百合组织培养及组织培养过程中的染色体行为
|
摘要
百合（LiLium spp）是单子叶植物亚纲百合科（LiLiaceae）百合属（Lilium）的所有种类的总称。百合的鳞茎含有丰富的营养，具有很高的食用价值；中医普遍认为百合入药具有补中益气、养阴润肺、止咳平喘的功效；百合也是一种名贵切花，其花形美丽、花色多样，是继鹤望兰和红掌之后的又一只切花新秀。百合的快繁主要有小鳞茎分株繁殖、鳞片扦插及组织培养。本实验利用百合属的兰州百合（Liliun davidii var.Unicolor（hong）cotton）及野百合（Lilium brownii F.E. brown ex Miellez）进行组织培养，从外植体的取材、消毒、初代培养物的建立、激素的种类及浓度的筛选、芽的增殖、根的诱导、移栽及组织培养过程中两种百合染色体的行为和B染色体进行了研究，初步提供了一组用于两种百合组织培养的培养基，阐述了组织培养过程中两种百合染色体的变化和B染色体的有无。
在两种百合的初代培养时，外植体在2001年春季取回。鳞片取回后用毛刷刷去泥土，流水冲洗24h。材料接种前在超净台上用75%酒精浸泡30s后，于0.1%升汞溶液中消毒13min，最后用无菌水冲洗5-6次。将消完毒的兰州百合及野百合外植体切成0.5cm*0.5cm的方块,分别接种于MS+0.6-1.0mg/lBA+0.2mg/LNAA及MS+0.1-0.5mg/lBA+0.1-0.2mg/LNAAH或0.5mg/lIAA的诱导培养基上，诱导率可高达70-100%，并发现外植体有以外层下部与鳞茎盘相连的部分为最佳外植体。
增殖培养时，兰州百合的最佳增殖培养基为MS+0.5mg/lBA+0.1mg/lNAA，而MS+0.6mg/lBA+0.1mg/lNAA及MS+1.0mg/lBA+0.1mg/lIAA也有较好的诱导效果，野百合的最佳增殖培养基为MS+1.0mg/lBA+0.1mg/LIAA,而MS+0.7mg/LBA+0.2mg/lNAA及MS+0.9mg/lBA+0.3mg/lNAA也较理想。在23-25℃，2000LX，16h光照，8h黑暗或12h黑暗，12h光照条件下培养，每28-32d左右继代一次，继代6次左右，增殖倍数可达4-5倍。
生根培养时，以1/2MS为基本培养基，附加0.1-0.2mg/lNAA或IAA可使根长的长而壮，活性炭对根的生长有利。根长至1-1.5cm时，开瓶炼苗3-5d后，移栽至盛有蛭石加腐殖土（1:1）的纸杯中，保持70%左右的湿度，60%的自然光，成活率可达80%以上，且移栽后长势良好。
组织培养时染色体存在一定的变异。愈伤组织中兰州百合及野百合的变异率分别为54.4%和49.4%，染色体数目变异范围分别为9-50和12-50条；而芽端分生组
织中兰州百合及野百合的变异率分别为17.5%和9.5%，说明愈伤组织中染色体的变异率高于芽端分生组织。激素对组织培养过程中染色体行为有明显的影响，单独使用一种激素如NAA引起的兰州百合及野百合染色体的变异率为3.5%及2.8%，而BA和NAA搭配引起的变异率普遍高于此值。另外不同种类及浓度的激素搭配引起的变异率也不相同。综合诱导率及变异率两种因素认为，No.5及No.11分别为兰州百合及野百合的最佳诱导培养基。组织培养时，继代次数不宜过多。实验中发现，继代次数增多导致染色体变异率的增加，染色体变异率增加使芽的增殖能力降低。兰州百合继代以4-5次较好，野百合以6次为好。不同基因型的百合在组织培养过程中染色体变异率无显著差异。根尖细胞中染色体变异率显著低于组织培养过程中各种组织的细胞。
对兰州百合及野百合的根尖细胞、愈伤组织及芽端分生组织细胞进行普通压片观察，发现野百合各种组织细胞中无B染色体被观察到；兰州百合的根尖、愈伤组织及芽端分生组织细胞中有B染色体，数目为1-2条。
This article research the tissue culture and cloning propagation and the chromosomal variation of two kinds of lilies —Lilium davidii Var.Unicolor(Hoog)Cotton and Lilium brownii F.E.Brown ex miellez.At the same time, we also observed the nuclear and the B-chromosome. The tissue culture and cloning propagation include the establishment of aseptic culture, bud induction and proliferation, selection of suitable hormone , rooting and acclimatization. The chromosomal variation include the change of chromosome number mainly.
