摘要
不定根发生既是植物器官分化的重要理论问题,又是无性繁殖和完整植株再生等林业重要实践的关键。成功地诱导不定根,对实现无性繁殖获得最大遗传增益以及转基因材料的完整植株再生都具有重要的实践意义。本研究以741杨试管苗为试材,系统地研究了影响不定根发生的因素,探讨最优化的不定根发生的研究体系,并且着重从激素调控、解剖学、IAA免疫组织化学定位等方面对不定根发生调控机制进行了研究。主要结果如下:
1.建立了高效、同步和稳定性好的741杨叶不定根发生体系。对741杨不同发育状态、不同外植体以及生长调节剂IBA水平进行了系统的研究,确定了以741杨生根试管苗上的叶为材料,在1/2MS附加0.5mg/L IBA培养基中进行生根诱导。利用这一体系,生根时间集中诱导后6-8天,第8天生根率达到100%,平均生根条数为3.7条/叶,重复试验的结果表明该体系具有较好的稳定性。
2.施用生长素极性运输抑制剂TIBA,明显地地抑制了741杨叶不定根发生,表明IAA极性运输在不定根发生中发挥重要作用。采取培养基中添加和叶片涂抹方法进行TIBA处理,TIBA为5mg/L时,第8天生根率为52.2%,比对照降低了47.8%;TIBA浓度为10mg/L时,第8天生根率为5.6%,比对照降低了94.4%;TIBA浓度为20mg/L时,第8天生根率为0,且叶柄基部长出了愈伤;TIBA浓度为100mg/L时,叶受到毒害,叶片变黄。因此,TIBA抑制不定根发生的较适宜浓度为10mg/L.
3.解剖学试验结果表明:741杨叶不定根属于诱生根原基类型,根原基起源于形成层,其发生过程为:形成层细胞纵向和横向分裂加快形成分生组织细胞群,分生组织进一步分裂分化形成根原基,根原基生长发育形成根,根穿过皮层突破表皮,形成不定根。
4.以741杨嫩茎和核桃幼胚为材料,优化和改进了木本植物IAA免疫胶体金定位体系。1)针对木本植物组织结构复杂、抗原往往深藏在蛋白质的网络中的特点,用尿素-胰蛋白酶联合消化技术对抗原进行了修复。使修复后的抗原充分暴露出来,抗体很容易接近; 2)提高了缓冲液中Triton X-100的用量。从而增强了细胞膜的通透性,使抗体更容易渗透到细胞内部并与IAA反应;3)在缓冲液中加入了2.5%牛血清白蛋白,消除了背景染色。牛血清白蛋白不仅能降低非特异性吸附,抑制非特异性染色,还可封闭抗血清中混杂的白蛋白抗体; 4)抗原抗体反应由4℃反应过夜改为37℃反应2h,缩短了反应时间,提高了效率。通过染色技术的改进,获得了很好的结果:敏感性高,特异性强,背景清晰,耗时少。
5. 741杨叶不定根发生过程中IAA免疫胶体金定位结果显示,不定根诱导阶段叶柄形成层有明显的IAA积累,即IAA在叶柄形成层中的积累与不定根的分化密切相关,施用10mg/L TIBA,明显地延缓了IAA在叶柄形成层中积累,延缓了生根时间,降低了生根率。具体的结果是:诱导前(0d),叶柄中IAA分布较少,表皮和皮层的金颗粒密度为4.8个/100um2,维管组织的金颗粒密度为5.1个/100um2;不定根诱导初期(1~3d),IAA主要分布在加厚的形成层及其周缘维管束,其金颗粒密度为216.8个/100um2;不定根原基形成阶段(3~5d),IAA大量积累在根原基细胞群,其金颗粒密度为345.7个/100um2;不定根伸长阶段(6~7d),IAA集中分布根尖生长点和根中柱鞘,其金颗粒密度分别为210.5和32.3个/100um2;施用生长素极性运输抑制剂TIBA,显著地推迟了生根时间,降低了生根率,TIBA施用后第3天,叶柄中的IAA含量仍然很少,表皮和皮层的金颗粒密度为6.9个/100um2,维管组织的密度为8.7个/100um2;第6天,IAA集中分布在维管组织,其金颗粒密度为150.2个/100um2;第8天,IAA集中分布在根原基,其金颗粒密度为300.5个/100um2;第10天,IAA集中分布在根尖和根维管,其金颗粒密度分别为220.4个/100 um2和30.1个/100um2。
6.利用免疫胶体金电镜技术对叶不定根发生过程中的IAA亚细胞分布进行了分析。在不定根诱导初期和根原基形成阶段,IAA主要分布在细胞壁、细胞质、液泡、质体、高尔基体及其囊泡中,没有发现在特定细胞器中积累的特点,间接反映出调控不定根的IAA是在其它组织器官运输而来,IAA质膜上的受体可能在调控不定根发生上发挥重要作用。
Adventitious root is an important topic both on the theoretical aspect of plant organogenesis and on the practical side of plant propagation and excised tissues or organs in vitro regeneration. Factors affecting rhizogenesis were investigated, technical-platform of adventitious root was optimized and established, and anatomical and IAA immunolocalized characteristics in the process of rhizogenesis were studied with Poplar 741 in vitro in this research. The main results were as follow:
