芥菜（Brassica juncea Coss.）离体培养及形态发生机制的初步研究
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摘要
本实验以芥菜的三个品种(圆大头菜、永胜大儿菜、容渝香芥菜)为材料,在以子叶和胚轴为外植体建立离体培养体系的基础上,对芥菜组织器官发生过程中各发育阶段的生理生化指标变化及可溶性蛋白质含量组分和过氧化物酶同工酶进行了研究,同时进行了组织细胞学观察。实验结果如下:
1芥菜离体培养体系的建立
不同品种的芥菜及培养基激素配比都会影响芥菜离体培养体系的建立。实验结果表明:芥菜种子消毒方法以采用5%NaClO溶液滴加2滴100%吐温-20,消毒15分钟最佳;种子无菌萌发培养基为:1/2MS+8.0g/L卡拉胶(pH5.8～6.0);最适愈伤组织诱导和分化的培养基为:MS+30g/L蔗糖+8.0g/L卡拉胶+0.1mg/L NAA+3.0mg/L 6-BA,当以子叶为外植体时,圆大头菜、永胜大儿菜、容渝香芥菜的分化率均为最高,分别为:72%、84%、76%;当以胚轴为外植体时上述三个品种的分化率分别为70%、72%、70%;最适生根培养基为MS+30g/L蔗糖+8.0g/L卡拉胶+0.1mg/LNAA,根系生长状态良好,生根率达到100%。
2可溶性蛋白质含量及组分的研究
具形态建成的愈伤组织中可溶性蛋白质平均含量为20.88mg╱g·FW,远远高于不具形态建成的愈伤组织中的平均含量12.08mg/g·FW,低水平的蛋白质表达量可能是使愈伤组织丧失分化能力,转变为不具形态建成的愈伤组织的一个重要原因。SDS-PAGE电泳结果显示,在具形态建成的愈伤组织增殖与分化过程中,有65 KDa、29 KDa、24 KDa、16 KDa的特异性蛋白质组分出现,其中16 KDa蛋白在不定芽分化后不再表达,推测是这4种特异性蛋白推动了愈伤组织的形态建成。在不具形态建成的愈伤组织中有50KDa和30KDa的蛋白质组分表达,可把这两种蛋白组分看作是芥菜子叶离体培养过程中不具形态建成的愈伤组织中的特异性蛋白。
3过氧化物同工酶酶谱的研究
通过过氧化物酶同工酶电泳,得到以下结果:具形态建成的愈伤组织的酶谱带比不具形态建成的愈伤组织的酶谱带颜色相对要浓,同时多出了C带和G带,其中G带是具形态建成的愈伤组织所特有的,C带是在不定芽分化后出现的,因此C带可作为不定芽分化的生化标记,D带在愈伤组织诱导期出现的,可作为脱分化开始的标志。
4生理生化变化的研究
两种愈伤组织随着培养时间的增加生理指标的差异越来越显著,具形态建成的愈伤组织比不具形态建成的愈伤组织内部代谢增强。外植体培养的第8天开始,具形态建成的愈伤组织中抗氧化酶(POD、SOD、CAT)活性、核酸(RNA、DNA)含量均高于不具形态建成的愈伤组织,呈明显的上升趋势,而可溶性糖和淀粉作为细胞中可利用的能源物质,在这个时期含量下降;不具形态建成的愈伤组织中以上各指标的变化则比较平缓。
5组织细胞学观察
利用石蜡切片技术对培养材料进行组织细胞学观察,结果表明,具形态建成的愈伤组织其表面和内部会逐渐形成两个分化中心,即分生细胞团和分生组织结节,前者将发育成为芽原基,后者将发育成为根原基。
This work aimed at the study of culturing three varieties of mustard (Yuandatoucai, Yongshengdaercai, Rongyuxiangjiecai), Starting with in vitro culture of cotyledons and hypocotyls of mustard established on the plant regeneration system. In the process of the organogenesis, this work mainly focused on the research of biochemical indicators, soluble proteins and peroxidase isoenzyme, and further research on the morphogenetic callus has been done at the cytohistology level. The main results are as follows:
1 The establishment of in vitro culture system in mustard
The result showed that different varieties, hormone concentration played important roles in mustard organogenesis. The optimal way of seed sterilization was 5% NaClO +tween-20 2 drops for 15min. The optimal culture medium for sterilitas plants was 1/2MS+8.0g/L Carrageenan (pH5.8~ 6.0); the appropriate medium for explants differentiation was MS+30g/L sucrose+8.0g/L Carrageenan+3.0mg/L 6-BA+0.1mg/L NAA, when the explants were cotyledons, the highest differentiation rate of three varieties (Yuandatoucai, Yongshengdaercai, Rongyuxiangjiecai)were 72%, 84%, 76%; and when the explants were hypocotyls, the highest differentiation rate were 70%, 72%, 70%. The best medium for rooting was MS+30g/L sucrose+8.0g/L Carrageenan+0.1mg/L NAA, with good root status and 100% rootage rate.
2 The studies on components and contents of the soluble proteins
The average content of soluble proteins in morphogenetic callus was 20.88 mg/g·FW, it was higher than the content in non-morphogenetic callus, which was 12.08 mg/g-FW. Therefore, the lower-level of the proteins might be one of the important reason that let morphogenetic callus lost the capacity of redifferentiation and turned into non-morphogenetic callus.The results of SDS-PAGE electrophoresis showed that different specific proteins found separately in different stages. There were four kinds of proteins (65KDa, 29KDa, 24KDa, 16KDa) expressed during callus differentiation. And the protein weight 16KDa didn't express after callus differentiated. These four kinds of proteins might be related with the morphochoresis of organs. The proteins weight 50KDa and 30KDa were detected in non-morphogenetic callus and not in morphogenetic callus.So, these two kinds of proteins can be used as markers for non-morphogenetic callus during mustard in vitro culture.
3 The studies on peroxidase isozyme
The peroxidase isozyme analyse was as follows: the thickness of isozyme bands of the morphogenetic callus was little stronger than those of the non-morphogenetic callus. The morphogenetic callus had C and G bands that the non-morphogenetic callus didn't have. C band appeared after callus differentiation, so it could be used as a biochemical marker for callus differentiation. D band appeared during callus induction, this might be a sign for the begining of explants dedifferentiation.
