杨树试管苗嫩茎生根过程中内源IAA的免疫金银定位

生长素类物质在木本植物生根过程中发挥重要作用。杨树生根与生长素的关系及生根过程中内源激素的变化已有大量报道, 而生根过程中生长素的组织定位分析则尚未见报道。该文应用免疫化学分析方法对741杨(Populus alba × (P. davidiana × P. simonii) × P. tomentosa)嫩茎生根过程中内源IAA在组织中的分布进行了研究。结果显示, 741杨的嫩茎在无外源激素的1/2MS培养基上诱导10天后可生根, 14天后生根率达100%。诱导前, 嫩茎基部组织中几乎没有IAA信号; 诱导8天后, 嫩茎基部维管组织中有大量的IAA积累, 而且中部的维管组织中也有明显的IAA信号(主要分布在韧皮部和维管形成层); 10天后, 形成不定根原基, 此时IAA主要分布在根原基; 12天后, 根原基分化成不定根并突破表皮, IAA在不定根中的分布主要集中在根尖和中柱。该文对741杨的嫩茎生根过程中IAA的组织分布特点及运输途径进行了讨论。     

金塔柏扦插不定根形成与内源激素的调控研究

金塔柏（Platycladus orientalis ‘Beverleyensis’）是重要的观赏树种。生长素（IAA）、玉米素（ZT）、脱落酸（ABA）和茉莉酸（JA）在金塔柏扦插不定根再生过程中起着重要的调控作用,但不同发育阶段内源激素的动态变化及其对不定根发生的影响仍不清楚。以金塔柏半木质化枝条为材料,采用连续组织切片技术观察了不定根发生过程,利用高效液相色谱串联质谱法检测了4种内源激素含量的动态变化。结果表明,金塔柏不定根原基起源于愈伤组织、髓射线、木质部、维管形成层、次生韧皮部、皮层、髓射线与形成层交界处等部位,属于多位点发生模式和多类型生根方式。在不定根形成过程中,随着愈伤组织的形成,IAA和ZT含量下降,ABA和JA含量升高;随着根原基的分化,IAA和ZT含量缓慢升高,ABA和JA含量下降;随着不定根形成与伸长,IAA、ZT、JA逐渐升高,ABA维持在低水平。激素平衡分析发现,IAA/ABA比值和IAA/JA比值下降、IAA/ZT比值上升利于愈伤组织的形成,反之利于根原基的诱导分化,而IAA/ABA比值升高,IAA/ZT和IAA/JA维持在较低水平利于不定根形成与伸长。研究结果为揭示不同内源激素对金塔柏扦插不定根再生的调节作用提供了依据。     

欧美杂种山杨微扦插不定根发生过程的解剖学研究

采用石蜡切片技术,以欧美杂种山杨插穗基部茎段为实验材料,连续解剖观察插穗不定根发生发育过程,分析根原基发生部位与扦插生根的关系。结果显示:欧美杂种山杨插穗不定根的发生过程分为4个时期,为根原基诱导期,不定根起始期、表达期和伸长生长期。根原基诱导期维管形成层产生具有分生组织特点的薄壁细胞;不定根起始期,维管形成层及附近的薄壁细胞脱分化,形成不定根原基发端细胞;不定根表达期,根原基发端细胞不断分裂成具有方向性的根原基,根原基穿过韧皮射线和皮层,向皮孔方向发展;不定根伸长生长期,根原基从皮孔伸出,其内部的维管系统开始发育,形成不定根。研究认为,欧美杂种山杨为皮部诱导生根类型,不定根原基起源于维管形成层区,起源部位单一,扦插难生根。     

微型月季插穗筛选及其生根生理和解剖结构研究

以微型月季Rosa chinensis品种‘淑女’和‘维纳斯’作为材料,探究未木质化插穗、半木质化插穗和木质化插穗的扦插生根效果;同时测定‘淑女’半木质化插穗内源激素含量的动态变化,观察其不定根原基起源的解剖结构。结果表明,插穗木质化程度影响扦插生根效果,两个月季品种的插穗扦插效果依次是：半木质化插穗>未木质化插穗>木质化插穗。‘淑女’半木质化插穗在第7 d产生不定根,其生根能力优于‘维纳斯’;‘淑女’半木质化插穗的IAA含量随时间呈递减趋势,ABA、GA和ZR含量则均呈先降低后升高趋势,插穗内源激素含量变化共同影响不定根的形成。通过解剖学观察,微型月季插穗中未发现潜伏根原基,属于诱发根原基类型,其不定根原基起源于韧皮部和维管形成层区域的薄壁细胞团。     