1. A stable, efficient, and synchromic technical-platform of rhizogenesis was established. Leaves, stems, leaf discs and petioles from rooted plantlets and subcultured cultures of Poplar 741 were taken to compare their rooting difference, and the effect of IBA concentration on the rhizogenesis was also studied. The achieved results were that leaves from rooted plantlets were obviously better than other explants in aspects like time of rooting, rooting rate and synchronism in 1/2MS medium with 0.5mg/L IBA. The adventitious root started at the 6th day, and the rooting rate was up to 100% at the 8th day, and the average number of root was 3.7 in this system.
2. TIBA application inhibited rhizogenesis of Poplar 741 leaves in vitro significantly, suggesting IAA polar transport plays an important role in the rhizogenesis. The rooting rate was 52.2% at the 8th day in the treatment of 5mg/L TIBA, decreased 47.8% compared with the control.The rooting rate was 5.6% at the 8th day in the treatment of 5mg/L TIBA, decreased 94.4% compared with the control. When TIBA increased to 20mg/L, the basal of petiole had no root and grew out callus. When the concentration of TIBA increased to 100mg/L, the leaves was hurt and the laminae get yellow. So the suitable concentration of TIBA to inhibit the adventitious root was 10mg/L.
3. Anatomic result showed: root primordium was induced root primordium in the Poplar 741 leaves.The root primordium originated from the cambium cells.The process of adventitious root was: the cambium cells divided to form cell groups, the cell groups developed into root primordium and the primordium cells grew and developed to adventitioud root.
4. The technique of immuno-gold localization of endogenous auxin was improved with embryo of walnut and poplar tender shoots. (1) considering the woody plants characteristics of perennial gowth, compound complexity, antigen hiding in the networks, urea together with trypsin was used to unmask the antigens of tissue sections. (2) the concentration of Triton X-100 in the buffer was improved to increase the permeability of cell membrane. (3) 2.5% BSA was added to the buffer to eliminate the background staining. (4) the reaction condition between antigen and antibody was at 37℃for 2h instead of 4℃overnight. The improved methods have characters of high sensitivity, strong speciality and clear background.
