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。     

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。     

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。     

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。     

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。     

黑果腺肋花楸扦插生根特性及不定根发生模式研究

【目的】为了解黑果腺肋花楸扦插不定根起源及发育过程,揭示其扦插生根机理。【方法】以‘福康源1号’当年生半木质化插穗为研究材料,利用水培扦插技术和石蜡切片法对不定根形成过程中插穗内部组织结构及外部形态的变化规律进行观察。【结果】在水培条件下,IBA处理扦插生根期为30~40 d,扦插过程中皮孔处10~15 d出现不定根,插穗切口处15~20 d出现不定根,生根速度、不定根数量及根长均优于对照,外源诱导可显著提高生根率和生根质量。扦插前的插穗内无潜伏根原基存在,不定根原基在插后形成;不定根形成为愈伤组织生根型和内部分生组织生根型。皮部产生的不定根起源于维管形成层、韧皮薄壁细胞或皮层;愈伤组织产生的不定根是由愈伤组织内的薄壁细胞团特化而成;叶隙或枝隙是形成不定根原基和产生愈伤组织的主要区域。【结论】扦插生根属于多位点发生模式,属于诱导生根型。     

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。     

侧柏扦插不定根发生模式研究

该研究以侧柏一年生硬枝插穗为实验材料,利用连续组织切片技术观察插穗不定根发生发育过程中的组织结构变化,分析插穗外部形态变化、不定根原基起源和不定根的形成过程,探讨侧柏插穗不定根发生模式和不定根的组织学起源。结果显示:侧柏扦插后可由愈伤组织、皮部诱导产生不定根,出现皮部生根、愈伤组织生根、愈伤组织兼具皮部生根3种类型;侧柏插穗中存在少量潜伏根原基,但插穗生根类型以诱导生根为主;不定根原基诱导产生于愈伤组织、木质部、形成层及次生韧皮部等部位。研究认为侧柏扦插生根属于多位点发生模式,不定根原基的组织学起源是愈伤组织、髓射线、射线原始细胞、尚未分化成熟的木质部细胞,通过人工诱导同时激活这些不定根起源位点能够显著提高生根率和生根质量。     

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.     

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.     

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.     

Indole-3-acetic acid accumulation during poplar rhizogenesis revealed by immunohistochemistry

Poplar hybrid 741 [Populus alba × (P. davidiana + P. simonii) × P. tomentosa] leaves were rooted within 8 d when cultured in vitro on 1/2 Murashige and Skoog (MS) medium. The spatial distribution of endogenous indole-3-acetic acid (IAA) in the rhizogenesis was investigated, using an immunohistochemical approach. In addition, the effect of 2,3,5-triiodobenzoic acid (TIBA) on IAA distribution was also analyzed. The results showed that a strong IAA signal was detected in the vascular bundles of the basal regions of the petioles 3 d after root induction. Furthermore, the signal in vascular bundles of the basal regions of the petioles was stronger than that of the middle regions of the petioles. Application of TIBA on lamina delayed both the accumulation of IAA in the vascular bundles and rhizogenesis. These data indicate that an endogenous IAA rise in vascular bundles is among the first signals leading to the rhizogenesis, and that it results from transportation of the hormone from the lamina of the leaf to the base of the petiole, rather than by in situ IAA generation.     

Adventitious rooting in hypocotyls of sunflower (Helianthus annuus) seedlings. IV. The role of changes in endogenous free and conjugated indole‐3‐acetic acid

We have studied the role of endogenous auxin on adventitious rooting in hypocotyls of derooted sunflower (Helianthus annuus L. var. Dahlgren 131) seedlings. Endogenous free and conjugated indole-3-acetic acid (IAA) were measured in three segments of hypocotyls of equal length (apical, middle, basal) by using gas chromatography-mass spectrometry with [¹³C₆]-IAA as an internal standard. At the time original roots were excised (0 h), the free IAA level in the hypocotyls showed an acropetally decreasing gradient, but conjugated IAA level increased acropetally; i.e. free to total IAA ratio was highest in the basal portion of hypocotyls. The basal portion is the region where most of root primordia were found. Some primordia were seen in this region within 24 h after the roots were excised. The quantity of free IAA in the middle portion of the hypocotyl increased up to 15 h after excision and then decreased. In this middle region there were fewer root primordia, and they could not be seen until 72 h. In the apical portion the amount of free IAA steadily increased and no root primordia were seen by 72 h. Surgical removal of various parts of the hypocotyl tissues caused adventitious root formation in the hypocotyl regions where basipetally transported IAA could accumulate. Reduction in the basipetal flow of auxin by N-1-naphthylphthalamic acid and 2,3,5-tri-iodobenzoic acid resulted in fewer adventitious roots. The fewest root primordia were seen if the major sources of endogenous auxin were removed by decapitation of the cotyledons and apical bud. Exogenous auxins promoted rooting and were able to completely overcome the inhibitory effect of 2,3,5-tri-iodobenzoic acid. Exogenous auxins were only partially able to overcome the inhibitory effect of decapitation. We conclude that in sunflower hypocotyls endogenously produced auxin is necessary for adventitious root formation. The higher concentrations of auxin in the basal portion may be partially responsible for that portion of the hypocotyl producing the greatest number of primordia. In addition to auxins, other factors such as wound ethylene and lowered cytokinin levels caused by excision of the original root system cuttings must also be important.     

