防风体细胞胚发生发育的研究
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摘要
体细胞胚发生途径是细胞全能性表达的最完全方式,深入研究体细胞胚的发生发育过程对于揭示细胞分化、发育、形态发生与合子胚发育等重大理论问题的机制以及真核细胞中基因表达与调节控制等具有十分重要的意义。
防风(Saposhnikovia divaricata(Turcz.)Schischk.)系伞形科多年生草本植物,为国家重点保护的野生药材之一。药用部位是植物的干燥根,具有解热、镇痛、镇静、抗炎等作用。目前关于防风体细胞胚发生发育的研究主要集中于超微结构以及核型分析等方面,关于组织培养中畸形胚的发生与控制、体细胞胚发生发育的生化基础以及基因表达和调控等方面尚未见相关报道。
本论文在组织培养的基础上首次探讨了防风畸形胚的发生和控制;观察到罕见的多年生植株组培苗的开花现象和畸形胚形成果实的现象,并初步探讨了其发生机制;研究了防风体细胞胚发生发育过程中抗氧化代谢系统、多糖等物质的含量动态变化;淀粉粒、蛋白体和脂体等后含物的动态分布等,为防风种质资源的保护以及体细胞胚的研究提供基础资料。
本实验以降低畸形胚的发生率为目的优化了防风的组织培养体系。研究发现防风体细胞胚的培养过程需要较复杂的培养基顺序变化。生长素是诱导胚性细胞发生的关键因素,细胞分裂素起协同作用,ABA能有效控制防风畸形胚的发生。分化培养基中的蔗糖浓度适当提高至4%～5%可增加渗透势,能减少玻璃化胚的发生和抑制早熟萌发的趋势。减少接种密度并将愈伤组织按其颗粒的自然状态分割成较小的块,或采用将愈伤组织悬浮培养24h后再转入固体培养基中,均能降低畸形胚的发生率。
长期继代培养使防风染色体数目发生变异,但是通过体细胞胚发生途径形成的再生植株仍大多是二倍体,说明染色体组的相对完整性是体细胞胚发生潜力实现的重要基础。
利用连续石蜡切片技术研究了防风体细胞胚发生发育的显微结构。发现体细胞胚与合子胚不同的是,胚柄分化不明显,说明该结构在体细胞胚发育中并非不可代替,推测离体培养条件与细胞的相互作用代替了胚柄与胚之间的相互作用,也可能是造成畸形体细胞胚的发生率远高于合子胚的原因之一。在发育过程中,体细胞胚始终与周围细胞存在明显界限,体细胞的壁显著加厚,而周围细胞处于半解体和解体状态。
利用半薄切片技术能较清楚的观察到细胞内淀粉粒、蛋白体和脂体等后含物的变化。在组织培养中淀粉粒的出现是胚性细胞开始分化的一个标记,淀粉的代
The way of somatic embryogenesis is the best expression of cell totipotency. Study on the somatic embryogenesis and development of Saposhnikovia divaricata is significant for the best understanding of mechanism of the differentiation, development, occurrence of morphology, zygote embryogenesis, the theory of gene modulation and expression as well.Sapcshnikovia divaricata (Turcz.) Schischk. belongs to Umbelliferae, perennial herbaceous, it is one of most critical natural medicines in China. Its root can be used to relieve pain and cold, and has the ability of antibacterial, anti-allergy as well as tranquilizer. Current research on the somatic embryogenesis of Saposhnikovia divaricata has been focused on the ultrastructure and the stability of chromosome. Both the problem of occurrence and control of abnormal embryo in tissue culture, mechanism of the biochemical and molecular haven't been studied and reported.The aim of this paper is to study the occurrence and growth of abnormal somatic embryos, and to observe and discuss the rare phenomena of blooming and bearing fruit in the abnormal somatic embryo of perennial herbaceous. The activities of superoxide dismutase (SOD), peroxidase (POD), polyoses have been studied in this thesis, and the distribution of ergastic substance such as starch, protein and oil have also been explored here. These studies just aim at keeping the germplasm resources of Saposhnikovia divaricata and providing the academic data for the field of somatic embryogenesis.The regeneration of tissue culture is optimized in order to control the occurrence of abnormal somatic embryos at first. The study shows that it is necessary to adjust the complex procedure of tissue culture and to seek the best combination of mediums. Auxin is key factor for initiating embryogenic cell, and cytokinin is cooperating. ABA can control abnormal somatic embryos efficiently. 4%-5% sucrose can be used to heighten osmotic pressure and decrease the rate of vitrification embryo and precociousness. In addition, lower the density of inoculation and divide the callus into granules according natural form, or utilize suspension culture for 24h all can decrease the rate of abnormal embryos.Though long-term subculture will cause chromosome variation, diploid plant is the