4 Studies on physiological and biochemical indexes
The physiological and biochemical indexes in two kinds of callus are obviously different during the culture process. The physiological and biochemical indexes of the morphogenetic callus had wider change range than those of the non-morphogenetic callus. When the callus had been cultivated for 8 days, the contents of nucleic acid (DNA, RNA), the antioxides enzyme (POD, SOD, CAT) activities of morphogenetic callus were higher than that of non-morphogenetic callus, and trends of those indexes were on the rise. At the same time, the contents of soluble sugars and starch were declined. While the changes of physiological and biochemical indexes of non-morphogenetic callus changed mildly.
5 Histocytological analysis
By the observation of paraffin wax slice, the morphogenetic cells of exterior and interior gradually formed two kinds of meristematic centres, which were the embryonic meristematic packed cells and the meristematic tubercles.The former would develop into primordial buds, and the latter would develop into primordial roots.
1芥菜离体培养体系的建立
不同品种的芥菜及培养基激素配比都会影响芥菜离体培养体系的建立。实验结果表明:芥菜种子消毒方法以采用5%NaClO溶液滴加2滴100%吐温-20,消毒15分钟最佳;种子无菌萌发培养基为:1/2MS+8.0g/L卡拉胶(pH5.8～6.0);最适愈伤组织诱导和分化的培养基为:MS+30g/L蔗糖+8.0g/L卡拉胶+0.1mg/L NAA+3.0mg/L 6-BA,当以子叶为外植体时,圆大头菜、永胜大儿菜、容渝香芥菜的分化率均为最高,分别为:72%、84%、76%;当以胚轴为外植体时上述三个品种的分化率分别为70%、72%、70%;最适生根培养基为MS+30g/L蔗糖+8.0g/L卡拉胶+0.1mg/LNAA,根系生长状态良好,生根率达到100%。
2可溶性蛋白质含量及组分的研究
具形态建成的愈伤组织中可溶性蛋白质平均含量为20.88mg╱g·FW,远远高于不具形态建成的愈伤组织中的平均含量12.08mg/g·FW,低水平的蛋白质表达量可能是使愈伤组织丧失分化能力,转变为不具形态建成的愈伤组织的一个重要原因。SDS-PAGE电泳结果显示,在具形态建成的愈伤组织增殖与分化过程中,有65 KDa、29 KDa、24 KDa、16 KDa的特异性蛋白质组分出现,其中16 KDa蛋白在不定芽分化后不再表达,推测是这4种特异性蛋白推动了愈伤组织的形态建成。在不具形态建成的愈伤组织中有50KDa和30KDa的蛋白质组分表达,可把这两种蛋白组分看作是芥菜子叶离体培养过程中不具形态建成的愈伤组织中的特异性蛋白。
3过氧化物同工酶酶谱的研究
通过过氧化物酶同工酶电泳,得到以下结果:具形态建成的愈伤组织的酶谱带比不具形态建成的愈伤组织的酶谱带颜色相对要浓,同时多出了C带和G带,其中G带是具形态建成的愈伤组织所特有的,C带是在不定芽分化后出现的,因此C带可作为不定芽分化的生化标记,D带在愈伤组织诱导期出现的,可作为脱分化开始的标志。
4生理生化变化的研究
两种愈伤组织随着培养时间的增加生理指标的差异越来越显著,具形态建成的愈伤组织比不具形态建成的愈伤组织内部代谢增强。外植体培养的第8天开始,具形态建成的愈伤组织中抗氧化酶(POD、SOD、CAT)活性、核酸(RNA、DNA)含量均高于不具形态建成的愈伤组织,呈明显的上升趋势,而可溶性糖和淀粉作为细胞中可利用的能源物质,在这个时期含量下降;不具形态建成的愈伤组织中以上各指标的变化则比较平缓。
5组织细胞学观察
利用石蜡切片技术对培养材料进行组织细胞学观察,结果表明,具形态建成的愈伤组织其表面和内部会逐渐形成两个分化中心,即分生细胞团和分生组织结节,前者将发育成为芽原基,后者将发育成为根原基。
This work aimed at the study of culturing three varieties of mustard (Yuandatoucai, Yongshengdaercai, Rongyuxiangjiecai), Starting with in vitro culture of cotyledons and hypocotyls of mustard established on the plant regeneration system. In the process of the organogenesis, this work mainly focused on the research of biochemical indicators, soluble proteins and peroxidase isoenzyme, and further research on the morphogenetic callus has been done at the cytohistology level. The main results are as follows:
1 The establishment of in vitro culture system in mustard
The result showed that different varieties, hormone concentration played important roles in mustard organogenesis. The optimal way of seed sterilization was 5% NaClO +tween-20 2 drops for 15min. The optimal culture medium for sterilitas plants was 1/2MS+8.0g/L Carrageenan (pH5.8~ 6.0); the appropriate medium for explants differentiation was MS+30g/L sucrose+8.0g/L Carrageenan+3.0mg/L 6-BA+0.1mg/L NAA, when the explants were cotyledons, the highest differentiation rate of three varieties (Yuandatoucai, Yongshengdaercai, Rongyuxiangjiecai)were 72%, 84%, 76%; and when the explants were hypocotyls, the highest differentiation rate were 70%, 72%, 70%. The best medium for rooting was MS+30g/L sucrose+8.0g/L Carrageenan+0.1mg/L NAA, with good root status and 100% rootage rate.
2 The studies on components and contents of the soluble proteins
The average content of soluble proteins in morphogenetic callus was 20.88 mg/g·FW, it was higher than the content in non-morphogenetic callus, which was 12.08 mg/g-FW. Therefore, the lower-level of the proteins might be one of the important reason that let morphogenetic callus lost the capacity of redifferentiation and turned into non-morphogenetic callus.The results of SDS-PAGE electrophoresis showed that different specific proteins found separately in different stages. There were four kinds of proteins (65KDa, 29KDa, 24KDa, 16KDa) expressed during callus differentiation. And the protein weight 16KDa didn't express after callus differentiated. These four kinds of proteins might be related with the morphochoresis of organs. The proteins weight 50KDa and 30KDa were detected in non-morphogenetic callus and not in morphogenetic callus.So, these two kinds of proteins can be used as markers for non-morphogenetic callus during mustard in vitro culture.
