]. ActaHorticulturae, 2000 ,530: 277-288