防风(Saposhnikovia divaricata(Turcz.)Schischk.)系伞形科多年生草本植物,为国家重点保护的野生药材之一。药用部位是植物的干燥根,具有解热、镇痛、镇静、抗炎等作用。目前关于防风体细胞胚发生发育的研究主要集中于超微结构以及核型分析等方面,关于组织培养中畸形胚的发生与控制、体细胞胚发生发育的生化基础以及基因表达和调控等方面尚未见相关报道。
本论文在组织培养的基础上首次探讨了防风畸形胚的发生和控制;观察到罕见的多年生植株组培苗的开花现象和畸形胚形成果实的现象,并初步探讨了其发生机制;研究了防风体细胞胚发生发育过程中抗氧化代谢系统、多糖等物质的含量动态变化;淀粉粒、蛋白体和脂体等后含物的动态分布等,为防风种质资源的保护以及体细胞胚的研究提供基础资料。
本实验以降低畸形胚的发生率为目的优化了防风的组织培养体系。研究发现防风体细胞胚的培养过程需要较复杂的培养基顺序变化。生长素是诱导胚性细胞发生的关键因素,细胞分裂素起协同作用,ABA能有效控制防风畸形胚的发生。分化培养基中的蔗糖浓度适当提高至4%～5%可增加渗透势,能减少玻璃化胚的发生和抑制早熟萌发的趋势。减少接种密度并将愈伤组织按其颗粒的自然状态分割成较小的块,或采用将愈伤组织悬浮培养24h后再转入固体培养基中,均能降低畸形胚的发生率。
长期继代培养使防风染色体数目发生变异,但是通过体细胞胚发生途径形成的再生植株仍大多是二倍体,说明染色体组的相对完整性是体细胞胚发生潜力实现的重要基础。
利用连续石蜡切片技术研究了防风体细胞胚发生发育的显微结构。发现体细胞胚与合子胚不同的是,胚柄分化不明显,说明该结构在体细胞胚发育中并非不可代替,推测离体培养条件与细胞的相互作用代替了胚柄与胚之间的相互作用,也可能是造成畸形体细胞胚的发生率远高于合子胚的原因之一。在发育过程中,体细胞胚始终与周围细胞存在明显界限,体细胞的壁显著加厚,而周围细胞处于半解体和解体状态。
利用半薄切片技术能较清楚的观察到细胞内淀粉粒、蛋白体和脂体等后含物的变化。在组织培养中淀粉粒的出现是胚性细胞开始分化的一个标记,淀粉的代
The way of somatic embryogenesis is the best expression of cell totipotency. Study on the somatic embryogenesis and development of Saposhnikovia divaricata is significant for the best understanding of mechanism of the differentiation, development, occurrence of morphology, zygote embryogenesis, the theory of gene modulation and expression as well.Sapcshnikovia divaricata (Turcz.) Schischk. belongs to Umbelliferae, perennial herbaceous, it is one of most critical natural medicines in China. Its root can be used to relieve pain and cold, and has the ability of antibacterial, anti-allergy as well as tranquilizer. Current research on the somatic embryogenesis of Saposhnikovia divaricata has been focused on the ultrastructure and the stability of chromosome. Both the problem of occurrence and control of abnormal embryo in tissue culture, mechanism of the biochemical and molecular haven't been studied and reported.The aim of this paper is to study the occurrence and growth of abnormal somatic embryos, and to observe and discuss the rare phenomena of blooming and bearing fruit in the abnormal somatic embryo of perennial herbaceous. The activities of superoxide dismutase (SOD), peroxidase (POD), polyoses have been studied in this thesis, and the distribution of ergastic substance such as starch, protein and oil have also been explored here. These studies just aim at keeping the germplasm resources of Saposhnikovia divaricata and providing the academic data for the field of somatic embryogenesis.The regeneration of tissue culture is optimized in order to control the occurrence of abnormal somatic embryos at first. The study shows that it is necessary to adjust the complex procedure of tissue culture and to seek the best combination of mediums. Auxin is key factor for initiating embryogenic cell, and cytokinin is cooperating. ABA can control abnormal somatic embryos efficiently. 4%-5% sucrose can be used to heighten osmotic pressure and decrease the rate of vitrification embryo and precociousness. In addition, lower the density of inoculation and divide the callus into granules according natural form, or utilize suspension culture for 24h all can decrease the rate of abnormal embryos.Though long-term subculture will cause chromosome variation, diploid plant is the
引文
[1] 常钰,刘涤,胡之壁,植物细胞和器官大规模培养研究进展,生物技术通报,2001,1,31-36.
