Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens III. Formation of IAA Conjugates in Cultured Carrot Tissues Transformed with Wild-type and Mutant Ti Plasmids

Inactivation of IAA was examined in cultured carrot tissuestransformed with Agrobacterium tumefaciens that harbored wild-type,aux^– or cyt^– Ti plasmids. IAA oxidase activities were similar among the three types oftransformed tissue. Metabolites of IAA were examined by followingthe fate of 3-indolyl-[l-^l4C]IAA. IAA conjugates were detectedin all transformed tissues as well as in hypocotyl segmentsof non-transformed carrot seedlings. The rate of formation ofIAA conjugates was ten times higher in the tissues transformedwith wild-type or cyt^– Ti plasmids than in the tissuestransformed with aux^– Ti plasmids. When the tissues transformedwith aux^– Ti plasmids were cultured on medium that containedIAA for 6 h, the rate of formation of IAA conjugates in thesetissues became as high as that in tissues transformed with wild-typeor cyt^– Ti plasmids. The tissues transformed with wild-type or cyt^– Ti plasmidsnot only synthesize larger amounts of IAA but also convert alarger amount of free IAA to conjugated IAA than do non-transformedand aux^– transformed tissues. Presumably, in carrot, theformation of IAA conjugates decreases the amount of free IAAin the transformed tissues that synthesize large amounts ofIAA and, consequently, the level of free IAA can be maintainedfairly constant. (Received June 2, 1989; Accepted May 23, 1990)     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens II. IAA Biosynthetic Activity of Cultured Carrot Tissues Transformed with Wild-Type and Mutant Ti Plasmids

IAA biosynthetic activity was examined in cultured carrot tissuestransformed with Agrobacterium tumefaciens harboring wild-type,aux^– or cyt^– Ti plasmids. In vitro IAAM hydrolaseactivities in tissues transformed with wild-type, and cyt^–Ti plasmids were 3.09 and 19.82 nmol/g proteins/30 min, respectively,but not detectable when aux^– Ti plasmids were used. Theactivity of IAA biosynthesis, determined by the incorporationof radioactivity into IAA in tissues fed with [¹⁴C]-tryptophan,was 34.13, 10.92 and 32.47 pmol/g fr wt/30 min in tissues transformedwith wild type, aux^– and cyt^– Ti plasmids, respectively.The incorporation of radioactivity into the IAAM fraction wasdetected only in the tissues transformed with wild type andcyt^– Ti plasmids. These results showed that the T-DNAencoded pathway of IAA biosynthesis was active in tissues transformedwith wild-type and cyt^h Ti plasmids, and that the activity ofIAA biosynthesis in those tissues was higher than that in tissuestransformed with the aux^– Ti plasmid. (Received March 16, 1988; Accepted July 31, 1988)     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens I. Auxin and Cytokinin Contents of Cultured Tissues Transformed with Wild-Type and Mutant Ti Plasmids

Carrot and tobacco plants were transformed with Agrobacteriumtumefaciens harboring wild-type, aux^– or cyt^– Tiplasmids. In tobacco, these wild and mutant Ti plasmids inducedthe formation of non-morphogenic galls, galls with shoots, andgalls with roots, respectively. In carrot, however, transformationwith any of these plasmids resulted in only the formation ofamorphous tumors. Determination of IAA and cytokinin contentsshowed that in tobacco, significantly high amounts of cytokininsor IAA are present in the cells transformed with Ti plasmidspossessing cytokinin or IAA biosynthetic genes, respectively.In carrot, cytokinin contents were also high in the cells transformedwith Ti plasmids having cytokinin biosynthetic genes, whereasIAA contents of the cells were similar regardless of the plasmidsused for transformation. These results suggest that the mechanism regulating IAA metabolismmay be different in tobacco and carrot. (Received June 25, 1987; Accepted February 1, 1988)     

Resemblance of growth substances to metal chelators with respect to their actions on duckweed growth

