Changes in Endogenous Ribosyl-trans-zeatin and IAA Levels in Relation to the Proliferation of Tobacco Crown Gall Cells

Changes in the contents of major endogenous plant hormones intobacco crown gall cells, namely IAA and ribosyl-trans-zeatin,during cell growth were examined using HPLC and ¹⁴C-labeledplant hormones. The content of IAA was high at the early logarithmicstage, while that of ribosyl-trans-zeatin was high at the middlelogarithmic stage. This suggests that cell growth is affectedfirst by IAA, then by ribosyl-trans-zeatin. ³ Present address: Department of Agricultural Chemistry, TottoriUniversity, Koyama, Tottori 680, Japan (Received July 13, 1981; Accepted September 11, 1981)     

Endogenous Levels of Gibberellins, IAA and Cytokinins in Tobacco Crown Gall Tissues of Different Morphologies

Endogenous levels of gibberellin (GA) as well as IAA and cytokininsin teratomas and unorganized crown gall tissues of tobacco wereexamined by GC-SIM (for GA and cytokinin) or HPLC with a fluorescencedetector (for IAA). Two different types of crown gall inducedby octopine-type and nopaline-type Ti-plasmids were used. Inboth types, GA contents were higher in shoot-forming teratomasthan in unorganized calluses, while IAA contents were higherin unorganized calluses. But cytokinin contents in octopine-typecells were higher in unorganized calluses than in teratomas,whereas the contents in nopaline-type cells were higher in teratomas.Our results suggest that there is not always a relationshipbetween the cytokinin/IAA balance and tobacco crown gall morphology,but GA production in tobacco tissues is closely related to itsdifferentiation. ⁴ Present address: Agency for the Assessment and Applicationof Technology (BPPT), Jakarta Pusat, Indonesia. (Received September 1, 1986; Accepted February 16, 1987)     

ent-Kaurene Synthesis and Endogenous Levels of Gibberellins in a Shoot Forming Tobacco Crown Gall Tissue

The in vitro ent-Mcaurene synthesizing capacity, as well asthe endogenous GA content of shoot-forming tobacco crown gallsinduced by a nopaline-type Ti plasmid, was studied. For determinationof the ent-kaurene synthesizing capacity, an HPLC procedurepreceded by sample clean-up was used and the GA content wasexamined by GC-SIM. Kaurene synthesis reached a maximum at thebeginning of the logarithmic phase of growth. There was a clearcorrelation between the ent-kaurene synthesizing capacity andthe content of C₂₀-GAs. It seems that gibberellin synthesisis related to growth and development of the tissue. The natureof the GAs identified suggests, that the GA metabolism mightbe an unusual one. (Received October 12, 1987; Accepted April 11, 1988)     

Endogenous Levels of Gibberellins, IAA and Cytokinins in Catharanthus Crown Gall Tissues of Different Tumor Types

Endogenous levels of gibberellins, auxins and cytokinins incrown gall tissues of Catharanthus roseus G. Don. (Vinca roseaL.) of two different tumor types, induced by a nopaline-typeand an octopine-type Ti-plasmid, were examined. The levels of gibberellins were higher in nopaline-type V208cells than in octopine-type V277 cells. GA₁₂ and GA₂₄ were identifiedfrom V208 cells. The level of IAA was 20-fold higher in V208cells than in V277 cells. The level of trans-ribosylzeatin washigher in V277 cells than in V208 cells, whereas trans-zeatinwas present at higher levels in V208 cells than in V277 cells. Our results revealed that the production of gibberellins, aswell as of IAA and cytokinins, in the crown gall tissues ofCatharanthus roseus differed between the octopine- and nopaline-typetumors, even though the crown galls apparently grow as unorganizedaggregates of cells in both cases. ⁴Present address: Facultad de Ciencias, Pontificia UniversidadJaveriana, Bogota, Colombia. (Received September 29, 1989; Accepted February 2, 1990)     

Changes in Morphology, Levels of Endogenous IAA and Protein Composition in Relation to the Development of Tobacco Genetic Tumor Induced in the Dark