[2] 周邦训,周淑训,美加两国植物药的临床应用简介,南京中医药大学学报,1998,14(4),254.
[3] 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.
[4] 周俊彦,植物体细胞在组织培养中产生胚状体,植物生理学报,1981,7(4),329-397.
[5] 姚敦义,张慧娟,王静之,植物形态发生学,北京:高等教育出版社,18-35.
[6] 黑住一昌,小川和朗,《植物细胞学》,北京:科学出版社,29-67.
[7] Steward FC, Mapes MO, Mears K, Growth and organized development of cultured cells. Ⅱ Organization in culture from freely suspended cells, American Joumal of Botany, 1958, (45), 705-708.
[8] J. Reinert. uber die kontrolle der Morphogenes und die Induktion von Adventiveembryonenan Gewebekulturen aus Karotten, Plant, 1959, 53, 318-333.
[9] 朱徵,植物组织培养中的胚状体,遗传学报,1978,5(1),79-88.
[10] 奚元龄,颜昌敬,植物细胞培养手册,北京:农业出版社,1982,26-36.
[11] 崔凯荣,邢更生,周公克,植物激素对体细胞胚胎发生的诱导与调节,遗传,2000,22(5),349-354.
[12] 王震宇,张春荣,李玲,细胞分裂素结合蛋白的研究进展,植物学通报,2000,17(6),520-527.
[13] L. Michalczuk, T. J. Cooke, Auxin levels at different stages of carrot somatic embryogenesis, Phytochernistry, 1992, 31, 1097-1103.
[14] 崔凯荣,裴新梧,秦琳等,ABA对枸杞体细胞胚发生的调节作用,实验生物学报,1998,31(2),195-201.
[15] J. Z. Dond, M. R. Perreas. Induced gene expression following uptake in embryogenic supension culture ofpiceaglauca, Plant Physiol, 1996, 34, 579-587.
[16] 朱玉贤,李毅,现代分子生物学:脱落酸参与的基因表达,北京:高等教育出版社,1997,248-251.
[17] 王傲雪,李景富,植物体细胞胚状体的诱导研究及应用,黑龙江农业科学,1992,2,39-41.
[18] 孙敬三,桂耀林,植物细胞工程实验技术,北京:科学出版社,1995,12-36.
[19] 崔凯荣,邢更生,周功克等,体细胞胚发生的生化基础,生命科学,2001, 13(1),28-33.
[20] 戴若兰,张玮,刑更生等,枸杞体细胞胚胎发生中蛋白质代谢动态的立体计量,西北植物学报,1999,19(2),266-269.
[21] 潘有福,王星,孟玉玲等,秦艽体细胞胚不同发育阶段几种同工酶的特性,西北植物学报,1994,15(5),6-10.
[22] 陈金慧,施季森,诸葛强等,植物体细胞胚胎发生机理的研究进展,南京林业大学学报(自然科学版),2003,27(1),75-80.
[23] 刑更生,崔凯荣,山仑等,植物体细胞胚发生的分子基础,遗传,1999,21(1),30-34.
[24] 邢更生,李杉,崔凯荣等,植物体细胞胚发生中的某些机理的探讨,自然科学进展,2000,10(8),684-692.