3 The studies on peroxidase isozyme
The peroxidase isozyme analyse was as follows: the thickness of isozyme bands of the morphogenetic callus was little stronger than those of the non-morphogenetic callus. The morphogenetic callus had C and G bands that the non-morphogenetic callus didn't have. C band appeared after callus differentiation, so it could be used as a biochemical marker for callus differentiation. D band appeared during callus induction, this might be a sign for the begining of explants dedifferentiation.
4 Studies on physiological and biochemical indexes
The physiological and biochemical indexes in two kinds of callus are obviously different during the culture process. The physiological and biochemical indexes of the morphogenetic callus had wider change range than those of the non-morphogenetic callus. When the callus had been cultivated for 8 days, the contents of nucleic acid (DNA, RNA), the antioxides enzyme (POD, SOD, CAT) activities of morphogenetic callus were higher than that of non-morphogenetic callus, and trends of those indexes were on the rise. At the same time, the contents of soluble sugars and starch were declined. While the changes of physiological and biochemical indexes of non-morphogenetic callus changed mildly.
5 Histocytological analysis
By the observation of paraffin wax slice, the morphogenetic cells of exterior and interior gradually formed two kinds of meristematic centres, which were the embryonic meristematic packed cells and the meristematic tubercles.The former would develop into primordial buds, and the latter would develop into primordial roots.
引文
[1]王蒂.植物组织培养[M].北京:中国农业出版社,2004:1-11,40
[2]沈海龙.植物组织培养[M].北京:中国林业出版社,2005:1
[3]曹孜义,刘国民.实用植物组织培养技术教程[M].兰州:甘肃科学技术出版社,1996:1-2
[4]朱至清.植物细胞工程[M].北京:化学工业出版社,2003:1-3
[5]颜昌敬.植物组织培养手册[M].上海:上海科学出版社,1990:1-3
[6]White J.A.Casette containing the bar gene of streptomyces hygroscopicus:aselectable Marker for plant transfromation[J].Nudeic Acids Res,1990,18:1062
[7]孙敬三.植物生物技术与作物改良[M].北京:中国科技出版社,1991:1-5
[8]Van P.Intron-mediated enhancement of gene expression in maize bleefrass[J].Plant Cell Rep,1996,(15):489-494
[9]Skoog F.,C.O.Mille.Chemical regulation of growth and organ formation in plant tissue cultured in Vitro[J].Symp.Soc.Exp.Biol.,1957,(11):118-130
[10]韩碧文,李颖章.植物组织培养中器官建成的生理生化基础[M].植物学通报,1993,10(2):1-6
[11]何道一,程炳嵩.植物组织培养材料分化与脱分化过程中的生理生化变化[J].山东农业大学学报,1992,23(3):27-31
[12]Skoog,F.and Tsui,C.Chemcial regulation of growth and bud formation in tobacco stem segments and callus culture in Vitro.Amer.J.bot.,1984,(35):782-787
[13]崔澂.植物激素与细胞分化及形成发生的关系[J].植物生理学通讯,1983,5(2):1-5
[14]金建平.观赏蕨的组织培养[J].植物生理学通讯,1991,27(1):71-73
[15]王冬梅.细胞分裂素类物质在植物组织培养中的作用机制[J].植物生理学通讯,1996,32(5):373-377
[16]王忠.植物生理学[M].北京:中国农业出版社,2000:323-330
[17]Wong,WC.In Vitro propagation of bananas(Musapp.):Initiation,proliferation and development of shoot tip culture on defined media[J].Plant Cell,Tissue and Organ Culture,1986,6(2):159
[18]叶叙丰.拟石莲花离休叶片的不定芽分化及其和内源激素状况的关系[J].植物生理学通讯,1989,4:26-30
[19]叶秀遴.石斛兰组织培养和细胞学观察[J].园艺学报,1995,22(3):303-304
[20]张松,李纪荣,李滨等.大葱组织培养体细胞胚胎发生的研究[J].园艺学报,1997,24(3):264-268
[21]伍宁丰,范云六.苏云金孢杆菌杀虫蛋白基因的杨树工程植株的建立[J].科学通报,1991,36(9):705-708
[22]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[J].山东大学党报(自然科学版),1995,30(1):105-111
[23]郑企成,朱光耀,陈文华等.小麦体细胞无性系籽粒胚乳醇溶蛋白的变异[J].核农学报,1991,5(3):134-138
[24]Dodds John H,Koberts Lorin W.Experiments in plant tissues culture.[M].Cambridge University Press,1995