5. The results of immuno-gold localization of IAA in the rhizogenesis showed that IAA was accumulated at the cambium during the induction stage. Application of 10mg/L TIBA delayed the accumulation of IAA. The details of the results were: little IAA was found in the basal of petiole before induction, the density of gold particles in cortex and vascular tissues were 4.8/100um2 and 5.1/100um2. At the beginning stage of induction, gold particles mainly distributed in vascular tissues and the density was 216.8/100um2. At the stage of root primordium formation, gold particles mainly distributed in the primordium and the density was 345.7/100um2. At the development stage of root, IAA mainly distributed in the root tip and stele, and the densitys were 210.5/100um2 and 30.1/100um2. At the 3th day after application TIBA, little IAA was found still in the basal of petiole, the density of gold particles in the cortex and vascular tissues were 6.9/100um2 and 8.7 /100um2. At the 6th day, gold particles maily distributed in the vascular tissues and the density was 150.2/100um2. At the 8th day, gold particles mainly distributed in the root primordium and the density was 300.5/100um2. At the 10th day, mainly distributed in the root tip and stele, and the density were 220.4/100um2 and 30.1/100um2
6 Subcellular localization of IAA in the rhizogenesis of Poplar 741 leaves was studied with immuno-gold microscope technique. Cytoplasm, cell wall, vacuole, plastid and golgi body were labeled with gold particles at the differentiation and primordium formation stages. It was not found that gole particles accumulate in the specific cell organ, indicating IAA regulated rhizogenesis was transported to the destination tissue from other tissues organs.
引文
[1] Blakesley D. Auxin Metabolish and Adventitious Root Initiation. In: Biology of Adventitious Root formation, Davis TD & Haissig BE(eds). Plenum Press, New York, 1994, P: 143-154
[2] Durski RS, Cohen JD, Slovin JP, Reinecke DM (1995). Auxin biosynthesis and metabolism. In Davies PJ (ed), Plant Hormones-physiology, biochemistry and molecular biology, Kluwer Academic Publishers, Dordrecht, P:39-65.
[3] Bak S, Tax F E, Feldmann KA, et al. CYP83B1, a cytochrome P450 at the metabolic branch point in auxin and indole glucosinolate biosynthesis in Arabidopsis thaliana. Plant Cell, 2001, 13(1): 101-112
[4] Zhao Y, Hull AK, Gupta NR, et al. Trp-dependant auxin biosynthesis in Arabidopsis: involvement of cytochrome P450s CYP79B2 and CYP79B3. Genes Dev, 2002, 16(23): 3100-3112
[5] Ge L, Chen H, Jiang JF, et al. Overexpression of OsRAA1 causes pleiotropic phenotypes in transgenic rice plants, including altered leaf, flower, and root development and root response to gravity. Plant Physiol, 2004, 135(3): 1502-1513
[6] Kim YS, Jung-Ki Min, Donghern Kim, et al. A soluble auxin-binding protein, ABP57. J. Biology Chemtary, 2001, 14(276): 10730-10736
[7] Xu M, Zhu L, Shou H,et al. A PIN1 Family Gene, OsPIN1, Involved in auxin-dependent adventitious root emergence and tillering in Rice. Plant Cell Physiol, 2005, 46(10): 1674-1681
[8] Goldfarb B, C Lanz-Garcia, ZG Lian, et al. Aux/IAA gene family is conserved in the gymnosperm, loblolly pine (Pinustaeda). Tree physiology, 2003, 23(17): 1181-1192
[9] Mallory AC, Bartel DP, Bartel B. MicroRNA-directed regulation of Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development and modulates expression of early auxin response genes. Plant Cell, 2005, 17(5): 1360-1375
[10] Sorin C, Bussell JD, Camus I, et al. Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1. Plant Cell, 2005, 17(5):1343-59
[11]李继华.扦插的原理与应用.上海:上海科学技术出版社. 1987
[12]哈特曼.HT.植物繁殖原理和技术.郑邢文译.北京:中国林业出版社. 1985
[13]刘桂丰.长白落叶松嫩枝扦插生根的解剖研究.东北林业大学学报, 1992,20(1):9-13.
[14]丘醒球,余倩珠.桉树插条生根解剖研究.林业科学研究, 1995, 8(2): 170-176.
[15]黄卓烈,林韶湘,谭绍满.桉树体内的生根抑制剂物质研究综述.林业科学研究, 1994,7(3): 319-324.
[16]韩碧文,李颖章.植物组织培养中器官建成的生理生化基础.植物学通报,1993,10(2): 1-6.