[25] Evans DA, Sharp WR, Flick CE, Growth and behaviour of cell cultures, Embryogenesis and Organogenesis, In: Plant Tissue Culture: Methods and Application in Agriculture, Thorpe TA ed, New York: Academic press.1981, 45-113.
[26] Sharp WR, Evans DA, Sndahl MR, Application of somatic embryogenesis to crop improvement, In: Plant Tissue Culture, FujiwaraAed, Tokyo, 1982, 759-763.
[27] Haccius B, Question of unicellular origin of non-zygotic embryos in callus cultures, Phytomorphology, 1978, 28, 74-81.
[28] WilliaMS EG, Maheswaran G, Somatic embryogenesis: factors influencing coordinated behaviour of cells as an embryogenic group, Ann Bot, 1986(57), 443-462.
[29] 《中国植物志》第55卷第3分册,北京:科学出版社,2001,220-222.
[30] 《秦岭植物志》第1卷第3分册,北京:科学出版社,1978,407-408.
[31] 赵希范,张亚杰,李长军,李桂样,野生防风人工种植技术,现代化农业,1994,9,17-19.
[32] 金东燮等,中国中药杂志,1990,15(2),10-15.
[33] 张贵君,张艳波,李影,我国生药防风近10年的研究概况,时珍国医国药,1997,8(1),73-75.
[34] 慈忠玲,陈惠民,防风悬浮培养中的胚状体发生,植物学报,1996,05,379-383.
[35] 慈忠玲,磁场对防风体细胞胚发生发育和亚显微结构的影响,植物生理学报,1999,25(3),309-312.
[36] 慈忠玲,陈惠民,防风体细胞在继代培养中的染色体变异,内蒙古大学学报(自然科学版),1995,26(3),339-341.
[37] 滕治杰,李忠林,伊洪光,防风的野生药材与其栽培品的鉴别,牡丹江医学院学报,2002,23(3),67-68.
[38] 刘忠双,赵桂琴,王庆余,防风的栽培技术.特种经济动植物,2003,6,28-29.
[39] 金光洙,李景道,朴惠善,防风化学成分的研究,中国中药杂志,1992,17(1),38.
[40] 肖永庆,李丽,杨滨,黄潞崎,防风化学成分研究,中国中药杂志,2001,26(2),117.
[41] 李丽,肖永庆,防风及其主要化学成分的TLC鉴定,中国药学杂志,2000,35(10),656-658.
[42] 慈忠玲,防风的核型分析,内蒙古林学院学报,1994,16(2),31-31.
[43] 马红.防风近年研究概述,中草药,1994,25(8),438-443.
[44] Kamada H, Hiroshi Harada, Changes in the endogenous level and effects of ABA during somatic embryogenesis of Daucus carrot L, Plants & Cell Physiol, 1981, 22(8), 1423-1429.
[45] 郭子彪,盖钧镒,内源激素IAA、ABA对大豆萌发子叶胚性愈伤组织诱导及其分化的调控,大豆科学,1997,16(3),194-198.
[46] 李卫,张春广,郭光沁,郑国锠,ABA诱导毛酸浆下胚轴体细胞胚状结构的直接发生,西北植物学报,2003,2,309-313.
[47] Van der Meulen RM, Visser K, Wang M. Effects of modulation of calcium levels and calcium fluxes on ABA-induced gene expression in barley aleurone, Plant Science, 1996, 117, 75-82.
[48] 程玉兰,刁丰秋,吴乃虎,黄美娟,蔗糖调控培养对胡萝卜体细胞胚内ABA水平的效应,植物学报,1999,41(7),761-765.
[49] 陶铭,组织培养中畸形胚状体及超度含水态苗的研究,西北植物学报,2001,21(5),1048-1058.
[50] 张宝红,李秀兰,李付广,等,棉花组织培养中畸形胚的发生和转化,作物学报,1996,22(1),107-111.
[51] JPA. Ortiz, G. Fama, RH. Vallejos, IN. Dahalac, Cytodifferentiation and cell organization in the somatic embryogenesis of wheat (Tritienm aestivum L.), Biocell, 1996, 20(1), 61-66.
[52] 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.
[53] 杨映根,桂耀林,唐巍,等,青扦愈伤组织在继代培养中的分化能力及染色体稳定性研究,植物学报,1994,36(12),934-939.