When the explants were collected in the spring, high induction rate can be achieved. Brushing the dust and washing the explants under ranning tap water to remove some of the dirt and contaminations, in 75%CH3CH2OH for 30 second and in 0.1%Hgcl2 for 13 minutes, rinsed 5-6 times with sterile water. The two kinds of explants were cultured on Murashige&Skoog medium(MS) added with 0.6-1.0mg/lN-benzyladenine(BA)+0.2mg/l naphthalane acetic(NAA)and added with 0.1-0.5mg/lBA+0.1-0.2mg/lNAA or indole-3-acetic(IAA).The induction rate can reached 70-100%.The best explants were the parts that were below and linked the scales.
During the buds poliferation,the best medium of L.davidii Var.Unicolor(Hoog)Cotton
is MS+0.5mg/lBA+0.1mg/lNAA, the best medium of L.brownii F.E.Brown ex miellez is MS+1.0mg/lBA+0.1mg/lIAA. It good to keep the culture at 23-25℃,photoperiod with a light intensity of 2,000Lx,keep 16 hours lighting and 8 hours darking or 12 hours lighting and 12hours darking on culture, to subculture every 28-32days.The poliferation can be reached 4-5 times.
One-half MS basal medium added 0.1-0.2mg/lNAA or IAA can make the root long and strong. The activated charcoal is good to root formation and growth. When the length of root is 1-1.5cm,lilies can be acclimatized. If the living condition is good, the survival rate were over 80%
During the tissue culture, we found the chromosomal variation in cells of two kinds of lilies. The variation rate of callus of L.davidii Var.Unicolor(Hoog)Cotton is 54.4%and the L.brownii F.E.Brown ex miellez is49.4%, the chromosome number varied from 9-50
and 12-50;the variation rate of buds of L.davidii Var.Unicolor(Hoog)Cotton and L.brownii F.E.Brown ex miellez are 17.5%and 19.5%,these show that the variation rate of callus is higher than buds. The hormone can affect the deeds of the chromosome. The variation rate caused by single hormone is lower than two kinds of hormone, the kind and intensity of hormone is different, the variation rate is different. We think that No.5and No.11 are best medium to two kinds of lilies when we thought over the induction rate and variation rate. The times of subculture should be limited because we found when the subculture times is increasing, the variation rate is higher and the differentiation capacity is lower. The best times of L.davidii Var.Unicolor(Hoog)Cotton is 4-5,the other is 6 times.
We observed the cells of root-tip, callus and bud of two kinds of lilies, we could not find the B-chromosome in cells of L. brownii F.E. Brown ex miellez, but find it in L.davidii Var.Unicolor(Hoog)Cotton,the number is 1-2.