Environmental and auxin regulation of wood formation involves members of the Aux/IAA gene family in hybrid aspen

Indole acetic acid (IAA/auxin) profoundly affects wood formation but the molecular mechanism of auxin action in this process remains poorly understood. We have cloned cDNAs for eight members of the Aux/IAA gene family from hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) that encode potential mediators of the auxin signal transduction pathway. These genes designated as PttIAA1-PttIAA8 are auxin inducible but differ in their requirement of de novo protein synthesis for auxin induction. The auxin induction of the PttIAA genes is also developmentally controlled as evidenced by the loss of their auxin inducibility during leaf maturation. The PttIAA genes are differentially expressed in the cell types of a developmental gradient comprising the wood-forming tissues. Interestingly, the expression of the PttIAA genes is downregulated during transition of the active cambium into dormancy, a process in which meristematic cells of the cambium lose their sensitivity to auxin. Auxin-regulated developmental reprogramming of wood formation during the induction of tension wood is accompanied by changes in the expression of PttIAA genes. The distinct tissue-specific expression patterns of the auxin inducible PttIAA genes in the cambial region together with the change in expression during dormancy transition and tension wood formation suggest a role for these genes in mediating cambial responses to auxin and xylem development.     

Early steps of adventitious rooting: morphology,hormonal profiling and carbohydrate turnover in carnation stem cuttings

The rooting of stem cuttings is a common vegetative propagation practice in many ornamental species. A detailed analysis of the morphological changes occurring in the basal region of cultivated carnation cuttings during the early stages of adventitious rooting was carried out and the physiological modifications induced by exogenous auxin application were studied. To this end, the endogenous concentrations of five major classes of plant hormones [auxin, cytokinin (CK), abscisic acid, salicylic acid (SA) and jasmonic acid] and the ethylene precursor 1‐aminocyclopropane‐1‐carboxylic acid were analyzed at the base of stem cuttings and at different stages of adventitious root formation. We found that the stimulus triggering the initiation of adventitious root formation occurred during the first hours after their excision from the donor plant, due to the breakdown of the vascular continuum that induces auxin accumulation near the wounding. Although this stimulus was independent of exogenously applied auxin, it was observed that the auxin treatment accelerated cell division in the cambium and increased the sucrolytic activities at the base of the stem, both of which contributed to the establishment of the new root primordia at the stem base. Further, several genes involved in auxin transport were upregulated in the stem base either with or without auxin application, while endogenous CK and SA concentrations were specially affected by exogenous auxin application. Taken together our results indicate significant crosstalk between auxin levels, stress hormone homeostasis and sugar availability in the base of the stem cuttings in carnation during the initial steps of adventitious rooting.     

Changes in auxin protectors and IAA oxidases during the rooting of chestnut shoots in vitro

Auxin protectors and IAA oxidase activity were comparatively analyzed in the upper and the lower parts of shoots of chestnut ( Castanea sativa Mill.) cultivated in vitro with indolebutyric acid (IBA) pretreatment. Rhizogenesis of the shoots is accompanied by an increase in auxin protectors in the lower parts and by a decrease of these protectors in the upper parts. Besides, the IAA oxidase activity declines in the basal parts during the rooting process while it increases in the upper ones. These biochemical events would enhance the IAA level in the rooting region of the shoots. In untreated, non-rooted cuttings, the IAA oxidase activity remains low in the upper parts and high in the basal parts of the shoots. The results thus indicate that the IBA treatment may control the endogenous auxin level of the cuttings, either through a direct regulation of the IAA oxidase system or more indirectly through the transport of auxin protectors.     

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