[54] 王喆之,李克勤,张大力,等,陆地棉胚性愈伤组织的变异及高频胚胎发生,植物学报,1994,36(5),331-338.
[55] S. Stim,, H. J. Jacobsen., Marker proteins for embryogenic differentiation patterns in peacallus, Plant cell Rep, 1987, 6, 50-54.
[56] R. W. Giroux, K. P. Pauls, Characterization of embryogenesis-ralated protein in alfalfa(Medieago satina), Physiologia Plantarum, 1996, 96(4), 585-592.
[57] 汪丽虹,王星,崔凯荣,等,石刁柏及党参体细胞胚发生中的淀粉代谢动态,植物学通报,1996,01,41-45.
[58] 韩碧文,胚状体发生的细胞学和生理生化研究,植物学通报,1989,6(1),1-4.
[59] 胡适宜,徐丽云,显示环氧树脂厚切片中多糖、蛋白质和脂类的细胞化学方法,植物学报,1990,32(11),841-846.
[60] 赵保路,氧自由基和天然抗氧化剂,北京:科学出版社,1999,133.
[61] Lei L, Effects of Ganoder MS polysaccharids on the activity of DNA polymerase in spleen cells stimulated by alloantigens in mice in vitro, Beijing Med. Univ., 1991, 23, 329-331.
[62] 许士凯,抗衰老药物学,北京:中国医药科技出版社,1999,615.
[63] 宋承吉,我国抗衰老中药、中成药研究现状,中成药,1992,14,38-40.
[64] 田庚元,植物多糖的研究进展,中国中药杂志,1995,20,441-443.
[65] 李江,陆蕴如,赵桂燕,防风多糖的研究,中草药,1999,30(9),652-653.
[66] 李莉,防风多糖抗肿瘤免疫促进作用的实验研究,中国肿瘤生物治疗杂志,1997,4(3),234.
[67] 娄志红,江延辉,孟祥才,野生防风与栽培防风多糖含量比较,特产研究, 2000,2(25),28-31.
[68] 张志良,植物生理学实验指导,北京:高等教育出版社,1990,35-78.
[69] 崔凯荣,任红旭,邢更生,王亚馥,枸杞组织培养中抗氧化酶活性与体细胞胚发生相关性研究,兰州大学学报,1998,34(3),93-99.
[70] 王爱国,大豆种子SOD研究,植物生理学报,1983,9,77-84.
[71] 李玉泉,宋占午,王莱,叶螨危害对豇豆叶片超氧化物歧化酶及过氧化氢酶活性的影响,西北师范大学学报(自),200l,37,62-65.
[2] 周邦训,周淑训,美加两国植物药的临床应用简介,南京中医药大学学报,1998,14(4),254.
[3] 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.
[4] 周俊彦,植物体细胞在组织培养中产生胚状体,植物生理学报,1981,7(4),329-397.
[5] 姚敦义,张慧娟,王静之,植物形态发生学,北京:高等教育出版社,18-35.
[6] 黑住一昌,小川和朗,《植物细胞学》,北京:科学出版社,29-67.
[7] Steward FC, Mapes MO, Mears K, Growth and organized development of cultured cells. Ⅱ Organization in culture from freely suspended cells, American Joumal of Botany, 1958, (45), 705-708.
[8] J. Reinert. uber die kontrolle der Morphogenes und die Induktion von Adventiveembryonenan Gewebekulturen aus Karotten, Plant, 1959, 53, 318-333.
[9] 朱徵,植物组织培养中的胚状体,遗传学报,1978,5(1),79-88.
[10] 奚元龄,颜昌敬,植物细胞培养手册,北京:农业出版社,1982,26-36.
[11] 崔凯荣,邢更生,周公克,植物激素对体细胞胚胎发生的诱导与调节,遗传,2000,22(5),349-354.
[12] 王震宇,张春荣,李玲,细胞分裂素结合蛋白的研究进展,植物学通报,2000,17(6),520-527.
[13] L. Michalczuk, T. J. Cooke, Auxin levels at different stages of carrot somatic embryogenesis, Phytochernistry, 1992, 31, 1097-1103.
[14] 崔凯荣,裴新梧,秦琳等,ABA对枸杞体细胞胚发生的调节作用,实验生物学报,1998,31(2),195-201.