在两种百合的初代培养时，外植体在2001年春季取回。鳞片取回后用毛刷刷去泥土，流水冲洗24h。材料接种前在超净台上用75%酒精浸泡30s后，于0.1%升汞溶液中消毒13min，最后用无菌水冲洗5-6次。将消完毒的兰州百合及野百合外植体切成0.5cm*0.5cm的方块,分别接种于MS+0.6-1.0mg/lBA+0.2mg/LNAA及MS+0.1-0.5mg/lBA+0.1-0.2mg/LNAAH或0.5mg/lIAA的诱导培养基上，诱导率可高达70-100%，并发现外植体有以外层下部与鳞茎盘相连的部分为最佳外植体。
增殖培养时，兰州百合的最佳增殖培养基为MS+0.5mg/lBA+0.1mg/lNAA，而MS+0.6mg/lBA+0.1mg/lNAA及MS+1.0mg/lBA+0.1mg/lIAA也有较好的诱导效果，野百合的最佳增殖培养基为MS+1.0mg/lBA+0.1mg/LIAA,而MS+0.7mg/LBA+0.2mg/lNAA及MS+0.9mg/lBA+0.3mg/lNAA也较理想。在23-25℃，2000LX，16h光照，8h黑暗或12h黑暗，12h光照条件下培养，每28-32d左右继代一次，继代6次左右，增殖倍数可达4-5倍。
生根培养时，以1/2MS为基本培养基，附加0.1-0.2mg/lNAA或IAA可使根长的长而壮，活性炭对根的生长有利。根长至1-1.5cm时，开瓶炼苗3-5d后，移栽至盛有蛭石加腐殖土（1:1）的纸杯中，保持70%左右的湿度，60%的自然光，成活率可达80%以上，且移栽后长势良好。
组织培养时染色体存在一定的变异。愈伤组织中兰州百合及野百合的变异率分别为54.4%和49.4%，染色体数目变异范围分别为9-50和12-50条；而芽端分生组
织中兰州百合及野百合的变异率分别为17.5%和9.5%，说明愈伤组织中染色体的变异率高于芽端分生组织。激素对组织培养过程中染色体行为有明显的影响，单独使用一种激素如NAA引起的兰州百合及野百合染色体的变异率为3.5%及2.8%，而BA和NAA搭配引起的变异率普遍高于此值。另外不同种类及浓度的激素搭配引起的变异率也不相同。综合诱导率及变异率两种因素认为，No.5及No.11分别为兰州百合及野百合的最佳诱导培养基。组织培养时，继代次数不宜过多。实验中发现，继代次数增多导致染色体变异率的增加，染色体变异率增加使芽的增殖能力降低。兰州百合继代以4-5次较好，野百合以6次为好。不同基因型的百合在组织培养过程中染色体变异率无显著差异。根尖细胞中染色体变异率显著低于组织培养过程中各种组织的细胞。
对兰州百合及野百合的根尖细胞、愈伤组织及芽端分生组织细胞进行普通压片观察，发现野百合各种组织细胞中无B染色体被观察到；兰州百合的根尖、愈伤组织及芽端分生组织细胞中有B染色体，数目为1-2条。
This article research the tissue culture and cloning propagation and the chromosomal variation of two kinds of lilies —Lilium davidii Var.Unicolor(Hoog)Cotton and Lilium brownii F.E.Brown ex miellez.At the same time, we also observed the nuclear and the B-chromosome. The tissue culture and cloning propagation include the establishment of aseptic culture, bud induction and proliferation, selection of suitable hormone , rooting and acclimatization. The chromosomal variation include the change of chromosome number mainly.
When the explants were collected in the spring, high induction rate can be achieved. Brushing the dust and washing the explants under ranning tap water to remove some of the dirt and contaminations, in 75%CH3CH2OH for 30 second and in 0.1%Hgcl2 for 13 minutes, rinsed 5-6 times with sterile water. The two kinds of explants were cultured on Murashige&Skoog medium(MS) added with 0.6-1.0mg/lN-benzyladenine(BA)+0.2mg/l naphthalane acetic(NAA)and added with 0.1-0.5mg/lBA+0.1-0.2mg/lNAA or indole-3-acetic(IAA).The induction rate can reached 70-100%.The best explants were the parts that were below and linked the scales.
During the buds poliferation,the best medium of L.davidii Var.Unicolor(Hoog)Cotton
is MS+0.5mg/lBA+0.1mg/lNAA, the best medium of L.brownii F.E.Brown ex miellez is MS+1.0mg/lBA+0.1mg/lIAA. It good to keep the culture at 23-25℃,photoperiod with a light intensity of 2,000Lx,keep 16 hours lighting and 8 hours darking or 12 hours lighting and 12hours darking on culture, to subculture every 28-32days.The poliferation can be reached 4-5 times.
