Overexpression of phytochrome A enhances the light-induced formation of adventitious shoots on horseradish hairy roots

A full-length cDNA of the gene for phytochrome A from Arabidopsis thaliana was fused with the 35S promoter of cauliflower mosaic virus (CaMV35S-PHYA) and introduced into horseradish (Armoracia rusticana Gaert., Mey. et Scherb.) hairy roots. The phytochrome level in hairy roots transformed with CaMV35SPHYA was about three times greater than that in normal hairy roots and the rate of light-induced formation of adventitious shoots was also higher in the hairy roots transformed with CaMV35SPHYA. These results indicate that the light-induced formation of adventitious shoots on horseradish hairy roots is closely related to the phytochrome level. Received: 11 August 1998 / Revision received: 21 October 1998 / Accepted: 20 November 1998     

Shoot RNA, cambrial activity and indolebutyric acid effectivity in seasonal rooting of juvenile and mature Ficus pumila cuttings

The seasonal influence on adventitious root formation was studied in woody leaf bud cuttings of Ficus pumila L., creeping fig. Juvenile cuttings rooted easily, whereas only mature cuttings treated with indolebutyric acid (IBA) exceeded 30% rooting. Greater rooting occurred in IBA-treated juvenile and mature cuttings than controls, regardless of the month each experiment was initiated. Seasonal changes influenced rooting in all treatments except IBA treated juvenile cuttings where percentage rooting was not affected. Higher vascular cambial activity and shoot RNA levels occurred in juvenile and mature forms during peak rooting periods. Highest RNA was recorded with juvenile materials during maximum rooting periods, while lowest RNA was observed in mature shoots during low rooting intervals.     

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.     

Thidiazuron influences the endogenous levels of cytokinins and IAA during the flowering of isolated shoots of Dendrobium

This study reports the effects of thidiazuron (TDZ) on the endogenous levels of indoleacetic acid (IAA), zeatin, zeatin riboside ([9R]Z), isopentenyladenine and isopentenyladenosine ([9R]iP) as well as structural changes in the apical meristem of Dendrobium Second Love shoots during flower induction and initial development in vitro. The results revealed that the presence of 1.8microM TDZ had a profound effect on the endogenous cytokinins (CKs) and IAA levels of the explants, when compared to those grown on a TDZ-free medium. A significant increase in CKs (especially [9R]iP and [9R]Z) and IAA in the first samples (taken at day 5) grown on TDZ-enriched medium was associated with flower induction, while a second increase in the level of these hormones after 25d of culture was related to flower development. The histological changes detected in the shoot apical meristem of explants grown in the presence of 1.8microM TDZ during 30d of culture are also described. Based on these findings, it is suggested that both auxin and CKs seem to be involved with the floral transition of Dendrobium Second Love in vitro. However, a possible direct effect of TDZ on flower formation is not discarded.     

Effects of temperature and gibberellin on the activity of endogenous cytokinin-like substances in leaves of Begonia x cheimantha

Changes in activity of endogenous cytokinin-like substances were examined in intact plants and excised leaves of Begonia x chemantha Everett cv. Prinsesse Astrid (Christimas Begonia) by means of the tobacco callus bioassay. Cytokinin activity in the leaves of intact plants was higher in plants grown at 18°C than in those grown at 21° or 24°C. In excised leaves, an increase in cytokinin activity was observed during the first 4 days following leaf detachment. However, after the seventh day cylokinin activity decreased again. This decrease was more profound in leaves exposed to 24°C than in those exposed to 18°C.
Treatment of detached leaves with gibberellic acid (2.8 m M ) caused an increase in measurable cytokinin activity. This increase was more profound in the zones of activity which correspond with zeatin glucosides on paper and Sephadex LH-20 chromatography. Additional zones of activity appeared after Sephadex chromatography. These were of a more slow moving nature with elution volumes corresponding to N^b-(Δ²-isopentenyl)adenine and its derivaties. Water-treated control leaves had higher activity in the regions corresponding to zeatin and zeatin riboside.     