[15] J. Z. Dond, M. R. Perreas. Induced gene expression following uptake in embryogenic supension culture ofpiceaglauca, Plant Physiol, 1996, 34, 579-587.
[16] 朱玉贤,李毅,现代分子生物学:脱落酸参与的基因表达,北京:高等教育出版社,1997,248-251.
[17] 王傲雪,李景富,植物体细胞胚状体的诱导研究及应用,黑龙江农业科学,1992,2,39-41.
[18] 孙敬三,桂耀林,植物细胞工程实验技术,北京:科学出版社,1995,12-36.
[19] 崔凯荣,邢更生,周功克等,体细胞胚发生的生化基础,生命科学,2001, 13(1),28-33.
[20] 戴若兰,张玮,刑更生等,枸杞体细胞胚胎发生中蛋白质代谢动态的立体计量,西北植物学报,1999,19(2),266-269.
[21] 潘有福,王星,孟玉玲等,秦艽体细胞胚不同发育阶段几种同工酶的特性,西北植物学报,1994,15(5),6-10.
[22] 陈金慧,施季森,诸葛强等,植物体细胞胚胎发生机理的研究进展,南京林业大学学报(自然科学版),2003,27(1),75-80.
[23] 刑更生,崔凯荣,山仑等,植物体细胞胚发生的分子基础,遗传,1999,21(1),30-34.
[24] 邢更生,李杉,崔凯荣等,植物体细胞胚发生中的某些机理的探讨,自然科学进展,2000,10(8),684-692.
[25] Evans DA, Sharp WR, Flick CE, Growth and behaviour of cell cultures, Embryogenesis and Organogenesis, In: Plant Tissue Culture: Methods and Application in Agriculture, Thorpe TA ed, New York: Academic press.1981, 45-113.
[26] Sharp WR, Evans DA, Sndahl MR, Application of somatic embryogenesis to crop improvement, In: Plant Tissue Culture, FujiwaraAed, Tokyo, 1982, 759-763.
[27] Haccius B, Question of unicellular origin of non-zygotic embryos in callus cultures, Phytomorphology, 1978, 28, 74-81.
[28] WilliaMS EG, Maheswaran G, Somatic embryogenesis: factors influencing coordinated behaviour of cells as an embryogenic group, Ann Bot, 1986(57), 443-462.
[29] 《中国植物志》第55卷第3分册,北京:科学出版社,2001,220-222.
[30] 《秦岭植物志》第1卷第3分册,北京:科学出版社,1978,407-408.
[31] 赵希范,张亚杰,李长军,李桂样,野生防风人工种植技术,现代化农业,1994,9,17-19.
[32] 金东燮等,中国中药杂志,1990,15(2),10-15.
[33] 张贵君,张艳波,李影,我国生药防风近10年的研究概况,时珍国医国药,1997,8(1),73-75.
[34] 慈忠玲,陈惠民,防风悬浮培养中的胚状体发生,植物学报,1996,05,379-383.
[35] 慈忠玲,磁场对防风体细胞胚发生发育和亚显微结构的影响,植物生理学报,1999,25(3),309-312.
[36] 慈忠玲,陈惠民,防风体细胞在继代培养中的染色体变异,内蒙古大学学报(自然科学版),1995,26(3),339-341.
[37] 滕治杰,李忠林,伊洪光,防风的野生药材与其栽培品的鉴别,牡丹江医学院学报,2002,23(3),67-68.
[38] 刘忠双,赵桂琴,王庆余,防风的栽培技术.特种经济动植物,2003,6,28-29.
[39] 金光洙,李景道,朴惠善,防风化学成分的研究,中国中药杂志,1992,17(1),38.
[40] 肖永庆,李丽,杨滨,黄潞崎,防风化学成分研究,中国中药杂志,2001,26(2),117.
[41] 李丽,肖永庆,防风及其主要化学成分的TLC鉴定,中国药学杂志,2000,35(10),656-658.
[42] 慈忠玲,防风的核型分析,内蒙古林学院学报,1994,16(2),31-31.
[43] 马红.防风近年研究概述,中草药,1994,25(8),438-443.