One-half MS basal medium added 0.1-0.2mg/lNAA or IAA can make the root long and strong. The activated charcoal is good to root formation and growth. When the length of root is 1-1.5cm,lilies can be acclimatized. If the living condition is good, the survival rate were over 80%
During the tissue culture, we found the chromosomal variation in cells of two kinds of lilies. The variation rate of callus of L.davidii Var.Unicolor(Hoog)Cotton is 54.4%and the L.brownii F.E.Brown ex miellez is49.4%, the chromosome number varied from 9-50
and 12-50;the variation rate of buds of L.davidii Var.Unicolor(Hoog)Cotton and L.brownii F.E.Brown ex miellez are 17.5%and 19.5%,these show that the variation rate of callus is higher than buds. The hormone can affect the deeds of the chromosome. The variation rate caused by single hormone is lower than two kinds of hormone, the kind and intensity of hormone is different, the variation rate is different. We think that No.5and No.11 are best medium to two kinds of lilies when we thought over the induction rate and variation rate. The times of subculture should be limited because we found when the subculture times is increasing, the variation rate is higher and the differentiation capacity is lower. The best times of L.davidii Var.Unicolor(Hoog)Cotton is 4-5,the other is 6 times.
We observed the cells of root-tip, callus and bud of two kinds of lilies, we could not find the B-chromosome in cells of L. brownii F.E. Brown ex miellez, but find it in L.davidii Var.Unicolor(Hoog)Cotton,the number is 1-2.
引文
[1]中国科学院 《中国植物志》编委会.《中国植物志》（第十四卷 百合科） 北京：科学出版社，1980 116-166
[2]龙雅宜，张金政，张兰年等，《百合—球根花卉之王》 北京：金盾出版社，1999 1-6
[3]《秦岭植物志》 第一卷. 种子植物（第一册）
[4]王刚，杜捷，甘肃百合种质资源及其利用 西北师范大学学报（自然科学版 2001专辑）102-104
[5]《中华人民共和国药典》
[6]王康才 《百合栽培新技术》 北京：中国农业出版社，1999
[7]李德美，赵祥云，《切花生产与保鲜技术问答》北京：中国农业出版社，1999
[8]金波，东惠茹， 《球根花卉》北京：中国农业出版社，1999
[9]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 185-187
[10]崔凯荣，戴若兰，《植物体细胞胚发生的分子生物学》北京：科学出版社 2000
[11]F.C.Steward,M.O.Mapes&K.Mears. 1958. Growth and Organized development of cultured cells Ⅱ organization in cultures growth from freely suspend cells. American Journal of Botany,45:705-708
[12]J.Reinert, uber die Kontrolle der Morphogenes und die Induktion von Adventive-embryonen an Gewebekulturen aus Karotten.1959.Plant,53:318-333
[13]郑企成，朱光耀，陈文华等，小麦体细胞无性系子粒胚乳醇溶蛋白的变异 核农学报 1991 5（3）：134-138
[14]鲍雪珍，张志忠，沈利爽等 葡萄胚性愈伤组织无性系的建立 保持和植株再生的研究 山东大学学报（自然科学学报），1995 30（1）：：105-111