[25]张献龙,唐克轩,林兴华等.植物生物技术[M].北京:科学出版社,2004:3-8
[26]许凤芹,刘桂茹,杨学举.植物组织培养研究进展[J].河北农业科学,2005,9(1):99-104
[27]瞿礼嘉,顾红雅,胡平等.现代生物技术导论[M].北京:高等教育出版社,1998:3-11
[28]崔德才,徐培文等.植物组织培养与工厂化育苗[M].北京:化学工业出版社.现代生物技术与医药科技出版中心.2003:1-15
[29]Nag K.K.Strect H.E.Carrot embryogenesis from frozen cultured cells[J].Nature.1973,(245):270-272
[30]牛小牧,黄美娟.人工种子的研究现况[J].生物工程进展,1990,10(1):18-25
[31]汤绍虎,唐佩华.甘薯人工种子研究[J].作物学报,1994,20(6):746-750
[32]唐巍,欧阳藩.马铃薯人工种子的研究[J].生物技术,1996,6(5):11-13
[33]张铭,黄华荣,魏小勇.植物人工种子研究进展[J].植物学通报,2000,17(5):407-411
[34]蒋亚华,谢如伟.扩大紫杉醇的生产途径[J].技术交流,2007,10:49-50
[35]黄百渠.植物体细胞遗传学简明教程[M].长春:东北师范大学出版社,1991
[36]韩碧文,刘淑兰.植物离体培养体细胞胚发生[J].植物生理学通讯,1988,(1):9-15
[37]张丕方,倪德祥,王富民等.百合鳞片离体培养诱导小鳞茎发生的研究[J].武汉植物学研究,1985,2:177-180
[38]朱微.植物组织培养中有胚状体[J].遗传学报,1978,5(1):79-87
[39]邹建.茄子(Solanum melongena L.)花药离体培养及其形态发生机制的初步研究[D].重庆:西南农业大学,2004
[40]王凯基,张丕方,倪德祥等.油橄榄组织培养的细胞组织学研究.植物学报,1979,21(2):127-131
[41]张丕方,倪德祥,王凯基.非洲紫罗兰叶片培养中胚状体发生的形态研究[J].植物学报,1982,24(3):282-285
[42]张德华,焦成瑾,王亚馥等.小麦体细胞胚胎发生及其合子胚比较[J].兰州大学学报,1994,30(增刊):55-59
[43]吕耀奎,陈天柱.小麦花粉无性系的建立及其胚状体的发生[J].实验生物学报,1984,17(1):13-23
[44]刘春明,姚郭义.陆地棉体细胞胚胎发生及其细胞组织学研究[J].植物学报,1991,33(5);378-384
[45]张新英,黄亚斌,尹光初等.离体培养下大豆体细胞胚胎发生的组织学研究[J].植物学报,1992,34(3):214-218
[46]郝建平,周小梅,李绍清.茴香组织培养中体细胞胚胎发生的组织细胞学研究[J].实验生物学报,1995,28(3):339-347
[47]崔凯荣,戴若兰.体细胞胚发生的分子生物学[M].科学出版社,2000
[48]L.J.Chen,D.S.Luthe.Analysis of protein from embryonic and nonembryonic rice(Oryzasativa L.)calli.Plant Science,1987:48
[49]Huang,B.Q..Search of moleular marker for the somatic embryogenesis in Arabidopsis thaliana L;Chinese Journal Botany,1989,1(1):25-34
[50]J.Z.Dong,D.I.Dunstan.Characterization of three heat-stock-protein genes and their developmental trgulation during somatic embryogengsis in white spruce[picea glauca(Moeneh)voss],Planta,1996,200:85-91
[51]陈雄,王星,王亚馥.激素对枸杞体细胞胚发生及可溶性蛋白质含量和组分的影响[J].西北植物学报,1995,15(5):26-30
[52]刘本叶,张艳萍,吴绛云等.非洲紫罗兰叶片脱分化过程中核酸蛋白质和淀粉含量动态的研究[J].生物技术,1995:5(1):12-15
[53]叶克难,李文雷,徐增富.苜蓿体细胞胚胎发生过程中DNA,RNA和蛋白质的合成动态[J].实验生物学 报,1992,25(4):403-411
[54]Sengupta C,Raghavan V.Somatic embryogenesis in carrot cell suspension[J].I.Patern of protein and nucleic acid synthesis,1980,31:247-258
[55]路铁钢,郑国铝.红豆草体细胞胚胎发生早期DNA,RNA和蛋白质的合成动态变化[J].植物学报,1989,31(10):757-767
[56]Z.R.Sung,R.Okimoto.Embryogenic proteins in somatic embryos of carrot[J].Proc.Natl.Acad.Sci.USA,1981,78:3683-3687
[57]L.J.Chen,D.S.Luthe.Analysis of protein from embryonic and nonembryonie rice(Oryza sativa L.)calli[J].Plant Science,1987,48:181-188
[58]P.F.Franz,N.C.A.De Ruijter,J.H.Schel.Isozyme as biochemical and cyological markers in embryogenic callus cultures of maize(Zea mavs L.)Plant Cell Rep,1989,8:67-70
[59]G.Hahne,J.E.Mayer,H.Lorz.Embryogenic and callus-speciffic proteins is somatic embryogenesis of the grass(Dactylis blomerate L.)[J].Plant Science,1988,55:267-279
[60]S.Stizn,H.J.Jacobsen.Marker protems for embryogenic differentiation partterns in pea callus[J].Plant Cell Rep,1987,6:50-54
[61]R.W.Girous,K.P.Pauls.Characterization of embryogenesis-related protein in alfalfa(Medieago sativa)[J].Physiologia Plantarum,1996,96(4):585-592
[62]王亚馥,崔凯荣,汪丽虹.小麦体细胞胚发生中蛋白质组分和过氧化物酶同工酶的变化[J].兰州大学学报(自然科学版),1993,29(3):189-193
[63]朱长甫,镰田博,何奕昆.胡萝卜(Daucus carota L.)胚性细胞蛋白的分享研究[J].实验生物学报,1997,30(1):13-18
[64]王凯基,张丕方,倪德祥等.几种木本植物组织培养的愈伤组织形成和器官发生[J].植物学报,1981,23(2):97-103
[65]崔凯荣,邢更生,周功克等.激素对体细胞胚发生的诱导与调节[J].遗传,2000,22(5):349-354
[66]李中奎,刘成远,权明清.梗稻蒲晚一号体细胞胚发生中的几种生理生化变化[J].武汉植物学院,1993,11(3):283-286
[67]杨和平,程井辰.马唐体细胞胚胎发生过程中生理变化的研究[J].武汉植物学研究,1991,9(4):306-312