杂种鹅掌楸插穗不定根发生与发育的解剖学观察

从解剖学角度着手,对杂种鹅掌楸〔Liriodendronchinense(Hemsl.)Sarg.×L.tulipiferaL.〕扦插过程中不定根的发生发育进行了研究。结果表明:杂种鹅掌楸插穗内未发现潜伏根原基。扦插后,不定根原基起源于维管形成层区,属于诱导生根类型。维管形成层恢复活动后,在不定根发生的部位附近形成1个明显的多薄壁细胞区域,在此区域不定根较容易发生。愈伤组织内没有发现根原基,愈伤组织在发育的过程中,内部细胞部分分化,并形成不规则的输导组织。大量的愈伤组织对不定根的发生有较强的抑制作用。杂种鹅掌楸插穗上不定根的发生可分为4个阶段:(1)维管形成层恢复活动,分裂出多层薄壁细胞;(2)维管形成层及附近的薄壁细胞脱分化,形成不定根原基发端细胞;(3)根原基发端细胞不断分裂成具有方向性的根原基,根原基穿过韧皮射线和皮层,向皮孔或下切口方向发展;(4)不定根从皮孔或下切口伸出,其内部的维管系统开始发育。     

核桃试管不定根的组织学研究

以核桃品种‘新早丰’试管嫩茎为试材,采用二步生根法诱导生根,对其试管苗不定根发生发育过程进行了解剖学研究。结果表明:核桃试管嫩茎内未发现潜伏根原基;诱导生根后,不定根原基起源于形成层,特别是髓射线正对的形成层部分,属于诱生根原基型;不定根上的侧根起源于中柱鞘细胞。核桃试管嫩茎不定根的发育过程可分为4个阶段:(1)形成层细胞分裂;(2)转变为分生组织细胞群(即根原始细胞);(3)细胞群发育成可见的根原基; (4)根原基内细胞继续分裂分化形成根尖的外形,其内发育出维管束,并向外生长,穿过皮层,突破茎表皮。在组织培养条件下长出的不定根内部解剖构造为典型的初生构造,移栽后68 d出现次生构造。另外,试管苗根毛出现与否及其发育状况受基质理化性质的影响,即生态条件可以改变组织发生及其形状。     

植物激素与不定根的形成

高水平的生长素可诱导不定根原基发生,高水平的脱落酸似乎有同样的作用,但效应不如生长素强;赤霉素似乎可增强生长素对不定根原基的诱导作用,却抑制脱落酸的诱导作用;细胞分裂素抑制不定根的发生;且上述激素处理都具有时效性;而乙烯似乎与不定根的发生无直接关系;ＳＡ和ＪＡ在不定根形成中可能只影响内源生长素和细胞分裂素的合成和代谢。     

不定根发生机理的研究进展

综述了近年来试管苗不定根发生机理的研究进展,在不定根发生过程中,专一基因的表达,内源激素水平的动态变化,酶活性的变化,多胺和钙水平以及碳水化合物水平的变化与不定根诱导,根原基形成和根发育生长有密切关系。自发性生根和诱导生根系统的植物或组织在不定根发生过程中存在较大的生理生化差异。通过了解不定根发生机理的研究动态,有助于更好地研究试管苗生根和试管苗商品化生产。     

红皮云杉茎的解剖结构与插条不定根形成的研究

１９９２年７－８月定时固定红皮云杉插条基部材料于ＦＡＡ液中,石蜡制片法室内解剖研究不定根的发生。结果表明：红皮云杉插条诱发根原基的来源有两种途径。一种是愈伤组织生根型,在愈伤组织的再生形成层处,或茎的维管形成层诱发根原基;另一种是非愈伤组织生根型,在插条切口处的维管形成层、皮层或初生木质部与次生木质部间的薄壁组织较深的部位,直接产生纵向不定根原始体,有的在距离切口０．１－０．５ｃｍ以上茎的维管形成层,维管形成层与木射线的交界处及叶隙等薄壁组织产生径向不定根。不同个体间产生的不定根数量及发育的早晚差异较大。     