外源IBA对无花果扦插苗生理特性及内源IAA生物合成途径的影响

【目的】随着区域栽培环境的变化,扦插苗生根难问题逐渐凸显。探讨适宜浓度吲哚丁酸(IBA）对无花果插穗生根萌芽、抗氧化性及生长素生物合成途径相关基因表达的影响,可为其应用于无花果的育种、繁殖、推广和种植提供理论依据。【方法】以‘波姬红’无花果品种硬枝为插穗,分析不同质量浓度IBA(0,30,45,60,90 mg/L)处理对插穗生根性状、抗氧化特性的影响,并对45 mg/L IBA处理及对照组的扦插枝条中段的腋芽进行转录组分析。【结果】（1）无花果插穗萌芽率和生根率在45 mg/L IBA处理时达到最大值,并与其他处理和对照差异显著。（2）随着IBA浓度的增加,插穗SOD和CAT活性先下降后上升,并均在45,60 mg/LIBA处理下显著低于对照,而POD活性无显著变化;各浓度IBA处理插穗中MDA和H2O2含量均显著高于对照,且45 mg/L IBA处理MDA显著低于其余处理。（3）45 mg/L IBA处理及对照组中共存在6 879个差异表达基因,KEGG富集显示有10个差异途径,GO富集分析表明生物学过程和分子功能为主要的生物学途径;与CAT、SOD相关的基因集中富集在过氧化物酶体通路上,POD相关基因则富集在苯丙烷生物合成通路中;IAA生物合成途径中代谢相关基因FcGH3显著上调表达,与信号转导相关基因FcAUX1、FcARG7和FcARF等显著下调表达。【结论】外源IBA处理会导致无花果插穗抗氧化酶和IAA生物合成途径中相关基因表达的差异变化,增强插穗抗逆性,促进插穗生根、萌芽、成苗,并以外源45 mg/L IBA促进效果最好。     

Levels of endogenous indole-3-acetic acid and indole-3-acetylaspartic acid during adventitious root formation in pea cuttings

Levels of endogenous indole-3-acetic acid (IAA) and indole-3-acetylaspartic acid (IAAsp) were monitored in various parts of leafy cuttings of pea ( Pisum sativum L. cv. Marma) during the course of adventitious root formation. IAA and IAAsp were identified by combined gas chromatography—mass spectrometry, and the quantitations were performed by means of high performance liquid chromatography with spectrofluorometric detection. IAA levels in the root forming tissue of the stem base, the upper part of the stem base (where no roots were formed), and the shoot apex remained constant during the period studied and were similar to levels occurring in the intact seedling. A reduction of the IAA level in the root regenerating zone, achieved by removing the shoot apex, resulted in almost complete inhibition of root formation. The IAAsp level in the shoot apex also remained constant, whereas in the stem base it increased 6-fold during the first 3 days. These results show that root initiation may occur without increased IAA levels in the root regenerating zone. It is concluded that the steady-state concentration is maintained by basipetal IAA transport from the shoot apex and by conjugation of excessive IAA with aspartic acid, thereby preventing accumulation of IAA in the tissue.     

Effects of auxin and irradiance on the rooting of cuttings of Pinus sylvestris

Abstract Seedlings of Pinus sylvestris L. were grown under controlled conditions (temperature 20°C, photoperiod 17 h) at two irradiances, 8 or 40 W m^-2. Hypocotyl cuttings were excised and rooted at different irradiances in tap water solutions of indolebutyric acid (IBA). The fastest rooting and highest rooting percentage were obtained with cuttings from stock plants grown at 8 W m^-2 and treated with 10^-5M IBA for 21 days. The concentration of 10^-4M IBA inhibited root formation. In comparable treatments rooting was always better in cuttings from stock plants grown at 8 W m^-2 than in cuttings from stock plants grown at 40 W m^-2. The irradiance during the rooting period had only a minor influence on rooting. When cuttings from plants irradiated with 40 W m^-2 were treated with 10^-5M IBA for 21 days the rooting percentage almost reached the same level as in untreated cuttings from stock plants given 8 W m^-2. In cuttings treated with IBA during the whole rooting period, rooting was depressed in comparison to untreated cuttings. Aeration of the 10^-4M IBA solution increased the rooting percentage, but aeration had no effect on untreated cuttings and on cuttings treated with lower IBA concentrations.     