When normal shoots which had been regenerated from tobacco genetictumor cultured in the dark were cut, tumorous tissues were againinduced on the cut segments. Morphological examination of thesegments, 5 days and 15 days after cutting, in a comparisonwith normally regenerated shoots, revealed active cell divisionin vascular-bundle tissue and formation of primordial teratoma-likestructures in the induced tumorous tissues. Endogeneous IAAremained at a constant, low level throughout the tumorigeneticprocess. Proteins from each step of tumorigenesis were separatedby two-dimensional electrophoresis on mini-gels and visualizedby silver staining. Twenty-seven polypeptides showed qualitativeor quantitative changes during the tumorigenetic process. Theseresults are discussed in relation to the development of tobaccogenetic tumor. (Received July 20, 1990; Accepted December 6, 1990)     

Dynamics of Endogenous IAA and Cytokinins during the Growth Cycle of Soybean Crown Gall and Untransformed Callus

The dynamics of the endogenous IAA and cytokinin levels duringthe growth cycle of two soybean crown gall lines (green andpale) induced by a nop⁺ Agrobacterium tumefaciens C58 strain,were compared with an untransformed soybean callus line. In both transformed tumor lines maximum cytokinin (essentiallyglucosyl-trans-zeatin) levels were attained in the beginningof the exponential growth phase, followed by a drastic decreasejust before the stationary phase was reached. Quantitativelythe green tumor line showed a 2–3 times higher cytokinincontent compared with the pale line. In the untransformed soybeancallus hardly any significant levels of cytokinin could be detected. Analysis of endogenous IAA levels showed no difference betweenthe two tumor lines and the untransformed callus tissue, allshowing a low and constant level throughout the entire growthcycle. The relevance of the endogenous accumulation of phytohormonesin relation to the hormone autotrophic growth of transformedsoybean tissue is discussed. ³ Senior Research Associate Nationaal Fonds WetenschappelijkOnderzoek (N.F.W.O.). ⁴ Recipient of an Instituut voor Wetenschappelijk Onderzoekin Nijverheid en Landbouw (I.W.O.N.L.) grant.     

Cytokinin Production in Relation to the Growth of Pea-root Callus Tissue

The natural occurrence of free cytokinins was examined in relationto the growth of serially propagated pea-root callus tissuecultures. The relatively slow-growing pea-root callus was harvestedat regular intervals throughout a 12-week period and fresh weight,dry weight, cell number, and cytokinin activity were determined.At the end of the culture period the fresh weight had increasedabout 34 times, the cell number about 100-fold, and the dryweight approximately 19 times over that found on inoculation.Purified ethanol extracts from pea-root callus were tested forcytokinin activity by the soybean callus bioassay. Cytokininactivity was detected in extracts made at all stages of growthtested. There was a sharp rise in cytokinin content during theearly period of culture. The peak in cytokinin activity establishedat the beginning of the phase of growth was associated witha high frequency in mitoses. As growth proceeded there was adecline in both cytokinin content and in the mitotic index.     

Growth Profile of Crown Gall Cells of Tobacco in Suspension Culture

In order to obtain a basic information of plant cell suspension culture as a step toward the development of large scale culture, culture conditions of crown gall cells (auxin non-requiring cells) were investigated. Addition of yeast extract to culture medium was significantly effective for the growth and cell dispersion.

In experiments on the ability of the cultured cells to utilize sugars as the carbon source, it was observed that galactose, added to the culture medium, markedly inhibited the cell growth.

Pasteurization of the medium containing fructose as carbon source made it brownish by Maillard reaction and the medium apparently restrained the cell growth. However, the fructose medium sterilized by filtration was excellent for the cell growth as well as sucrose or glucose medium. In a jar fermentor, even the glucose medium became brownish by heat sterilization and the brown colored medium restrained the cell growth. Under optimum conditions, the doubling time was 1.1 day in exponential phase and 2.0 g of cell (dry weight) per 100 ml culture was obtained as the maximum yield.     