胡杨不定根原基发生的分生细胞结构特征及内源激素变化分析

以胡杨实生苗去根插穗为试材,采用显微技术、气质(GC/MS)联用技术和同工酶分析技术观察和研究了外源3-吲哚丁酸(IBA)对其不定根原基(干细胞)形成的影响.结果表明:(1)胡杨插穗培养在无IBA的培养基上,36 h时有大核细胞发生,60 h时大核细胞聚集成团形成根原基,72 h时生成不定根;而添加了IBA的培养基上插穗培养60 h时大核细胞分散,出现许多薄壁细胞,72 h时薄壁细胞变大,没有根原基的形成和根的发生,表现出细胞组织愈伤化,不再分化出干细胞.添加外源IBA抑制了胡杨插穗不定根形成.(2)组织化学观察显示,在无IBA的培养基上的插穗,60 h时具双环状核仁的干细胞细胞质浓,富含蛋白质;而此时添加了IBA的插穗,具双环状核仁的细胞细胞质很少,蛋白质含量也少.(3)整个胡杨不定根形成的过程中,内源激素IAA和ABA可能作为诱导根原基发生的重要信号分子;内源激素IAA和GA3处于一个较稳定状态,有利于不定根的形成.(4)过氧化物酶(POD)2b酶带的持续表达有利于根原基的诱导;淀粉酶在60 h时表达增强,是根原基发育的标志;根原基诱导时不需要酯酶,而根原基发育时需要酯酶.     

木本植物内源生长素免疫胶体金定位分析技术的研究

以木本植物杨树(Populus sp.)和核桃(Juglans regia L.)为材料,对内源生长素免疫胶体金定位技术在固定、烤片、免疫染色、显色等关键环节进行了改进优化与验证.结果显示,优化后适合木本植物定位方法的主要技术要点是:在染色中,通过采用尿素-胰蛋白酶联合消化技术和增加牛血清白蛋白处理大大地改善了抗原修复结果,提高了染色的敏感性和特异性.利用优化后的方法对核桃幼胚和杨树嫩茎诱导生根过程中的吲哚乙酸(IAA)进行定位研究,发现杨树试管嫩茎生根过程中,形成层及周缘维管束有很强的IAA信号,核桃子叶生根中,胚中有很强的IAA信号,胚根中有半圆形、胚芽中有>"形强信号区,胚轴信号较弱,胚根信号最强.研究表明,与传统免疫染色方法相比,优化后的方法对木本植物生长素定位具有敏感性高,特异性强,银颗粒明显,背景清晰,耗时少等特点.     

Subcellular localization of endogenous IAA during poplar leaf rhizogenesis revealed by in situ immunocytochemistry

Poplar 741 [Populus alba × (P. davidiana + P. simonii) × P. tomentosa] leaves were rooted within 8 days when cultured on 1/2 MS medium. The subcellular localization of endogenous indole-3-acetic acid (IAA) in the rhizogenesis was investigated, using an immunocytochemical approach. The results of IAA subcellular localization revealed organelle-specific distribution. Three days after root induction, IAA in vascular cambium cells of the basal region of the petiole was distributed mainly in the plasma membrane, endoplasmic reticulum (ER), and nucleus, with a lesser amount in the cytoplasm. In phloem of the basal region of the petiole, IAA was detected in the plasma membrane and ER of the companion cell and in the plasma membrane of the sieve element. In xylem of the basal region of the petiole, no IAA gold particles were labeled. In mesophyll cells IAA was distributed in the chloroplast starch grains before root induction, and the amount in the chloroplast starch grains increased after 3 days after root induction. This suggests that the plasma membrane and nucleus of cambium cells may be the target sites where IAA performs its physiological activities during poplar leaf rhizogenesis. IAA polar transport from lamina mesophyll to the basal region of the petiole during rhizogenesis is mediated by phloem. The starch grains of mesophyll chloroplasts appeared to accumulate IAA and may be a source of IAA during poplar leaf rhizogenesis. Novel and direct evidence regarding the function of IAA during rhizogenesis is provided in this study.     