[15]徐子勤，贾敬芬，豆科植物的体细胞杂交 植物生理学通讯，1997，33（4）：288-291
[16]L.Martinelli, and G.Mandoline. Genetic transformation and regeneration of transgenetic plant in grape vine (vitis rupestris).Theor.Appl.Genet.,1994,88:621-678
[17]张松,李红蓉,李滨等,分葱组织培养体细胞胚发生的研究,园艺学报,1997,24(3):264-268
[18]伍宁丰,范云六,苏云金芽孢杆菌杀虫蛋白的杨树工程植株的建立,科学通报,1991,(9):705-708
[19]P.Avihai,P.L.Ofra,M.Abu-Abied&D.Holland,Establishment of an Agrobacterium-mediated transformation system for grape:The role of antioxidants during grape-Agrobacterium interations,
Nature Biotechnology,1996,14:624-628
[20]C.J.J.M Raemakers,E.Jacobsen,R.G.F.Visser, Secondaty somatic embryogenesis and applications in plant breeding, Euphytical,1995,81:93-107
[21]Debergh pc and pe, read, Micropagation. In:P.C.Debergh and R.H.Zimmerman(Ends) Micropropa-gation,Kluwer Academic Publishers,Netherlands,1991,1-13
[22]赵祥云,王树栋,陈新露等, 《百合》北京：中国农业出版社 2000
[23]汪有良，南京百合快速繁殖，江苏林业科技，1995（1）：35-36，43
[24]丁兰，刘国安，田卫东等，新铁炮百合组织培养和快速繁殖研究，西北师范大学学报（自然科学版），2001，37（1）：80-82
[25]朴日子，李华英等，百合试管苗快速繁殖技术的研究，延边大学农学学报，1999，21（4）：310-312
[26]黄敏玲，陈诗林，透百合离体快速繁殖，植物生理学通讯，1993，29（6）：437
[27]马宪红，丁冰等，毛百合的胚培养，东北林业大学学报，1997，25（1）：26-28
[28]陈善娜，高建丽，郑志英， 百合子房的组织培养， 云南大学学报（自然科学版）1997，19（4）：374-375
[29]刘幼琪，杨毅等，湖北百合离体繁殖中的形态发生， 湖北大学学报，1995，17（2）214-219
[30]许智宏，植物生理学和生物工程， 植物生理学通讯，1994（3）：22
[31]Larkin P G,Scowcroft W R,T A G,1981,60:197-214
[32] Larkin P G,Scowcroft W R,T A G,1984,67:443-445
[33]商效民，细胞生物学杂志，1984，（1）：5-12
[34]Murashige J, Plant Physiol,1974,25:135-166
[35]Green G E, Hort Sci,1977,12:7-10
[36]Skirvin R M, J Amer Soc Hort Sci,1976,10:67-74
[37]Seter P,et al, Plant Sci Lett,1982,25:345-352
[38]王关林，方宏筠， 体细胞无性系变异的遗传机制，辽宁师范大学自然科学学报，1991，14（2）：168-171
[39]罗士韦，细胞生物学杂志，1982，4（2）：1-9
[40]胡含，中国科学，1980，5：485-491
[41]Ahloowalin B S,Plant cell culture,Canada,Calgary,1978:162
[42]Brettel R S,et al.T A G,198058:55-58
[43]胡含， 遗传学报，1978，5（1）：23-29 （二手资料）
[44]Gamberg O L,et al.Plant Sci Lett,1977,10:67-74
[45]heiuz D J,et al,Crop Sci,1969,9:346-348
[46]Krishnamurthi M,et al.Technol,1974,15:130-137
[47]Banks M S, et al. Plant Sci Lett,1976,7:417-427
[48]贾敬芬， 植物学报，1981，23：17
[49]Orten T J,T A G,1980,56:101-112
[50]褚圻， 遗传学，上海：上海教育出版社，1980，6-10
[51]McCoy T J,et al.Can J Genet Cytol,1982,24:37-50
[52]Siminvitch L.Cell,1976,7:1-11
[53]Carlsonp,Sci,1973,180:1366-1368
[54]Cullis C A, et al. The Plant Genome,Norway,Norwich,1980,91-97
[55]Siegel A, Modification of the Information Content of Plant Cell, Netherlands Amsterdam,1975,15-26
[56]Whitfeld R R,Bottmley W,Ann Rev Plant Physiol,1983,34:279-310