Quantification of Intact Cytokinin Glucosides in Datura innoxia Crown Gall Tissue by Desorption Chemical Ionisation Mass Spectrometry

Cytokinin glucosides are routinely quantified as their aglycones produced by enzymic or chemical hydrolysis. It is, however, important to be able to measure their levels per se. The present paper illustrates the use of desorption chemical ionisation mass spectrometry coupled with stable isotope dilution for the determination of intact, underivatized N- and O- glucosyl conjugates of cytokinins in Datura innoxia crown gall tissue. A total of six glucosyl conjugates were determined; the two N-glucosides, zeatin-7-glucoside and zeatin-9-glucoside, were present in higher quantities than the O-glucosyl derivatives of zeatin, dihydrozeatin and their ribosides.     

Studies on Auxin Protectors X. Protector Levels and Lignification in Sunflower Crown Gall Tissue

The lignification and differentiation of phloem fibers in sunflower stems is inhibited by growing crown gall tumors. Crown gall tumor tissue has previously been shown to contain large quantities of auxin protectors. Since auxin protectors are antioxidants which inhibit peroxidase-catalyzed reactions, and since the formation of lignin is known to involve a peroxidase-catalyzed reaction, an investigation was undertaken to examine the relationship between auxin protectors and lignification in sunflower crown gall tissue. Sunflower crown gall tissue placed into media low in mineral content, rapidly lignifies. In the low mineral media, protectors appear in the medium within an hour or two, implying that endogenously-synthesized protectors rapidly leak out of the tissue. In control media, the tissue neither lignified appreciably, nor did it exhibit an excessive amount of protector release. The addition of Ca²⁺ to the low mineral medium markedly slowed, but did not entirely prevent lignification; similarly Ca²⁺ markedly slowed the release of protector into the low mineral medium. Auxin protectors added to the low mineral medium did not inhibit lignification apparently because, in the medium, the protectors are rapidly oxidized to quinones. The addition of catechol, a substance which mimics protector, also failed to inhibit lignification and also formed a colored compound in the medium suggesting o-qui none formation. In contrast, dithiothreitol, a strong anti-oxidant which upon oxidation does not form a strong oxidant (such as o-quinone), when added to the low mineral medium does inhibit lignification. It is suggested that in the in vitro situation lignification and senescence occurs in low mineral media because the protectors leak out rapidly causing the cell's metabolism to favor peroxidase-catalyzed oxidations including those leading to lignification, while in the in vivo situation the excess protectors produced by crown gall tumor tissue diffuse into surrounding tissue, maintaining a reduced state in such tissues and thereby inhibiting differentiation and lignification. The synthesis of large quantities of protectors by the tumor tissue therefore could account for the anaplasia of the bundle caps observed in sunflower internodes in the vicinity of growing crown gall tumors.     

Determination by Gas Chromatography-Mass Spectrometry of [N(5)]Adenine Incorporation into Endogenous Cytokinins and the Effect of Tissue Age on Cytokinin Biosynthesis in Datura innoxia Crown Gall Tissue

In this study gas chromatographic-mass spectrometric techniques have been used to identify and quantify the metabolic incorporation of [¹⁵N₅]adenine into zeatin and its metabolites by 3-week-old Datura innoxia Mill, crown gall tissue. In a parallel study the levels of endogenous cytokinins were also determined by the stable isotope dilution technique using deuterium (²H)-labeled internal standards. Incorporation levels of the [¹⁵N₅]adenine after 8 hours of incubation, expressed as a percentage of the endogenous cytokinins, were as follows: zeatin (1.0%), zeatin riboside (1.5%), and zeatin riboside 5′-phosphate (10.2%). These results are consistent with those observed in complementary experiments using [U-¹⁴C]adenine, and support the proposal that the cytokinin biosynthesis occurs primarily at the nucleotide level. The effect of tissue age on cytokinin biosynthesis, determined by [U-¹⁴C]adenine incorporation into cytokinins by tissues at varying growth stages, indicated a steady increase with time reaching maximal synthesis at five weeks following subculture after which the level of ¹⁴C incorporation into cytokinins declined.     