[17] Gatineau F, JG Fouche, C Kevers, JF Hausman, T Gaspar. Quantitative variations of indolyl compounds including IAA, IAAsp and serotonin in walnut microcuting during root induction. Bio Plant, 1997, 39(1): 131-137.
[18] Heloir MC, Kevers C, Hausman JF et al. Changes in the concentrations of auxins and polyamines during root of in-vitro-propagated walnut shoot. Tree Physiology, 1996, 16(5): 515-519.
[19] Heloir MC, Kevers C, Hausman JF et al. Changes in the concentrations of auxins and polyamines during root of in-vitro-propagated walnut shoot. Tree Physiology, 1996, 16(5): 515-519.
[20]谷瑞升,蒋湘宁,郭仲琛.植物离体培养中器官发生机制的研究进展.植物学通报,1996,16(3):238-244.
[21] Marks TR, SE Simpson. Interaction of explant type and indole-3-butyric acid during rooting in intro in a range of difficult and easy-to-root woody plants. Plant Cell Tissue and Organ Culture, 2000, 1(62): 65-74.
[22] Centeno ML, Rodrigue A, Ffeito I et al. Relationship between endogenous auxin and cytokinin level and morphogenic responses in Actinidia deliciosa tissue cultures. Plant Cell Reports, 1997, 1(16): 58-62.
[23]裴东,袁丽钗,奚声柯.核桃品种试管幼茎生根的研究.林业科学,2002, 38(2):32-37.
[24]裴东,袁丽钗,蒋湘宁,谷瑞升.核桃子叶不定根发生调控的研究.林业科学,2003, 39(6):33-39.
[25] Blakesley D. Auxin Metabolish and Adventitious Root Initiation. In: Biology of Adventitious Root formation, Davis TD & Haissig BE(eds). Plenum Press, New York, 1994, P143-154
[26] Liu ZH, IC Hsiao, YW Pan. Effect of naphthalene acetic acid on endogenous IAA, peroxidases and auxin oxidase in hypocotyls cuttings of soybean during root formation. Bot. Bull. Acad.Sin., 1996, 52(12): 57-61.
[27]裴东,袁丽钗.核桃等木本植物不定根发生研究进展.干果研究进展,北京:中国林业出版社,2001,234-238
[28] Alion R. The induction of vascular tissues by auxin and cytokinin. In: Davies PJ, editor. Plant hormones: physiology, biochemistry and molecular biology, 2nd ed. Dordrecht;Kluwer Academic Publishers, 1995, 531-546.
[29] Lomax TL, Muday GK, Rubery PH. Auxin transport. In PJ Davies, ed, . Plant Hormones: Physiology, Biochemistry and Molecular Biology. Kluwer Academic Publishers, Dordrecht, The Netherlands, 1995, 509-530.
[30] Avery GS. Differential distribution of phytohormone in the developing leaf of Nicotiana, and its relation to polarized growth. Bull Toray Culb, 1935, 6(62): 313-330.
[31] Bartel B. Auxin biosynthesis. Annu Rev Plant Physiol and Plant Mol Biol, 1997, 48: 51-66
[32] Orna AK, Cheng JC, Chen LJ. Indole acetic acid distribution coincides with vascular differentiation pattern during Arabidopsis leaf ontogeny. Plant Physiology, 2002, 130(1): 199-209.
[33] Alion R, Feigenbaum P, Kalev N, Rozovsky S.. Hormonal control of vascular differentiation in plant: the physiological basis of cambium ontogeny and xylem evolution. In: Savidge RA, Barnett JR, Napir R, editors. Cell and molecular biology of wood formation. Oxford: BIOS Scientific Publishers, 2000, 223-236.
[34] Sachs T. The control of the patterned differentiation of vascular tissues. Adv Bot Res, 1981, 9: 151-262.
[35] Sachs T. Intrgrating cellar and organismal aspects of vascular differentiation. Plant Cell Swati Basu, Haiguo Sun, Leigh Brian. Early embryo development in Fucus distichus is auxin sensitive. Plant Physiology. 2002, 130: 292-302.