[57]许智宏，遗传，1985，7（6）37-40（二手资料）
[58]Bayliss M W, Nature,1973,246:529-530
[59]Gengenbach B G,et al. Crop Nat Acad Sci,1977,74:513-517
[60]Kasperbauer M J,et al.Crop Sci,1979,19:457-460
[61]Hoffmann F, Production of Natural Compounds by Cell Culture Methods,G D R, Munchen,1978,319-329
[62]Thomas e,et al.Z Pflanzenzucht,1979,82:1-30
[63]Jacobsen E, Plant Cell Tissue and Organ Culture,1981,1:77-84
[64]Karp A,et al. T A G,1982,63:256-272
[65]Ruiz M L,et al. Protplasma,1982,111:83-86
[66]王关林，植物学报，1989，31；（7）495-504
[67]Bayliss M W,Int Rev Cytol,1980,11A113-114
[68]Barbier M,et al. Plant,1980,30:321-344
[69]Novak F J,Cytologia,1981,46:371-380
[70]Yeoman M M,et al.Int Cytol Suppl,1980,11A:1-21
[71]Singh M R,Cytologia,1982,47:419-426
[72]黄娇香，遗传，1981：（3）31-32（二手资料）
[73]杨宁，美国大杏仁砧木微繁殖技术研究，2000年硕士论文
[74]杨汉民，王亚馥，王仑山等，五种植物组织培养物的染色体变异，植物体细胞无性系变异与育种，江苏科学技术出版社 189-196
[75]汪丽虹，杨汉民，枸杞再生植株不同发育途径中染色体变异的研究，植物学报，8（增刊）：61-64
[76]张金文，曹孜义，崔建垣，继代十七次的玉米花粉胚性细胞系的异常核和异常分裂观察，实验生物学报，30（4）：407-415
[77]R.Roth,I.Ebert,&J.Schrnidl,Trisomy associated with loss of maturation capacity in a long-term embryogenic culture of abies alba,Theoretical and Applied Genetics,1997,9(3):353-358
[78]An Lijia,He Menyuan and Hao Shui, Variation of Differentation Capacity of Maize Pollen Clones and Instability of Chromosome Number, Journal of Northeast Normal University,1986,(3):111-116
[79]Jones R N,Rees.h, London Ltd; Academic Press IHC INC,B Chromosome,1982
[80]李懋学，张赞平，《作物染色体及其研究技术》北京：中国农业出版社，1990
[81]Gupta P K, Tsuchiya T. Chromosome Engineer in Plant:Genetic.Breeding.Evolution. New York: Elsevier Science Publishing Company INC,1991.141-157
[82]Mendelson D,Zohary D, Behaviour and transmission of supernumerary chromosome in Aegilops speltoides. Heredity,1972,29:329-339
[83]Hewitt G M, The intergation of supernumerary chromosome into the orthopteran genome. Cold Spring Harb Symp Quant Biol,1973c,38:183-194
[84]Zheng Y R, Yang Q L et al. Studies on the origin of B-chromosomes in rye. Science in China(series B).1991,34:1186-1197
[2]龙雅宜，张金政，张兰年等，《百合—球根花卉之王》 北京：金盾出版社，1999 1-6
[3]《秦岭植物志》 第一卷. 种子植物（第一册）
[4]王刚，杜捷，甘肃百合种质资源及其利用 西北师范大学学报（自然科学版 2001专辑）102-104
[5]《中华人民共和国药典》
[6]王康才 《百合栽培新技术》 北京：中国农业出版社，1999
[7]李德美，赵祥云，《切花生产与保鲜技术问答》北京：中国农业出版社，1999
[8]金波，东惠茹， 《球根花卉》北京：中国农业出版社，1999
[9]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 185-187
[10]崔凯荣，戴若兰，《植物体细胞胚发生的分子生物学》北京：科学出版社 2000