亚硒酸钠对烟草冠瘿组织生长的影响及其与内源激素的关系

低浓度(0.1～1μmol/L)亚硒酸钠(Na_2-ScO_s)对烟草冠瘿组织的生长有促进作用;但浓度较高时(5～20μmol/L)不仅对生长有抑制作用,同时能促使其内源吲哚乙酸(IAA)和玉米素(ZA)含量下降,脱落酸(ABA)含量增加,IAA氧化酶活性增强。适宜浓度的外源2,4-D,IAA,6-BA,ZA都可部分缓解较高浓度Na_2SeO_3对生长的抑制作用,其中2,4-D,6-BA的缓解能力分别大于IAA和ZA。Na_2SeO_3还引起烟草冠瘿组织过氧化物同工酶谱发生显著变化,这种变化可被适宜浓度的外源6-BA逆转,并向正常组织方向转化,缓解较高浓度Na_2SeO_3对生长的抑制作用与逆转其过氧化物同工酶谱变化的6-BA浓度范围完全一致。     

Effect on Root Growth of Endogenous and Applied IAA and ABA: A Critical Reexamination

Applications of indole-3yl-acetic acid (IAA) and abscisic acid (ABA) were done on two-day-old intact maize (cv LG 11) roots. The effect of the treatment on the root growth depends on their initial elongation rate. The slow growing roots were all inhibited by exogenous IAA and ABA at any concentrations used whereas for the fast growing roots their elongation was promoted by these two hormones at low concentrations. Quantitative analyses of endogenous IAA and ABA were performed using the gas chromatography-mass spectrometry technique. Detection and quantification of endogenous IAA and ABA were done on the zone of the root implicated in elongation. These techniques were achieved by electron impact on the IAA-Me-heptafluorobutyryl derivative and by negative ion chemical ionization with NH₃ on the ABA-Me ester derivative. A negative correlation between the growth and the endogenous content of these two hormones was obtained. ABA presented a larger range of endogenous level than IAA on the whole population of roots tested. When using applied IAA and ABA at different concentrations the same differentiating effect on the growth was observed. This allowed us to conclude that for identical concentrations, IAA has a more powerful effect on root elongation than ABA. Present results are discussed in relation to previous data related to the role of IAA and ABA in the growth and gravireaction of maize roots.     

重穗型杂交水稻籽粒灌浆过程中强势和强势颖花内源IAA、ABA和GA水平的动态状况

研究了重穗型杂交水稻培矮 6 4s/E3 2的灌浆过程和强、弱势颖花中内源IAA、ABA和GA1 GA3水平的动态状况。籽粒发育过程中不同内源激素水平高低依次为 :IAA >GA1 GA3>ABA。IAA和ABA水平在强势颖花中较高而GA1 GA3水平在弱势颖花中较高。 3种激素水平的变化与谷粒增重速率之间均存在正相关 ,两个最高的相关系数值分别存在于单位鲜重样本的IAA含量(ng/gFW ) 与籽粒鲜重的增重速率之间 (r =0 .82 1 8 )和单个籽粒IAA含量 (ng/grain)与籽粒干重的增重速率之间 (r =0 .8485 )。推测启动和维持籽粒灌浆过程可能需要较高的IAA水平 ;ABA可能具有促进籽粒中同化物的累积和种子成熟的作用 ;GA1 GA3可能具有保持弱势颖花活性的特殊作用     

The Effects of Mechanical Impedance to Growth on the Levels of ABA and IAA in Root Tips of Zea mays L.

Extraction and analytical methods have been refined and newones devised to allow precise determinations by GC-EC of thelevels of abscisic acid (ABA) and indol-3ylacetic acid (IAA)in samples of maize root tips as small as 1.0 g fr. wt. Seminalroots of 5-d-old maize seedlings grown in normal (bulk density1200 kg m^–3) and compacted (bulk density 1600 kg m^–3)sand/garden loam mixtures have been examined. Seminal rootsfrom compacted soil had an average length of about 40% of thatof control roots and were much thicker. The ABA levels in 10mm tips of impeded roots (c. 25–35 ng g^–1 fr.wt.)did not differ significantly from those of normal root tipson both a fresh and dry weight basis. The levels in 0–1mm tips were approximately double those in the remaining 1–10mm zones. IAA levels were increased by about 3 times in impededroots (176.3 as compared with 52.4 ng g^–1 fr.wt) and itis concluded that this response is likely to be the main causeof the morphological and growth changes brought about by soilcompaction.     