[44] Kamada H, Hiroshi Harada, Changes in the endogenous level and effects of ABA during somatic embryogenesis of Daucus carrot L, Plants & Cell Physiol, 1981, 22(8), 1423-1429.
[45] 郭子彪,盖钧镒,内源激素IAA、ABA对大豆萌发子叶胚性愈伤组织诱导及其分化的调控,大豆科学,1997,16(3),194-198.
[46] 李卫,张春广,郭光沁,郑国锠,ABA诱导毛酸浆下胚轴体细胞胚状结构的直接发生,西北植物学报,2003,2,309-313.
[47] Van der Meulen RM, Visser K, Wang M. Effects of modulation of calcium levels and calcium fluxes on ABA-induced gene expression in barley aleurone, Plant Science, 1996, 117, 75-82.
[48] 程玉兰,刁丰秋,吴乃虎,黄美娟,蔗糖调控培养对胡萝卜体细胞胚内ABA水平的效应,植物学报,1999,41(7),761-765.
[49] 陶铭,组织培养中畸形胚状体及超度含水态苗的研究,西北植物学报,2001,21(5),1048-1058.
[50] 张宝红,李秀兰,李付广,等,棉花组织培养中畸形胚的发生和转化,作物学报,1996,22(1),107-111.
[51] JPA. Ortiz, G. Fama, RH. Vallejos, IN. Dahalac, Cytodifferentiation and cell organization in the somatic embryogenesis of wheat (Tritienm aestivum L.), Biocell, 1996, 20(1), 61-66.
[52] 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.
[53] 杨映根,桂耀林,唐巍,等,青扦愈伤组织在继代培养中的分化能力及染色体稳定性研究,植物学报,1994,36(12),934-939.
[54] 王喆之,李克勤,张大力,等,陆地棉胚性愈伤组织的变异及高频胚胎发生,植物学报,1994,36(5),331-338.
[55] S. Stim,, H. J. Jacobsen., Marker proteins for embryogenic differentiation patterns in peacallus, Plant cell Rep, 1987, 6, 50-54.
[56] R. W. Giroux, K. P. Pauls, Characterization of embryogenesis-ralated protein in alfalfa(Medieago satina), Physiologia Plantarum, 1996, 96(4), 585-592.
[57] 汪丽虹,王星,崔凯荣,等,石刁柏及党参体细胞胚发生中的淀粉代谢动态,植物学通报,1996,01,41-45.
[58] 韩碧文,胚状体发生的细胞学和生理生化研究,植物学通报,1989,6(1),1-4.
[59] 胡适宜,徐丽云,显示环氧树脂厚切片中多糖、蛋白质和脂类的细胞化学方法,植物学报,1990,32(11),841-846.
[60] 赵保路,氧自由基和天然抗氧化剂,北京:科学出版社,1999,133.
[61] Lei L, Effects of Ganoder MS polysaccharids on the activity of DNA polymerase in spleen cells stimulated by alloantigens in mice in vitro, Beijing Med. Univ., 1991, 23, 329-331.
[62] 许士凯,抗衰老药物学,北京:中国医药科技出版社,1999,615.
[63] 宋承吉,我国抗衰老中药、中成药研究现状,中成药,1992,14,38-40.
[64] 田庚元,植物多糖的研究进展,中国中药杂志,1995,20,441-443.
[65] 李江,陆蕴如,赵桂燕,防风多糖的研究,中草药,1999,30(9),652-653.
[66] 李莉,防风多糖抗肿瘤免疫促进作用的实验研究,中国肿瘤生物治疗杂志,1997,4(3),234.
[67] 娄志红,江延辉,孟祥才,野生防风与栽培防风多糖含量比较,特产研究, 2000,2(25),28-31.
[68] 张志良,植物生理学实验指导,北京:高等教育出版社,1990,35-78.
[69] 崔凯荣,任红旭,邢更生,王亚馥,枸杞组织培养中抗氧化酶活性与体细胞胚发生相关性研究,兰州大学学报,1998,34(3),93-99.
[70] 王爱国,大豆种子SOD研究,植物生理学报,1983,9,77-84.
[71] 李玉泉,宋占午,王莱,叶螨危害对豇豆叶片超氧化物歧化酶及过氧化氢酶活性的影响,西北师范大学学报(自),200l,37,62-65.