The role of the shoot apex in controlling rhizogenesis in vitro

This paper reports that rhizogenesis in woody plant species in vitro was mediated through the basipetal transport of auxin from the shoot apex. This can directly induce roots in easy-to-root species such as Betula pendula, but was dependent upon an interaction with exogenous auxin in more difficult-to-root species such as Daphne cneorum, and to a lesser extent in Quercus robur. Shoot apex removal reduced rhizogenesis in Quercus, and inhibited it in Daphne, even in the presence of exogenous auxin, whereas rooting in Betula was unaffected. That basipetally transported auxin modulates rhizogenesis was demonstrated by the inhibition of root induction in Betula shoots by the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA), and by the substitution of indole-3-acetic acid (IAA) for a bud in Betula internodal sections.Abbreviations IAA indole-3-acetic acid - IBA indole-3-butyric acid - TIBA 2,3,5-triiodobenzoic acid - MS Murashige and Skoog medium - WPM woody plant medium     

Increased endogenous indole-3-acetic acid:abscisic acid ratio is a reliable marker of Pinus massoniana rejuvenation

ABSTRACT

Pinus massoniana is a recalcitrant tree species for rooting in vitro. We rejuvenated 26-year-old P. massoniana trees by successive grafting. Rooting rates of rejuvenated shoots were > 83.1% after rooting induction. We compared endogenous levels of indole-3-acetic acid (IAA), abscisic acid (ABA), gibberellins (GAs) and zeatin-riboside (ZR), and the rhizogenesis ability of axillary shoots of mature and rejuvenated materials in vitro, i.e., somaplants and grafts. Enhancement of the rooting ability of mature materials in vitro following somatic embryogenesis or repeated grafting onto juvenile rootstocks was accompanied by increased IAA and GAs levels, and by decreased ABA levels in scions used as starting material for micropropagation in vitro. Successive subcultures did not influence the rooting ability of shoots from untreated mature material. Rooting ability of shoots in vitro, however, gradually increased with subculture frequency during repeated subculturing in grafting materials. The IAA:ABA ratio in shoots in vitro after grafting five times, and consequently capable of root organogenesis, was higher than in shoots of untreated mature material incapable of root organogenesis in vitro. A high IAA:ABA ratio was detected in scions of somaplants that were capable of rooting in vitro despite subculture times. We found that the endogenous IAA:ABA ratio is a reliable marker for the recovery of root organogenesis in vitro after rejuvenating treatments for mature P. massoniana trees.     

In vitro direct rhizogenesis from Gerbera jamesonii Bolus leaf

The present report describes an original protocol for in vitro direct induction of roots from leaf explants of gerbera for the first time. Since gerbera has immense potential as a premium cut-flower, the major attempts were made on in vitro mass propagation chiefly through in vitro multiple shoot proliferation or callus regeneration. Nevertheless, rhizogenesis could be impending an unattempted method with its yet-to-be known advantages. In our study, the optimum conditions for direct root induction from leaf explants were assessed employing tissue culture technique. Leaves were inoculated to MS medium containing no or variable auxin sources and concentrations namely, 2,4-dichlorophenoxyacetic acid, indole-3-acetic acid (IAA), indole-3-butyric acid or α-naphthaleneacetic acid for root induction. It was evident that the maximum root induction (with a frequency of 92.6 %) occurred on MS media fortified with 1.5 mg l^?1 IAA, wherein root induction was observed as early as 11 days of culture and an average of ~19 roots with ~13 mm length was obtained from 4 cm² leaf segment after 45 days of culture. Stereo microscopic observation revealed the induction of roots and gradual developmental stages of rhizogenesis. The efficiency of direct root induction without any interim growth stages (such as, callus or shoots) in our study offers a reproducible system that could provide a model protocol for more comprehensive developmental studies on root growth.     