[68]郑晓峰,黄百渠.几种植物体细胞胚胎发生标记蛋白的研究[J].植物学报,1994,36(3):175-180
[69]陈婷,栾恒淳,李力等.水稻悬浮体细胞胚胎发生及其特异蛋白[J].东北师大学报自然科学版,2000,32(4):52-58
[70]Franz P F.Isozymes as biochemical and cytochemicao markers in embryogeneic callus cultures of maize glutamate-dehydroge-nase,peroxidase and acid phosphatase isoemzyme application[J].Plant Cell Rep,1989(8):67-70
[71]杜胜利,韩毅科,丛颖等.黄瓜离体雌核发育的过程及其早期生化变化研究[J].南开大学学报,2003,36(2):27- 30
[72]李效宇,刘熔山.小麦幼穗胚性愈伤组织诱导及分化过程中内源激素的作用[J].植物生理学通讯,1994,30(4):255-260
[73]胡忠,丁惠宾,王星等.宁夏枸杞器官发生和体细胞胚发生过程中DNA、RNA和蛋白质合成动态的比较研究[J].实验生物学报,1998,31:403-411
[74]路铁刚,王义琛,郑国锠等.红豆草体细胞胚发生早期过氧化物酶和酸性磷酸酶同工酶的研究[J].西北植物学报,1990,10(1):17-22
[75]刘天磊,江晓雯,王仑山.苜蓿组织培养中球形胚发生时特异蛋白和同工酶分析[J].西北植物学报,2002,22(3):625-628
[76]Gui K,Xing G,Wang Y.Effect of hydmgen peroxide on somatic embryogenesis of Lycium barum L.[J].Plant Science,1999,146:9-16
[77]詹园凤,吴震,金潇潇等.大蒜体细胞胚胎发生过程中抗氧化酶活性变化及某些生理特征[J].西北植物学报,2006,26(9):1799-1802
[78]庄东红,杜虹.大白菜子叶培养过程中POD同工酶和可溶性蛋白质含量的变化[J].汕头大学学报(自然科学版),2002,17(1):65-68
[79]徐桂芳,牛玉仙,唐定台等.四种愈伤组织中吲哚乙酸氧化酶和过氧化物酶的活性与同工酶的关系[J].植物学报,1983,25(6):551-555
[80]汪丽虹,王亚馥,杨汉民等.伊贝母组织培养形态发生过程中几种糖类含量及淀粉酶和蔗糖酶活力动态研究[J].西北植物学报,1991,11(8):194-198
[81]范昌发,郭骁才,贾敬芬等.植物细胞中淀粉代谢与离体形态发生途径决定的关系[J].华北农学报,2000,15(4):52-57
[82]G.M.Hart,F.Menis.Trans-acting factors involved in tobacco β-1,3-glicanase gene expression[J].Plant Physiol,1991,96(1):96-98
[83]刘淑兰,陈正华.芝麻体细胞胚胎发生及其内源激素和可溶性蛋白的变化[J].农业生物技术学报,1994,2(2):44-49
[84]郑玉梅,李育,王世旄.芥蓝薄细胞层培养中愈伤组织和器官形成的组织细胞学观察[J].华南农业大学学报,1994,15(4):18-22
[85]中国科学院上海植物生理研究所细胞室编译.植物组织和细胞培养[M].上海:上海科学技术出版社,1978
[86]由翠荣,曲复宁,崔龙波等.仙客来(Cyclamen persicum Mill)组织培养中不定芽形态发生的细胞细胞学研究[J].烟台大学学报(自然科学与工程版),2002,15(4):273-279
[87]林双荣,王庆华,梁丽琨等.TDZ诱导花生幼叶的不定芽和体细胞胚发生的组织学观察[J].植物研究,2003,23(2):169-173
[88]侯丙凯,鲍学珍,于惠敏等.葡萄离体培养中胚状全发和的研究[J].山东大学学报,1997,32(2):208-212
[89]Pareek L.K.,Chandra N.,Growth and differentiation in Brassica juncea tissues grown in vitro[J].Proc Ind Sc Cong,1972(59):372-373
[90]Hui L.H.,Zee S.Y.,In vitro plant formation from hypocotyls and cotyledons of leaf-mustard cabbage(brassica juncea Coss)Zeitschrift fur Pflanzenphysiologie,1978,89(1):77-80
[91]George L.,Rao P.S.,In vitro induction of pellen embryos and plantlets in Brassica juncea through anther culture[J].Plant Sci lett,1982,26(1):111-116
[92]Chatterjee G.,Regeneration of plantlets from mesophyll protoplasts of Brassica juncea(L)[J].Czern.Plant Cell Reports,1985,4(5):245-247
[93]雷建军,陈世儒,郭余龙等.茎用芥菜原生质体培养及植株再生[J].植物学报,1991,33(2):91-97
[94]雷建军,陈世儒,郭余龙.叶用茎菜叶片原生质体再生植株[J].园艺学报,1992,19(1):52-56
[95]余小林,曹家树,陈石头等.提高榨菜离体培养植株再生频率[J].细胞生物学杂志,2004,26:439-443
[96]黄菊辉,周长久,秦凤琴等.发根农杆菌的Ri T-DNA对茎用芥菜的遗传转化[J].植物学报,1994,36(12):911-917
[97]Lillo Cand,Shahin E.A.Rapid regeneration of cabbage ptoplasts[J].HortScience,1986,21:315-317
[98]李文彬,陈正华,宋玉华等.芥菜型油菜原生质体再生成植株的研究[J].遗传学报,1986,13:184-187
[99]陈利萍,徐春霞,李春顺.影响茎有芥菜愈伤组织诱导和植株再生的因素[J].植物生理学通讯,2005,41(6):758-760
[100]孔娟.芥菜类蔬菜高频再生体系的建立及硝酸银的作用[D].杭州,浙江大学,2002
[101]徐春霞.茎用芥菜不同再生体系的建立及遗传稳定性分析[D].杭州,浙江大学,2005
[102]刘佩瑛.中国芥菜[M].北京,中国林业出版社,1996:1-7
[103]陈竹君,张明方,汪炳良.榨菜胞质雄性不育及其农艺性状的研究[J].园艺学报,1995,22(1):46-52
[104]郭尧君.蛋白质电泳实用技术[M].北京:科学出版社,1999:123-125
[105]陈毓荃.生物化学实验方法与技术[M].科学出版社,2002:120-124
[106]李合生.植物生理生化实验原理和技术[M].北京:北京高等教育出版社,2001:214-217
[107]郑国铝.生物显微技术[M].北京:人民教育出版社,1978
[108]王武.油菜素内脂对陆地棉体细胞胚胎发生的影响[J].植物生理学通讯,1992,28(10):15-18
[109]赵成章,孙宗修.提高粳稻体细胞培养绿苗频率的研究[J].中国水稻科学,1986,1(1):19-25
[110]雷建军,曹必好,郭余龙等.用茎瘤芥再生芽离体筛选抗S-(2-氨已基)-L-半胱氨酸变异研究[J].西南农业大学学报,1995,17(2):98-100
[111]王慧芳.甘蓝组织培养再生、转化及筛选系统的优化研究[D].山西农业大学,2004