Effect of abscisic acid on endogenous IAA, auxin protector levels and peroxidase activity during adventitious root initiation in Vigna radiata cuttings

Endogenous levels of free and conjugated IAA, auxin protectors (Prs) and peroxidase (PER) activity and their relation to adventitious root initiation (ARI) were investigated at the potential sites of adventitious rooting in relation to exogenous application of 250 μM ABA during the first 120 h after treatment. Cuttings from 7-day-old mung bean [Vigna radiata (L.) Wilcz.] seedlings were treated with 125, 250, and 500 μM ABA for 24 h. ABA significantly stimulated ARI but extremely inhibited epicotyl growth as compared to control. Free and conjugated IAA were measured by reversed-phase high performance liquid chromatography while Prs and PER activities were measured spectrophotometrically. The present results also indicate that endogenous free IAA levels peaked later in ABA-treated cuttings than that in control, suggesting that ABA extended the length of the induction phase of rooting process in treated cuttings and that might explain the significant delay of the appearance of roots at the treated cuttings. Higher level of IAA conjugates was found in ABA-treated cuttings than that in untreated ones. Pr level also peaked later in ABA-treated cuttings than that in control, indicating that ABA extended the period of Pr activity. An initial temporary decrease of PER activity was found in associating with high levels of free IAA and Prs during most of the primary events, while the opposite occurred during the secondary events of adventitious rooting process in both treated and untreated cuttings. Thus, ABA may stimulate ARI in mung bean Vigna radiata cuttings by regulating the concentration and /or activities of endogenous IAA, Prs, and PER activity in favor of inducing a large number of adventitious roots at their potential sites of adventitious rooting.     

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。     

Variation among and within clones in formation of roots and shoots during micropropagation of Campanula isophylla

It is known that treatments enhancing shoot formation often suppress root formation and vice versa. It would be of interest to know if such negative correlations between formation of roots and shoots were also present among genetically different plants, given the same treatment, to ensure that selection for superior shoot formation would not lead to inadvertent decreases in the capacity for root formation. Height and dry weight of micropropagated shoot clusters and the numbers of shoots and roots were measured in 95 seedling clones. Within clones, shoot size was negatively correlated with number of shoots and positively correlated with number of roots. Among clones, however, the number of shoots was not correlated with the size of shoots, but positively correlated with the number of roots. While it is difficult to devise treatments that simultaneously optimize the initiation of roots and shoots, it is thus possible to select for fast-growing clones without compromising root formation.Abbreviations CM clonal means - DCM deviation from clonal means     

Synergistic interaction between coumarin 1,2-benzopyrone and indole-3-butyric acid in stimulating adventitious root formation in Vigna radiata (L.) Wilczek cuttings: I. Endogenous free and conjugated IAA and basic isoperoxidases

Cuttings from 7-day-old Vigna radiata seedlings were treated for 24 h with various concentrations of coumarin and/or indole-3-butyric acid (IBA), applied either alone or in combination, in order to stimulate adventitious root formation (ARF). The effects of treatment on endogenous free and conjugated indole-3-acetic acid (IAA), basic peroxidase (basic PER) activity and its isoperoxidases analysis and their relation to ARF were then investigated at the potential rooting sites during the first 96 h after application. Simultaneously, combined treatments acted synergistically in inducing more adventitious roots in treated cuttings than in those treated with coumarin or IBA individually, as compared with the control. Endogenous free IAA increased transiently in treated cuttings as compared with the control and the maximum increase occurred with the combined treatment. This suggests that coumarin and IBA may act synergistically in increasing the endogenous free IAA level during the induction phase of rooting to initiate more roots. Likewise, higher level of conjugated IAA was also found in treated cuttings than in untreated ones, during the primary events of ARF, with the maximum level occurring in the combined treatment. Comparison of the dynamics of conjugated IAA and activity of basic PERs led to conclusion that the former but not the latter is responsible for downregulation of endogenous IAA levels significantly during the primary events of ARF. A sharp increases in basic PERs occurred during the secondary events of ARF, suggesting their role in root initiation and development rather than root induction.     

NAA、IBA和PP333对怀山药试管苗生长发育的影响

就不同生长调节剂对怀山药试管苗生长发育的影响进行了系统的研究。结果表明 :( 1 )NAA、IBA和PP3 3 3 均能诱导根的生成 ,但根形成的时间、发生方式及发达程度均不相同。低浓度的NAA( 0 .1～ 0 .5mg.L 1)有利于根的生成 ,但高浓度的NAA(≥ 2mg .L 1)则易形成愈伤组织 ,且随着浓度的升高 ,愈伤组织化程度变大 ,根多来源于愈伤组织 ;IBA( 0 .1～ 2mg .L 1)对根的生成较为有利 ,其中以IBA 1mg .L 1的生根效果较好 ;PP3 3 3 ( 0 1～ 8mg.L 1)有利于根的生成 ,根形成的最早、最多 ,且随着浓度的升高 ,根更加粗壮发达。 ( 2 )PP3 3 3 抑制试管苗的纵向伸长生长 ,使株高降低 ,但却显著地促进了根系的发育 ,使试管苗生长健壮 ,叶色浓绿 ,叶片增多。这种效应随着PP3 3 3 浓度的升高而加强。从培养壮苗的角度来看 ,PP3 3 3 ( 2～ 4mg.L 1)是最佳浓度     