[36] Alion R. Role of auxin and sucrose in the differentiation of sieve and tracheary elements in plant tissue cultures. Planta, 1980, 150: 255-263.
[37] Alion R. Differentiation of vascular tissues. Annu Rev Plant Physiol. 1987, 38: 179-204.
[38] Lersten NR. . Sieve tube in foliar vein endings: review and quantitative survey of Rudbeckia laciniata(Asteraceae). Amer J Bot, 1990, 77: 1132-1141
[39] Horner HT, Lersten NR, Wirth C L. Quantitative survey of sieve tube distribution in foliar terminal veins of ten dicot spesies. Amer J Bot., 1994, 81: 1267-1274.
[40] Raven PH, Evert RE, Eichhorn SE. Biology of Plants, 6th ed. New York: Freeman and Company Worth Publishers. 1999, P: 944.
[41] Alion R. Foliar and axial aspects of vascular differentiation hypotheses and evidence. J Plant Growth Regul, 2001, 20: 22-34.
[42] Sieburth LE. Auxin is required for leaf xein pattern in Arabidopsis. Plant Physiol, 1999, 121: 1179-1190.
[43] Swati B, Sun HG, Brian L. Early embryo development in Fucus distichus is auxin sensitive. PlantPhysiology, 2002, 130: 292-302.
[44]周云龙.植物生物学.北京:高等教育出版社, 1999
[45] Dewitte W, Van O, H. Probing the distribution of plant hormones by immunocychemistry. Plant Growth Regulation, 2001, 33: 67-74.
[46] Faulk WP, Taylor GM. An immunocolloid method for the electron microscrope. Immunochem-itry. 1971, 8(11): 1081-1083
[47] Romano EL, Stolinski C, Hughes - Jones NC. An antiglobalin reagent-labelled with coUoidal gold for use in electron reagent labelled with colloidal gold for use in electron microscopy. Immunochemistry, 1974, 11(8): 521-22.
[48] Geoghegan WD, Ackerman GA. Adsorption of borseradish peroxidase,ovomucoid and anti immunoglobulin to colloidal gold for the indirect detection of conranavalin A,wheat germ agglutinin and goat antihuman immunoglobalin G on cell surfaces at the electron microscopic level. J Histochem Cytochem, 1977, 25 (11): 1187~1200.
[49] Holgate CS, Jackson P, Cowen PN, et al. Immunogold silver staining:new method of immunostaining with enhanced sensitivity. J Histochem Cytochem, 1983, 31 (7): 938-944
[50] Ohimiya A, Tateki Hayashi, Norio Kakiuchi. Immuno-gold localization of IAA in peach seedings. Plant Cell Physiol, 1990, 31(5): 711-715
[51] Pence VC, Caruso JL. Immunoassay methods of plant hormone arialysis.New York: Martinus Nijhoff Publishers, 1987: 25l
[52] Jia wen suo , Huang cong lin. Immuno-gold localization of IAA in the leaf cells of vicia faba. Acta Botanica Sinica,1997, 39(7): 596-600
[53]陈以峰,梁世平,杨弘远,周燮.受精前后烟草卵细胞内玉米素和三类酸性植物激素分布的免疫电镜观察.植物学报,1999,41(11): 1145-1149
[54] L. B. Vysotskaya, S. Yu. Veselov, D. S. Veselov,V. N. Filippenko,E. A. Ivanov,I. I. IvanovG. R. Kudoyarova. Immunohistological Localization and Quantification of IAA in Studies of Root Growth Regulation. Russian Journal of Plant Physiology, 2007, 54(6): 827~832.
[55]汪向彬,王震,段俊华,崔克明.构树去木质部后维管组织再生中内源IAA浓度的变化及其组织定位.植物学报,1999, 41(12):1327-1331
[56] Cui KM, Wang XB, Duan JH. Change in the concentrationand tissue localization of endogenous IAA during the regeneration after girdling in Broussonetia papyrifera (L.) Vent. Acta ScientiarumNaturalium Universitatis Pekinensis,2000,36:495-502.