[112]Fazekas G.A.,Sedmach P.A.and Palmer M.V.Genetic and enviromental effects on in vitro shoot regeneration from cotyledon explants of Brassicajuncea[J].Plant Cell,Tissue and Organ Culture,1986,6:177-180
[113]J.Z.Dong,D.I.Dunstan,Expression of abundant mRNAs during somatic embryogenesis of white spruce[Picea glauca(Moench)Voss][J].Plant,1996,199:459-466
[114]王熊,罗士韦.烟草组织培养过程中过氧化物同工酶的变化[J].植物生理学报,1981,7(1):73-82
[115]白守信,苏文辉,梁红玉.豌豆组织培养中器官的分化生长及过氧化物酶同工酶及核酸含量的变化[J].西北植物学报,1985,5(3):192-198
[116]徐桂芳,牛玉仙,张静兰等.哈密瓜子叶脱分化和再分化过程中几种脱氢酶同工酶的研究[J].植物学报,1983,25(2):131-135
[117]Kovhha j.,Lavee S.,Spiegel-Roy P.differences in peroxidase activity and isnenzymes in embryogenic and bryogezic "Shamouti" orange ovular callus lines[M].Plant Cell.Physiol,1987,28(8):463-467
[118]高述民,陆帼一,杜慧芳.大蒜胚状体发育分化特异蛋白和某些生理生化变化[J].植物生理学通讯,2005,37(3):207～210
[2]沈海龙.植物组织培养[M].北京:中国林业出版社,2005:1
[3]曹孜义,刘国民.实用植物组织培养技术教程[M].兰州:甘肃科学技术出版社,1996:1-2
[4]朱至清.植物细胞工程[M].北京:化学工业出版社,2003:1-3
[5]颜昌敬.植物组织培养手册[M].上海:上海科学出版社,1990:1-3
[6]White J.A.Casette containing the bar gene of streptomyces hygroscopicus:aselectable Marker for plant transfromation[J].Nudeic Acids Res,1990,18:1062
[7]孙敬三.植物生物技术与作物改良[M].北京:中国科技出版社,1991:1-5
[8]Van P.Intron-mediated enhancement of gene expression in maize bleefrass[J].Plant Cell Rep,1996,(15):489-494
[9]Skoog F.,C.O.Mille.Chemical regulation of growth and organ formation in plant tissue cultured in Vitro[J].Symp.Soc.Exp.Biol.,1957,(11):118-130
[10]韩碧文,李颖章.植物组织培养中器官建成的生理生化基础[M].植物学通报,1993,10(2):1-6
[11]何道一,程炳嵩.植物组织培养材料分化与脱分化过程中的生理生化变化[J].山东农业大学学报,1992,23(3):27-31
[12]Skoog,F.and Tsui,C.Chemcial regulation of growth and bud formation in tobacco stem segments and callus culture in Vitro.Amer.J.bot.,1984,(35):782-787
[13]崔澂.植物激素与细胞分化及形成发生的关系[J].植物生理学通讯,1983,5(2):1-5
[14]金建平.观赏蕨的组织培养[J].植物生理学通讯,1991,27(1):71-73
[15]王冬梅.细胞分裂素类物质在植物组织培养中的作用机制[J].植物生理学通讯,1996,32(5):373-377
[16]王忠.植物生理学[M].北京:中国农业出版社,2000:323-330
[17]Wong,WC.In Vitro propagation of bananas(Musapp.):Initiation,proliferation and development of shoot tip culture on defined media[J].Plant Cell,Tissue and Organ Culture,1986,6(2):159
[18]叶叙丰.拟石莲花离休叶片的不定芽分化及其和内源激素状况的关系[J].植物生理学通讯,1989,4:26-30
[19]叶秀遴.石斛兰组织培养和细胞学观察[J].园艺学报,1995,22(3):303-304
[20]张松,李纪荣,李滨等.大葱组织培养体细胞胚胎发生的研究[J].园艺学报,1997,24(3):264-268
[21]伍宁丰,范云六.苏云金孢杆菌杀虫蛋白基因的杨树工程植株的建立[J].科学通报,1991,36(9):705-708
[22]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[J].山东大学党报(自然科学版),1995,30(1):105-111
[23]郑企成,朱光耀,陈文华等.小麦体细胞无性系籽粒胚乳醇溶蛋白的变异[J].核农学报,1991,5(3):134-138
[24]Dodds John H,Koberts Lorin W.Experiments in plant tissues culture.[M].Cambridge University Press,1995
[25]张献龙,唐克轩,林兴华等.植物生物技术[M].北京:科学出版社,2004:3-8
[26]许凤芹,刘桂茹,杨学举.植物组织培养研究进展[J].河北农业科学,2005,9(1):99-104
[27]瞿礼嘉,顾红雅,胡平等.现代生物技术导论[M].北京:高等教育出版社,1998:3-11
[28]崔德才,徐培文等.植物组织培养与工厂化育苗[M].北京:化学工业出版社.现代生物技术与医药科技出版中心.2003:1-15
[29]Nag K.K.Strect H.E.Carrot embryogenesis from frozen cultured cells[J].Nature.1973,(245):270-272
[30]牛小牧,黄美娟.人工种子的研究现况[J].生物工程进展,1990,10(1):18-25
[31]汤绍虎,唐佩华.甘薯人工种子研究[J].作物学报,1994,20(6):746-750
[32]唐巍,欧阳藩.马铃薯人工种子的研究[J].生物技术,1996,6(5):11-13
[33]张铭,黄华荣,魏小勇.植物人工种子研究进展[J].植物学通报,2000,17(5):407-411
[34]蒋亚华,谢如伟.扩大紫杉醇的生产途径[J].技术交流,2007,10:49-50
[35]黄百渠.植物体细胞遗传学简明教程[M].长春:东北师范大学出版社,1991
[36]韩碧文,刘淑兰.植物离体培养体细胞胚发生[J].植物生理学通讯,1988,(1):9-15
[37]张丕方,倪德祥,王富民等.百合鳞片离体培养诱导小鳞茎发生的研究[J].武汉植物学研究,1985,2:177-180
[38]朱微.植物组织培养中有胚状体[J].遗传学报,1978,5(1):79-87
[39]邹建.茄子(Solanum melongena L.)花药离体培养及其形态发生机制的初步研究[D].重庆:西南农业大学,2004