Action spectrum for light-induced formation of adventitious shoots in hairy roots of horseradish

An action spectrum was determined for lightinduced formation of adventitious shoots in hairy roots of horseradish (Armoracia rusticana Gaert., Mey. et Scherb.) cultured in vitro. Near ultraviolet (350–400 nm), blue (440–460 nm) and red light (600–680 nm) were most effective in inducing formation of adventitious buds. Farred light (730 nm) inhibited the promotive effect of all three wavelength regions. These results are consistent with induction by phytochrome(s) of adventitious shoots in hairy roots of horseradish.     

Caffeic acid affects early growth, and morphogenetic response of hypocotyl cuttings of mung bean (Phaseolus aureus)

Caffeic acid (CA) is one of the most common cinnamic acids ubiquitously present in plants and implicated in a variety of interactions including allelopathy among plants and microbes. This study investigated the possible interference of CA with root growth and the process of rhizogenesis in hypocotyl cuttings of mung bean (Phaseolus aureus=Vigna radiata). Results indicated that CA (0-1000 microM) significantly suppressed root growth of mung bean, and impaired adventitious root formation and root length in the mung bean hypocotyl cuttings. Further investigations into the role of CA in hampering root formation indicated its interference with the biochemical processes involved in rooting process at the three stages - root initiation (third day; RI), root expression (fifth day; RE), and post-expression (seventh day; PE) - of rhizogenesis. CA caused significant changes in the activities of proteases, peroxidases (PODs), and polyphenol oxidases (PPOs) during root development and decreased the content of total endogenous phenolics (TP) in the hypocotyl cuttings. The enhanced activity of PODs and PPOs, though, relates to lignification and/or phenolic metabolism during rhizogenesis; yet their protective role to CA-induced stress, especially during the PE phase, is not ruled out. At 1000 microM CA, where rooting was significantly affected, TP content was very high during the RI phase, thus indicating its non-utilization. The study concludes that CA interferes with the rooting potential of mung bean hypocotyl cuttings by altering the activities of PODs and PPOs and the endogenous TP content that play a key role in rhizogenesis.     

黄瓜和绿豆下胚轴不定根发生的研究

在MS基本培养基上,黄瓜和绿豆幼苗的下胚轴切段培养4d时即可见不定根发生。下胚轴不同部位切段的发根能力不同。下胚轴切段反插时比正插时发根快1-2d,发根率也高于正插的;0.01-0.05mg/L的NAA还诱导下胚轴切段在形态学上端发根。TIBA对正插或反插的下胚轴切段的不定根发生都有抑制作用。结果提示,生长素极性运输活性对不定根形成起着重要作用。     

Ethylene accelerates the breakdown of cytokinins and thereby stimulates rooting in Norway spruce hypocotyl cuttings

Cuttings were taken from 4-week-old seedlings of Norway spruce ( Picea abies L. Karst.) raised at two different irradiation levels. Rooting experiments showed that root formation was increased by the ethylene formed by adding 1-aminocyclopropane-1-carboxylic acid ACC or Ethrel, especially in the slowly rooting cuttings grown under high light (HL). Cobaltousion. an ethylene synthesis inhibitor, delayed rooting, especially in the easily rooted cuttings grown under low light (LL).
Compounds isolated from the cuttings using immunoaffinity chromatography, on a column with antibodies against cytokinins, and separated by HPLC decreased in amount during the first week of the rooting period. An increase in ethylene production accelerated this process, especially in cuttings grown under HL, whereas cobaltous ion delayed it. We suggest that ethylene stimulates rooting by enhancing the degradation of cytokinins.     