Restart of Lignification in Micropropagated Walnut Shoots Coincides with Rooting Induction

The lignin content of walnut shoots did not change during in vitro shoot multiplication. Lignin content started to increase as soon as shoots were passed to a rooting medium with auxin. Exogenous auxin (applied for rooting) caused a transient elevation of the endogenous free indoleacetic acid (IAA) content with a simultaneous decrease of peroxidase activity. These events typically marked the completion of the rooting inductive phase (before any visible histological event, that is before the cell divisions beginning the rooting initiation phase). This meant that either the given exogenous auxin or the endogenous IAA has served as signal for the stimulation of lignification. Continued increase of lignification in the shoots required completion of root formation; this increase indeed was slown down when root emergence did not occur. It was further shown that lignification varied conversely to the content of the soluble phenol content, itself apparently being related to the activity of phenylalanine ammonia-lyase activity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Immuno-cytochemical localization of indole-3-acetic acid during induction of somatic embryogenesis in cultured sunflower embryos

Immature zygotic embryos of sunflower (Helianthus annuus L.) produce somatic embryos when cultured on medium supplemented with a cytokinin as the sole source of exogenous growth regulators. The timing of the induction phase and subsequent morphogenic events have been well characterized in previous work. We address here the question of the role of endogenous indole-3-acetic acid (IAA), since auxins are known to have a crucial role in the induction of somatic embryogenesis in many other culture and regeneration systems. The fact that in the sunflower system no exogenous auxin is required for the induction of somatic embryos makes this system very suitable for the study of the internal dynamics of IAA. We used an immuno-cytochemical approach to visualize IAA distribution within the explants before, during and after the induction phase. IAA accumulated transiently throughout cultured embryos during the induction phase. The detected signal was not uniform but certain tissues, such as the root cap and the root meristem, accumulated IAA in a more pronounced manner. IAA accumulation was not restricted to the reactive zone but the kinetics of endogenous variations strikingly mimic the pulse of IAA that is usually provoked by exogenous IAA application. The direct evidence presented here indicates that an endogenous auxin pulse is indeed among the first signals leading to the induction of somatic embryogenesis.     

Tissue-specific localization and dynamic changes of endogenous IAA during poplar leaf rhizogenesis revealed by in situ immunohistochemistry

Poplar 741 [Populus alba × (P. davidiana + P. simonii) × P. tomentosa] leaves were rooted within 8 days when cultured on 1/2 MS medium. The spatial distribution of endogenous indole-3-acetic acid (IAA) and its dynamic changes in the rhizogenesis were investigated, using an immunohistochemical approach. Anatomical analyses showed that the root primordia arose from vascular cambium cells in the basal regions of the petioles of the leaves. Before root induction, immunostaining patterns showed a basipetally decreasing gradient of IAA along the leaves. Three days after induction, the IAA immunostaining pattern observed along the leaves was high at both ends and low in the middle. And IAA in the basal regions of the petiole was distributed mainly in the vascular bundles. Localized application of 2,3,5-triiodobenzoic acid (TIBA) on laminas of the leaves delayed the accumulation of IAA in the vascular bundles of the basal regions of the petioles, but not in the mesophyll of the laminas. These data indicate that an accumulation of IAA in the vascular bundles of the basal regions of the petioles induces the occurrence of rhizogenesis of poplar leaves. And IAA accumulated in the vascular bundle of the basal region of the petiole results from its polar transportation from mesophyll of the laminas, rather than by in situ IAA generation.     

Ornithine and arginine decarboxylases and polyamine involvement during in vivo differentiation and in vitro dedifferentiation of Datura innoxia leaf explants

The effects of exogenous ornithine, arginine and polyamines added to media leading to root, callus or bud initiation of Datura innoxia Mill. leaf explants growing in vitro were examined. Ornithine and arginine decarboxylase activities (ODC, EC 4.1.1.17; ADC, EC 4.1.1.19) as well as endogenous polyamine levels were also determined during the course of in vivo differentiation of the leaves and their subsequent in vitro dedifferentiation under rooting, callusing, or budding conditions. Decarboxylase activities were determined by measuring the ¹⁴CO₂ released and the polyamines were quantified after dansylation by thin-layer chromatography. In vivo, ODC and ADC activities decreased from shoots to young to old leaves. In vitro, synergistic effects between ornithine and indole-3-acetic acid on rhizogenesis were detected, while arginine was not effective. Exogenous putrescine also acted synergistically with auxin by promoting root growth. A close relationship was found between rhizogenesis, ODC activity and increase in endogenous putrescine and spermidine levels. ODC increased during the induction and time course of cell dedifferentiation and seemed to support these events, while ADC seemed to support only the later events involving redifferentiation.