[40]王凯基,张丕方,倪德祥等.油橄榄组织培养的细胞组织学研究.植物学报,1979,21(2):127-131
[41]张丕方,倪德祥,王凯基.非洲紫罗兰叶片培养中胚状体发生的形态研究[J].植物学报,1982,24(3):282-285
[42]张德华,焦成瑾,王亚馥等.小麦体细胞胚胎发生及其合子胚比较[J].兰州大学学报,1994,30(增刊):55-59
[43]吕耀奎,陈天柱.小麦花粉无性系的建立及其胚状体的发生[J].实验生物学报,1984,17(1):13-23
[44]刘春明,姚郭义.陆地棉体细胞胚胎发生及其细胞组织学研究[J].植物学报,1991,33(5);378-384
[45]张新英,黄亚斌,尹光初等.离体培养下大豆体细胞胚胎发生的组织学研究[J].植物学报,1992,34(3):214-218
[46]郝建平,周小梅,李绍清.茴香组织培养中体细胞胚胎发生的组织细胞学研究[J].实验生物学报,1995,28(3):339-347
[47]崔凯荣,戴若兰.体细胞胚发生的分子生物学[M].科学出版社,2000
[48]L.J.Chen,D.S.Luthe.Analysis of protein from embryonic and nonembryonic rice(Oryzasativa L.)calli.Plant Science,1987:48
[49]Huang,B.Q..Search of moleular marker for the somatic embryogenesis in Arabidopsis thaliana L;Chinese Journal Botany,1989,1(1):25-34
[50]J.Z.Dong,D.I.Dunstan.Characterization of three heat-stock-protein genes and their developmental trgulation during somatic embryogengsis in white spruce[picea glauca(Moeneh)voss],Planta,1996,200:85-91
[51]陈雄,王星,王亚馥.激素对枸杞体细胞胚发生及可溶性蛋白质含量和组分的影响[J].西北植物学报,1995,15(5):26-30
[52]刘本叶,张艳萍,吴绛云等.非洲紫罗兰叶片脱分化过程中核酸蛋白质和淀粉含量动态的研究[J].生物技术,1995:5(1):12-15
[53]叶克难,李文雷,徐增富.苜蓿体细胞胚胎发生过程中DNA,RNA和蛋白质的合成动态[J].实验生物学 报,1992,25(4):403-411
[54]Sengupta C,Raghavan V.Somatic embryogenesis in carrot cell suspension[J].I.Patern of protein and nucleic acid synthesis,1980,31:247-258
[55]路铁钢,郑国铝.红豆草体细胞胚胎发生早期DNA,RNA和蛋白质的合成动态变化[J].植物学报,1989,31(10):757-767
[56]Z.R.Sung,R.Okimoto.Embryogenic proteins in somatic embryos of carrot[J].Proc.Natl.Acad.Sci.USA,1981,78:3683-3687
[57]L.J.Chen,D.S.Luthe.Analysis of protein from embryonic and nonembryonie rice(Oryza sativa L.)calli[J].Plant Science,1987,48:181-188
[58]P.F.Franz,N.C.A.De Ruijter,J.H.Schel.Isozyme as biochemical and cyological markers in embryogenic callus cultures of maize(Zea mavs L.)Plant Cell Rep,1989,8:67-70
[59]G.Hahne,J.E.Mayer,H.Lorz.Embryogenic and callus-speciffic proteins is somatic embryogenesis of the grass(Dactylis blomerate L.)[J].Plant Science,1988,55:267-279
[60]S.Stizn,H.J.Jacobsen.Marker protems for embryogenic differentiation partterns in pea callus[J].Plant Cell Rep,1987,6:50-54
[61]R.W.Girous,K.P.Pauls.Characterization of embryogenesis-related protein in alfalfa(Medieago sativa)[J].Physiologia Plantarum,1996,96(4):585-592
[62]王亚馥,崔凯荣,汪丽虹.小麦体细胞胚发生中蛋白质组分和过氧化物酶同工酶的变化[J].兰州大学学报(自然科学版),1993,29(3):189-193
[63]朱长甫,镰田博,何奕昆.胡萝卜(Daucus carota L.)胚性细胞蛋白的分享研究[J].实验生物学报,1997,30(1):13-18
[64]王凯基,张丕方,倪德祥等.几种木本植物组织培养的愈伤组织形成和器官发生[J].植物学报,1981,23(2):97-103
[65]崔凯荣,邢更生,周功克等.激素对体细胞胚发生的诱导与调节[J].遗传,2000,22(5):349-354
[66]李中奎,刘成远,权明清.梗稻蒲晚一号体细胞胚发生中的几种生理生化变化[J].武汉植物学院,1993,11(3):283-286
[67]杨和平,程井辰.马唐体细胞胚胎发生过程中生理变化的研究[J].武汉植物学研究,1991,9(4):306-312
[68]郑晓峰,黄百渠.几种植物体细胞胚胎发生标记蛋白的研究[J].植物学报,1994,36(3):175-180
[69]陈婷,栾恒淳,李力等.水稻悬浮体细胞胚胎发生及其特异蛋白[J].东北师大学报自然科学版,2000,32(4):52-58
[70]Franz P F.Isozymes as biochemical and cytochemicao markers in embryogeneic callus cultures of maize glutamate-dehydroge-nase,peroxidase and acid phosphatase isoemzyme application[J].Plant Cell Rep,1989(8):67-70
[71]杜胜利,韩毅科,丛颖等.黄瓜离体雌核发育的过程及其早期生化变化研究[J].南开大学学报,2003,36(2):27- 30
[72]李效宇,刘熔山.小麦幼穗胚性愈伤组织诱导及分化过程中内源激素的作用[J].植物生理学通讯,1994,30(4):255-260
[73]胡忠,丁惠宾,王星等.宁夏枸杞器官发生和体细胞胚发生过程中DNA、RNA和蛋白质合成动态的比较研究[J].实验生物学报,1998,31:403-411
[74]路铁刚,王义琛,郑国锠等.红豆草体细胞胚发生早期过氧化物酶和酸性磷酸酶同工酶的研究[J].西北植物学报,1990,10(1):17-22
[75]刘天磊,江晓雯,王仑山.苜蓿组织培养中球形胚发生时特异蛋白和同工酶分析[J].西北植物学报,2002,22(3):625-628
[76]Gui K,Xing G,Wang Y.Effect of hydmgen peroxide on somatic embryogenesis of Lycium barum L.[J].Plant Science,1999,146:9-16