Micropropagation of Romulea minutiflora, Sisyrinchium laxum and Tritonia gladiolaris — Iridaceae with ornamental potential

Three species of the Iridaceae with ornamental potential were micropropagated with the intention of producing propagules more rapidly for possible commercialization. Shoot induction from in vitro germinated seedlings of Romulea minutiflora was obtained with 5.4 μM α-naphthaleneacetic acid (NAA) and 23.2 μM kinetin. Shoot explants formed corms best with 3.4 or 17 μM paclobutrazol, and one incidence of in vitro flowering was observed. Sisyrinchium laxum shoot explants produced more and healthier multiple shoots with meta-topolin (mT) than with 6-benzyladenine (BA). Rooting was best in control (no hormone) cultures, and addition of NAA and indole-3-acetic acid (IAA) inhibited root formation and growth of shoot explants, and formed short, stunted roots. Roots produced by indole-3-butyric acid (IBA) were morphologically most similar to those produced in control cultures. Liquid-shake culture of shoots did not lead to meristemoid formation, despite supplementation with various growth regulators (mT, GA₃ or paclobutrazol). Low temperature (10-20 °C) induced corm formation in Tritonia gladiolaris shoot cultures, while corm formation was completely inhibited above 20 °C. Increasing temperature from 10 °C to 15 °C and from 15 °C to 20 °C increased corm mass significantly. Paclobutrazol (3.4 μM), GA₃ (2.9 μM), NAA (5.4 μM ) or methyl jasmonate (4.5 μM ) could not induce corm formation at 25 °C, while at 15 °C, NAA and methyl jasmonate inhibited corm formation. These experiments successfully demonstrate the ease with which different genera of the Iridaceae can be multiplied in in vitro systems.     

Invertase and sucrose synthase activity, carbohydrate status and endogenous IAA levels during Citrus leaf development

Levels of activity of the sucrose catabolizing enzymes, acid invertase (EC 3.2.1.26) and sucrose synthase (EC 2.4.1.13), were measured during development of new leaves of Citrus sinensis (L.) Osbeck cv. Shamouti. Soluble acid invertase showed a peak activity of 32 nkat (g fresh weight)⁻¹ at ca 60% of full leaf expansion and rapidly declined toward and after full expansion. There was no concomitant increase in an insoluble form of the enzyme. Sucrose synthase activity, measured in the synthesis direction, declined from 33% of full leaf expansion [10 nkat (g fresh weight)⁻¹] 10, and following, full expansion. Highest sucrose synthase activity, measured in the cleavage direction, was 6 nkat (g fresh weight)⁻¹ and showed little change during development. Acid invertase has a K_m of 5 m M for sucrose, while sucrose synthase had a K_m of 118 m M for sucrose. Changes in acid invertase activity correlated with changes in the reducing sugar:sucrose ratio. These results suggest that soluble acid invertase activity is the primary enzyme responsible for sucrose catabolism in the expanding Citrus leaf. Changes in leaf expansion rate and invertase activity did not correlate positively with changes in endogenous free IAA level, as determined by enzyme linked immunoassay.     

The effect of 4-chlororesorcinol on the endogenous levels of IAA,ABA and oxidative enzymes in cuttings

Treatment of bean cuttings with 4-chlororesorcinol (4-CR), known to increase the number of roots and extend their distribution, prevented the accumulation of free indol-3-yl-acetic acid (IAA) in the hypocotyls within 24 h after cutting preparation. In mung bean there was no change in the distribution (upper half vs. 1 ower half of the hypocotyl) of IAA within the hypocotyl as a result of the treatment. In bean cuttings the treatment with 4-CR prevented the accumulation of IAA in the bottom of the cutting. Oxidation of IAA as a measure of IAA oxidase activity in bean was enhanced appreciably by 4-chlororesorcinol. The level of abscisic acid in mung bean, on the other hand, remained 3–4 fold higher than in the control, yet still about 50% lower than the zero time level. In untreated mung bean cuttings the activity of peroxidase increased after cutting preparation. In contrast, the activity of peroxidase in 4-Cr-treated cuttings was consistently lower. In order to relate to the effect of exogenously applied auxin the level of peroxidase was measured also in indol-3-yl-butyric acid-treated cuttings. The overall peroxidase activity in IBA-treated cuttings was not affected. However, when assaying for the different isozymes the drop in peroxidase activity was most evident in the inducible basic isoperoxidases both in 4-CR and IBA treatments. It appears that the exposure to 4-CR exerts an effect that is similar to that of exogenously applied auxin, affecting the activity of basic peroxidases and enhancing the oxidation of endogenous IAA, thus allowing the organization of the primordia.Abbreviations ABA - abscisic acid - 4-CR - 4-chlororesorcinol - IAA - indol-3-yl-acetic acid - IBA - indol-3-yl-butyric acid