[57] LI Nian-Hua , TONG Zhe, LU Shan- Fa. Immunohistochemical Analysis of IAA in Anthers of the Photoperiod- sensitive Genic Male- sterile Rice. Acta Botanica Sinica, 2000, 42 (10):1045-1050
[58]卢善发.离体茎段嫁接体内IAA的免疫组织化学定位.科学通报,2000,45(8):856-860
[59]王幼群,韩静,林金星.紫丁香叶柄离区IAA的免疫组织化学定位.植物学报,2001,43(2):213-216.
[60]候智霞.草莓花芽形成和果实发育过程中IAA及ABP1的免疫化学分析,中国农业大学博士学位论文,2004
[61] Zavala ME,Brandon DI. Localization of a phytohormone using immunocytochemistry.J Cell Biol, 1983, 97(4): 1235
[62] Eberle J, W ang TL, Cook S et al. Immunoassays and ultrastructural localizafon of isopent- enyla denine and related cytokinins using monoclonal antibodies. Planta, l987, 172-289
[63] Sossountzov L, Maldiney R, Sotta B et al.1rnmunocytochemical localization of cytokinins in Craigella tomato and a sideshootless mutant. Planta, 1988, 175-291
[64] Annie Jacqmard, Nathalie Detry, Walter Dewitte Harry Van Onckelen, Georges Bernier. In situ localisation of cytokinins in the shoot apical meristem of sinapis albs at floral transition. Planta, 2002, 214:970-973.
[65] P. Moncaleán, C. López-Iglesias, B. Fernández ,A. Rodríguez. Immunocytochemical Location of Endogenous Cytokinins in Buds of Kiwifruit (Actinidia deliciosa) during the First Hours of in vitro Culture. The Histochemical Journal, 2004,33(7):403-411
[66] Sossountzov L, Maldiney R, Sotta B et al. Immunoelectron-microscopy localization of ABA with colloidal gold on lowicryl-embedded tissues of Chnopodium polyspermum L. Planta, l986, l68(4): 471-481
[67] Welbaum G E, Hill D R. Freezing tolerance protein composition and abscisic acid localization and content of pea epicotyl shoot and root tissue in response to temperature and water stress. Journal of Experimental Botany, 1997, 48: 643-654
[68] Pastor A, Alegre L. Abscisic acid immunolocalization and ultrastructural changes in water-stressed lamender (Lavandula stoechsas L) plants. Physiologia Plantarum, 1999, 105: 272-279
[69]贾文锁,王学臣,娄成后.蚕豆(Vicia faba L.)叶肉细胞中ABA的胶体金免疫电镜定位.植物生理学报,1994, 20(4): 380-384
[70]王学臣,贾文锁.水分胁迫下蚕豆(Vicia faba L.)气孔关闭与叶片细胞中ABA区隔化与再分配的关系.植物生理学报,1995, 21(4): 324-328
[71] Dapeng Zhang, Conglin Huang and Wensuo Jia。Immunogold electron-microscopy localization of abscisic acid in flesh cells of grape berry (Vitis vinifera L.×Vitis labrusca L. cv Kyoho). Scientia Horticulturae, 1999, 81(2): 189-198
[72]孟祥红,王建波,利容千.光敏细胞质不育小麦花药发育过程中ABA免疫电镜定位.武汉大学学报(理学版), 2001, 47(6): 775-781
[73] Yamaguchi I, Weiler E W. In: Takahashi N, Phinney BO, MacMillan (eds) Gibberellins. Springer Verlag, 1991,146-165
[74]孟祥红,王建波,利容千.光敏胞质不育小麦花药发育过程中GA1 + 4分布的免疫电镜研究.中国农业科学,2002 ,35 (6) : 596-599
[75]魏丽,蒋湘宁,裴东.不定根发生分子调控机制的研究进展.生命科学, 2006, 18(3): 266-272.
[76] Krisantini, S; Johnston, M; Williams, R R; Beveridge, C. Adventitious root formation in Grevillea (Proteaceae), an Australian native species. Scientia Horticulturae, 2006, 107(2): 171-175.