Changes in the Levels of IAA,ABA, and Cytokinins in Wheat Seedlings Infected with Tilletia caries

The seedling growth and the content of endogenous phytohormones in wheat seedlings were estimated 3, 6, and 9 days after infection with the bunt pathogen (Tilletia caries) (DC.)TUL. The infection of a pathogen-susceptible species Triticum aestivum L. and a resistant species T. timopheevii Zhuk. resulted, respectively, in an increase and a decrease in the seedling growth and the IAA content as compared to the control. The cytokinin content increased in both species, and the increase in T. timopheevii was more rapid. The pathogen-induced increase in auxin content is suggested to enhance fungal invasion of plants. In the susceptible species, a high ABA concentration was retained for a longer period of time and could act as a factor of virulence. At the same time, in the resistant species, an increase in ABA content was transient and seems to trigger plant defense mechanisms.     

Changes in Endogenous Cytokinin Levels During Abscission and Senescence of Streptocarpus Leaves

The cytokinin levels of butanol and aqueous extracts of proximalportions of Streptocarpus leaves showed very little change duringthe summer and early autumn months, whereas those of the distalportions changed markedly. These changes appear to be closelyrelated to the formation of abscission layers and the subsequentsenescence of the distal part of the leaves. It is postulatedthat the increase in butanol-soluble cytokinins during Januarymay induce abscission, while the decrease in February appearsto be directly related to senescence of the distal portionsof the leaves.     

Suppression of the Tumorous State in Crown Gall Teratomas of Tobacco A Clonal Analysis

Two types of experimental approaches designed to test the possible involvement of chimeras in the persistent but readily reversible suppression of the tumorous state and in a recovery from that state in crown gall teratomas of tobacco were investigated. These studies showed that the tumorous state was suppressed in specialized cells present in the different tissues derived from each of the three layers of the apical meristem of teratoma-derived shoots of clonal origin. Although differences existed in the exogenous requirements needed for the reestablishment of the tumorous state in the different specialized cell types present in the different tissues, all again acquired a capacity for autonomous growth when appropriately stimulated. No evidence was found for the existence of periclinal or sectoral chimeras in teratoma-derived shoots of a single cell origin. Chimerism was, however, found to exist in one noncloned teratoma-derived shoot. Analysis of more than 500 clones derived from leaf mesophyll cells of cloned teratoma shoots showed that the vast majority of them (> 99%) were tumorous, while all of the 144 seed-grown plants from these shoots had completely recovered from the tumorous state. The results of these studies suggest that (a) a teratoma shoot does not depend for its development on the presence of significant numbers, if any, of cells that had recovered from the tumorous state; (b) the tumorous state may be suppressed in even the most highly specialized cells; (c) some special mechanism exists in the meiotic process for the elimination of all or most of the foreign DNA from transformed cells.     

Changes in the Levels of IAA and ABA in Cucumber Leaves under Progressive Soil Drought

Changes in the IAA and ABA contents in cucumber (Cucumis sativus L.) leaves during adaptation to drought were studied. An increase in the water-retaining capacity and heat resistance of leaves indicating the onset of adaptation occurred when the leaf growth has been already suppressed. There was a transient increase in the ABA content during the initial stage of adaptation. An increased IAA content was maintained for a longer period, throughout about two-third of the adaptation period. A second increase in the ABA content was observed before the onset of leaf permanent wilting, when IAA content already decreased. Our data suggest that not only ABA, but also IAA are involved in the development of defense responses during the adaptation to drought.     

几种木本植物插穗生根与内源IAA,ABA的关系

迄今为止,传统的插条繁殖仍是林业和园林工作者获得良种无性系和培育苗木的重要途径。在影响插穗不定根形成内外因素中,植物内源激素水平和生长调节剂应用占有重要地位。已知第一个根原基细胞的分裂依赖于内源生长素或外源的生长调节剂(Hartmann 1983,Haissig 1974),低浓度的ABA(1.26～20μg/ml)能促进