[11]F.C.Steward,M.O.Mapes&K.Mears. 1958. Growth and Organized development of cultured cells Ⅱ organization in cultures growth from freely suspend cells. American Journal of Botany,45:705-708
[12]J.Reinert, uber die Kontrolle der Morphogenes und die Induktion von Adventive-embryonen an Gewebekulturen aus Karotten.1959.Plant,53:318-333
[13]郑企成，朱光耀，陈文华等，小麦体细胞无性系子粒胚乳醇溶蛋白的变异 核农学报 1991 5（3）：134-138
[14]鲍雪珍，张志忠，沈利爽等 葡萄胚性愈伤组织无性系的建立 保持和植株再生的研究 山东大学学报（自然科学学报），1995 30（1）：：105-111
[15]徐子勤，贾敬芬，豆科植物的体细胞杂交 植物生理学通讯，1997，33（4）：288-291
[16]L.Martinelli, and G.Mandoline. Genetic transformation and regeneration of transgenetic plant in grape vine (vitis rupestris).Theor.Appl.Genet.,1994,88:621-678
[17]张松,李红蓉,李滨等,分葱组织培养体细胞胚发生的研究,园艺学报,1997,24(3):264-268
[18]伍宁丰,范云六,苏云金芽孢杆菌杀虫蛋白的杨树工程植株的建立,科学通报,1991,(9):705-708
[19]P.Avihai,P.L.Ofra,M.Abu-Abied&D.Holland,Establishment of an Agrobacterium-mediated transformation system for grape:The role of antioxidants during grape-Agrobacterium interations,
Nature Biotechnology,1996,14:624-628
[20]C.J.J.M Raemakers,E.Jacobsen,R.G.F.Visser, Secondaty somatic embryogenesis and applications in plant breeding, Euphytical,1995,81:93-107
[21]Debergh pc and pe, read, Micropagation. In:P.C.Debergh and R.H.Zimmerman(Ends) Micropropa-gation,Kluwer Academic Publishers,Netherlands,1991,1-13
[22]赵祥云,王树栋,陈新露等, 《百合》北京：中国农业出版社 2000
[23]汪有良，南京百合快速繁殖，江苏林业科技，1995（1）：35-36，43
[24]丁兰，刘国安，田卫东等，新铁炮百合组织培养和快速繁殖研究，西北师范大学学报（自然科学版），2001，37（1）：80-82
[25]朴日子，李华英等，百合试管苗快速繁殖技术的研究，延边大学农学学报，1999，21（4）：310-312
[26]黄敏玲，陈诗林，透百合离体快速繁殖，植物生理学通讯，1993，29（6）：437
[27]马宪红，丁冰等，毛百合的胚培养，东北林业大学学报，1997，25（1）：26-28
[28]陈善娜，高建丽，郑志英， 百合子房的组织培养， 云南大学学报（自然科学版）1997，19（4）：374-375
[29]刘幼琪，杨毅等，湖北百合离体繁殖中的形态发生， 湖北大学学报，1995，17（2）214-219
[30]许智宏，植物生理学和生物工程， 植物生理学通讯，1994（3）：22
[31]Larkin P G,Scowcroft W R,T A G,1981,60:197-214
[32] Larkin P G,Scowcroft W R,T A G,1984,67:443-445
[33]商效民，细胞生物学杂志，1984，（1）：5-12
[34]Murashige J, Plant Physiol,1974,25:135-166
[35]Green G E, Hort Sci,1977,12:7-10
[36]Skirvin R M, J Amer Soc Hort Sci,1976,10:67-74
[37]Seter P,et al, Plant Sci Lett,1982,25:345-352
[38]王关林，方宏筠， 体细胞无性系变异的遗传机制，辽宁师范大学自然科学学报，1991，14（2）：168-171
[39]罗士韦，细胞生物学杂志，1982，4（2）：1-9
[40]胡含，中国科学，1980，5：485-491
[41]Ahloowalin B S,Plant cell culture,Canada,Calgary,1978:162
[42]Brettel R S,et al.T A G,198058:55-58
[43]胡含， 遗传学报，1978，5（1）：23-29 （二手资料）
[44]Gamberg O L,et al.Plant Sci Lett,1977,10:67-74
[45]heiuz D J,et al,Crop Sci,1969,9:346-348
[46]Krishnamurthi M,et al.Technol,1974,15:130-137
[47]Banks M S, et al. Plant Sci Lett,1976,7:417-427