[77] Fatma Koyuncu; Fikri Balta. Adventitious root formation in leaf-bud cuttings of tea (camellia sinensis L.). Pakistan Journal of Botany, 2004, 36(4): 763-768.
[78] LU Shang-Fa, ZHAO Han-Yan, WEI Jian-Hua, et al. Establishment of in Vitro Regeneration System of Triploid Chinese White Poplar. Acta Botanica Sinica, 2001, 43(4): 435-437.
[79] Guo H S, Xie Q, Fei J F, et al. MicroRNA Directs mRNA Cleavage of the Transcription Factor NaCl to Downregulate Auxin Signals for Arabidopsis Lateral Root Development. Plant Cell, 2005, 17(5): 1376-1386.
[80] Sedira, M, Butler, E, Gallagher, T et al. Verification of auxin-induced gene expression during adventitious rooting in rolB-transformed and untransformed apple Jork 9. Plant Science, 2005, 168(5): 1193-1198.
[81] Brinker M; van Zyl L; Liu WB. Microarray analyses of gene expression during adventitious root development in Pinus contorta. Plant physiology, 2004, 135(3): 1526-1539.
[82]陈正华.木本植物组织培养及其应用.北京:高等教育出版社, 1986: 141-196.
[83] Orna AK, Cheng JC, Chen LJ.. Indole acetic acid distribution coincides with vascular differentiation pattern during Arabidopsis leaf ontogeny. Plant Physiology, 2002, 130: 199-209.
[84]杨世杰.植物生物学.北京:科学出版社, 2002: 104-234.
[85]裴东,袁丽钗,谷瑞升等.核桃子叶不定根发生调控的研究.林业科学, 2003, 39(6): 33-39.
[86]李小方,何玉科,汤章城.生长素和模拟微重力效应对大白菜不定根形态发生的影响.实验生物学报, 2000, 33(2): 179-185.
[87]陈新建陈梅英赵会杰. 1998.免疫学技术在植物科学中的应用.北京:中国农业大学出版社
[88]杨军,赵洁,杨弘远,植物激素的免疫细胞化学定位. 2002,细胞生物学杂志,24(5): 257-262
[89]张军,韩碧文.植物激素的免疫化学定位.植物生理学通讯, 1993, 29(1): 65-68
[90] Aloni R, Schwalm K, Langhans M, Ullrich CI. Gradual shifts in sites of free auxin-productionduring , leaf-primordium development and their role in vascular differentiation and leafmorphogenesis in Arabidopsis. Planta, 2003, 216: 841-853
[91] Geldner N, Friml J, Stierhof Y D, Ju rgens q Palme K. Auxin transport inhibitors block PIN1 cycling and vesicle trafficking. Nature,2001, 413: 425-428.
[92] Aloni R. Foliar and axial aspects of vascular differentiation: hypotheses and evidence. J Plant Growth Regul, 2001, 20:22-34
[93] Jones AM, Im KH, Savka MA expressed auxin binding protein et al. Auxin-dependent cell expansion mediated by over 1. Sci, 1998, 282: 1114-1116.
[94] Jutta Ludwig-Muller, Amy Vertocnik, Christopher D. Town. Analysis of indole-3-butyricacid-induced adventitious root formation on Arabidopsis stem segments. Journal of Experimental Botany, 2005, 56(418): 2095–2105.
[95] Thomas C., Bronner, R., Molinier J., Prinsen E., Van Onckelen, H. and Hahne G. Immuno-cytochemical localization of indole-3-acetic acid during induction of somatic embryogenesis incultured sunflower emb. Planta, 2002, 215: 577-583.
[96] Shi, L, Miller, L and Moore, R. Immunocytochemical localization of indole-3-acetic acid in primary roots of Zea mays. Plant Cell Environment, 1993, 16: 967-973.
[97]刘新,贾文锁,王幼群,娄成后,张蜀秋.伤胁迫对蚕豆叶片中茉莉酸分布的影响.分子细胞生物报, 2007, 40(3): 233-238