[77]詹园凤,吴震,金潇潇等.大蒜体细胞胚胎发生过程中抗氧化酶活性变化及某些生理特征[J].西北植物学报,2006,26(9):1799-1802
[78]庄东红,杜虹.大白菜子叶培养过程中POD同工酶和可溶性蛋白质含量的变化[J].汕头大学学报(自然科学版),2002,17(1):65-68
[79]徐桂芳,牛玉仙,唐定台等.四种愈伤组织中吲哚乙酸氧化酶和过氧化物酶的活性与同工酶的关系[J].植物学报,1983,25(6):551-555
[80]汪丽虹,王亚馥,杨汉民等.伊贝母组织培养形态发生过程中几种糖类含量及淀粉酶和蔗糖酶活力动态研究[J].西北植物学报,1991,11(8):194-198
[81]范昌发,郭骁才,贾敬芬等.植物细胞中淀粉代谢与离体形态发生途径决定的关系[J].华北农学报,2000,15(4):52-57
[82]G.M.Hart,F.Menis.Trans-acting factors involved in tobacco β-1,3-glicanase gene expression[J].Plant Physiol,1991,96(1):96-98
[83]刘淑兰,陈正华.芝麻体细胞胚胎发生及其内源激素和可溶性蛋白的变化[J].农业生物技术学报,1994,2(2):44-49
[84]郑玉梅,李育,王世旄.芥蓝薄细胞层培养中愈伤组织和器官形成的组织细胞学观察[J].华南农业大学学报,1994,15(4):18-22
[85]中国科学院上海植物生理研究所细胞室编译.植物组织和细胞培养[M].上海:上海科学技术出版社,1978
[86]由翠荣,曲复宁,崔龙波等.仙客来(Cyclamen persicum Mill)组织培养中不定芽形态发生的细胞细胞学研究[J].烟台大学学报(自然科学与工程版),2002,15(4):273-279
[87]林双荣,王庆华,梁丽琨等.TDZ诱导花生幼叶的不定芽和体细胞胚发生的组织学观察[J].植物研究,2003,23(2):169-173
[88]侯丙凯,鲍学珍,于惠敏等.葡萄离体培养中胚状全发和的研究[J].山东大学学报,1997,32(2):208-212
[89]Pareek L.K.,Chandra N.,Growth and differentiation in Brassica juncea tissues grown in vitro[J].Proc Ind Sc Cong,1972(59):372-373
[90]Hui L.H.,Zee S.Y.,In vitro plant formation from hypocotyls and cotyledons of leaf-mustard cabbage(brassica juncea Coss)Zeitschrift fur Pflanzenphysiologie,1978,89(1):77-80
[91]George L.,Rao P.S.,In vitro induction of pellen embryos and plantlets in Brassica juncea through anther culture[J].Plant Sci lett,1982,26(1):111-116
[92]Chatterjee G.,Regeneration of plantlets from mesophyll protoplasts of Brassica juncea(L)[J].Czern.Plant Cell Reports,1985,4(5):245-247
[93]雷建军,陈世儒,郭余龙等.茎用芥菜原生质体培养及植株再生[J].植物学报,1991,33(2):91-97
[94]雷建军,陈世儒,郭余龙.叶用茎菜叶片原生质体再生植株[J].园艺学报,1992,19(1):52-56
[95]余小林,曹家树,陈石头等.提高榨菜离体培养植株再生频率[J].细胞生物学杂志,2004,26:439-443
[96]黄菊辉,周长久,秦凤琴等.发根农杆菌的Ri T-DNA对茎用芥菜的遗传转化[J].植物学报,1994,36(12):911-917
[97]Lillo Cand,Shahin E.A.Rapid regeneration of cabbage ptoplasts[J].HortScience,1986,21:315-317
[98]李文彬,陈正华,宋玉华等.芥菜型油菜原生质体再生成植株的研究[J].遗传学报,1986,13:184-187
[99]陈利萍,徐春霞,李春顺.影响茎有芥菜愈伤组织诱导和植株再生的因素[J].植物生理学通讯,2005,41(6):758-760
[100]孔娟.芥菜类蔬菜高频再生体系的建立及硝酸银的作用[D].杭州,浙江大学,2002
[101]徐春霞.茎用芥菜不同再生体系的建立及遗传稳定性分析[D].杭州,浙江大学,2005
[102]刘佩瑛.中国芥菜[M].北京,中国林业出版社,1996:1-7
[103]陈竹君,张明方,汪炳良.榨菜胞质雄性不育及其农艺性状的研究[J].园艺学报,1995,22(1):46-52
[104]郭尧君.蛋白质电泳实用技术[M].北京:科学出版社,1999:123-125
[105]陈毓荃.生物化学实验方法与技术[M].科学出版社,2002:120-124
[106]李合生.植物生理生化实验原理和技术[M].北京:北京高等教育出版社,2001:214-217
[107]郑国铝.生物显微技术[M].北京:人民教育出版社,1978
[108]王武.油菜素内脂对陆地棉体细胞胚胎发生的影响[J].植物生理学通讯,1992,28(10):15-18
[109]赵成章,孙宗修.提高粳稻体细胞培养绿苗频率的研究[J].中国水稻科学,1986,1(1):19-25
[110]雷建军,曹必好,郭余龙等.用茎瘤芥再生芽离体筛选抗S-(2-氨已基)-L-半胱氨酸变异研究[J].西南农业大学学报,1995,17(2):98-100
[111]王慧芳.甘蓝组织培养再生、转化及筛选系统的优化研究[D].山西农业大学,2004
[112]Fazekas G.A.,Sedmach P.A.and Palmer M.V.Genetic and enviromental effects on in vitro shoot regeneration from cotyledon explants of Brassicajuncea[J].Plant Cell,Tissue and Organ Culture,1986,6:177-180
[113]J.Z.Dong,D.I.Dunstan,Expression of abundant mRNAs during somatic embryogenesis of white spruce[Picea glauca(Moench)Voss][J].Plant,1996,199:459-466
[114]王熊,罗士韦.烟草组织培养过程中过氧化物同工酶的变化[J].植物生理学报,1981,7(1):73-82
[115]白守信,苏文辉,梁红玉.豌豆组织培养中器官的分化生长及过氧化物酶同工酶及核酸含量的变化[J].西北植物学报,1985,5(3):192-198
[116]徐桂芳,牛玉仙,张静兰等.哈密瓜子叶脱分化和再分化过程中几种脱氢酶同工酶的研究[J].植物学报,1983,25(2):131-135
[117]Kovhha j.,Lavee S.,Spiegel-Roy P.differences in peroxidase activity and isnenzymes in embryogenic and bryogezic "Shamouti" orange ovular callus lines[M].Plant Cell.Physiol,1987,28(8):463-467
[118]高述民,陆帼一,杜慧芳.大蒜胚状体发育分化特异蛋白和某些生理生化变化[J].植物生理学通讯,2005,37(3):207～210