[48]贾敬芬， 植物学报，1981，23：17
[49]Orten T J,T A G,1980,56:101-112
[50]褚圻， 遗传学，上海：上海教育出版社，1980，6-10
[51]McCoy T J,et al.Can J Genet Cytol,1982,24:37-50
[52]Siminvitch L.Cell,1976,7:1-11
[53]Carlsonp,Sci,1973,180:1366-1368
[54]Cullis C A, et al. The Plant Genome,Norway,Norwich,1980,91-97
[55]Siegel A, Modification of the Information Content of Plant Cell, Netherlands Amsterdam,1975,15-26
[56]Whitfeld R R,Bottmley W,Ann Rev Plant Physiol,1983,34:279-310
[57]许智宏，遗传，1985，7（6）37-40（二手资料）
[58]Bayliss M W, Nature,1973,246:529-530
[59]Gengenbach B G,et al. Crop Nat Acad Sci,1977,74:513-517
[60]Kasperbauer M J,et al.Crop Sci,1979,19:457-460
[61]Hoffmann F, Production of Natural Compounds by Cell Culture Methods,G D R, Munchen,1978,319-329
[62]Thomas e,et al.Z Pflanzenzucht,1979,82:1-30
[63]Jacobsen E, Plant Cell Tissue and Organ Culture,1981,1:77-84
[64]Karp A,et al. T A G,1982,63:256-272
[65]Ruiz M L,et al. Protplasma,1982,111:83-86
[66]王关林，植物学报，1989，31；（7）495-504
[67]Bayliss M W,Int Rev Cytol,1980,11A113-114
[68]Barbier M,et al. Plant,1980,30:321-344
[69]Novak F J,Cytologia,1981,46:371-380
[70]Yeoman M M,et al.Int Cytol Suppl,1980,11A:1-21
[71]Singh M R,Cytologia,1982,47:419-426
[72]黄娇香，遗传，1981：（3）31-32（二手资料）
[73]杨宁，美国大杏仁砧木微繁殖技术研究，2000年硕士论文
[74]杨汉民，王亚馥，王仑山等，五种植物组织培养物的染色体变异，植物体细胞无性系变异与育种，江苏科学技术出版社 189-196
[75]汪丽虹，杨汉民，枸杞再生植株不同发育途径中染色体变异的研究，植物学报，8（增刊）：61-64
[76]张金文，曹孜义，崔建垣，继代十七次的玉米花粉胚性细胞系的异常核和异常分裂观察，实验生物学报，30（4）：407-415
[77]R.Roth,I.Ebert,&J.Schrnidl,Trisomy associated with loss of maturation capacity in a long-term embryogenic culture of abies alba,Theoretical and Applied Genetics,1997,9(3):353-358
[78]An Lijia,He Menyuan and Hao Shui, Variation of Differentation Capacity of Maize Pollen Clones and Instability of Chromosome Number, Journal of Northeast Normal University,1986,(3):111-116
[79]Jones R N,Rees.h, London Ltd; Academic Press IHC INC,B Chromosome,1982
[80]李懋学，张赞平，《作物染色体及其研究技术》北京：中国农业出版社，1990
[81]Gupta P K, Tsuchiya T. Chromosome Engineer in Plant:Genetic.Breeding.Evolution. New York: Elsevier Science Publishing Company INC,1991.141-157
[82]Mendelson D,Zohary D, Behaviour and transmission of supernumerary chromosome in Aegilops speltoides. Heredity,1972,29:329-339
[83]Hewitt G M, The intergation of supernumerary chromosome into the orthopteran genome. Cold Spring Harb Symp Quant Biol,1973c,38:183-194
[84]Zheng Y R, Yang Q L et al. Studies on the origin of B-chromosomes in rye. Science in China(series B).1991,34:1186-1197