Action patterns of IAA, KIN and GA on the growth of Lemna gibbaG3 are similar to those reported for agents chelating both cupricand ferric ions. Relatively high doses of growth substances,e.g.10^–6 M IAA or KIN and ^–4 M GA, inhibit developmentof photoperiodically induced flower buds and antagonisticallypromote frond multiplication; whereas, at relatively low doses,e.g. 10^–9 M IAA or KIN and ^–5 M GA, they acceleratethe process occurring in the latter half of the induction periodto enhance flower induction. Complex forming abilities of IAA,KIN and GA with cupric and ferric ions are demonstrated spectrophotometrically.Moreover, the ferrous ion-dependent oscillatory change in reproductiveand vegetative photophilies of duckweed is eliminated by KINbut not by IAA and GA. Of the three growth substances tried,KIN alone shows an affinity for ferrous ions. (Received April 5, 1971; )     

Promotion of Plant Cell Division by an Epidermal Growth Factor

In some specified treatments, an epidermal growth factor (EGF)promoted adventitious root formation in epicotyl cuttings ofVigna angularis. The number of the roots induced in cuttingstreated with 0.1 mg liter^-1 EGF during the first 24 h and with210^-4 M IAA during the second 24 h was 15% greater than thatof the roots in cuttings treated without EGF and with IAA. Analysisof the optimum timing of EGF application was performed by dividingthe first 24 h period into three sequential 8 h periods (0–8h, 8–16 h and 16–24 h). The most effective timeperiods in terms of the root formation were 8–16 h and16–24 h. The 0–8 h period was ineffective with respectto the formation. When carrot suspension cells were culturedfor 15 days at a very low cell density (1,000 cells/3 ml Murashigeand Skoog's medium) with more than 0.1 mg liter^-1 EGF, cellnumbers were 72% higher than those cultured without EGF. Theseresults suggest that EGF promotes cell division of plants. (Received October 5, 1992; Accepted May 24, 1993)     

Identification of conjugated IAA in carrot crown gall as indole-3-acetylaspartic acid (IAAsp) by LC/MS

In carrot crown gall cells transformed with Ti plasmids, Ti-derived IAA biosynthetic genes are transcribed and translated, followed by overproduction of IAA. However, the newly synthesized IAA is immediately metabolized to IAA-amino acid conjugate, and the content of endogenous free IAA is maintained at a low level. In this study, IAA-amino acid conjugate in carrot tissues transformed with Ti plasmids was identified as indole-3-acetylaspartic acid (IAAsp) by using frit-fast atom bombardment liquid chromatography/mass spectrometry (LC/MS).     

The Uptake of Growth Substances: XIII. DIFFERENTIAL UPTAKE OF INDOL-3YL-ACETIC ACID THROUGH THE EPIDERMAL AND CUT SURFACES OF ETIOLATED STEM SEGMENTS

The patterns of uptake of indol-3yl-acetic acid (IAA-2-¹⁴C)by etiolated stem segments of varying lengths have been examined,employing tissues excised from (a) the first and third internodesof Pisum sativum, (b) the top and base of the hypocotyl of Gossypiumhirsutum, and (c) the mesocotyl of Avena sativa. For all species,concentrations (10^–5–10^–3 M) and times upto 24 h, there is a steady accumulation of radioactivity inthe segments. For equal volumes of tissue uptake is inverselycorrelated with segment length but for extending tissues theinitial enhanced extension growth is independent of length;that is there is no direct linkage between the rate of extensionand auxin content. Comparisons between segments with free andsealed ends established that over 24 h some 57–73 percent of the IAA enters via the cut surfaces. Initially, thepercentage is greater; expressed as a rate per unit of surfacethe differences between cut and epidermal surfaces can reach28-fold. The rate of entry through the epidermal surface islinearly proportional to the external concentration but thisdoes not hold for cut surfaces. The addition of streptomycin,synthalin, cetyltrimethylammoniumbromide (CTAB), and chitosanto the external medium does not promote uptake of IAA by Pisumsegments; indeed synthalin is markedly inhibitory. With Gossypiumsynthalin causes little inhibition. Larger depressive effectswere induced for entry via the cut surfaces. On entry the IAAis rapidly metabolized and the rate of conversion is higherfor segments with sealed ends. These findings are discussedin relation to (a) differences in the mechanisms determiningthe uptake of IAA and other auxins, (b) cell extension and thedistribution of auxin in the tissues.     

Effects of indoleacetic, p-chlorophenoxyisobutyric and 2,4,6-trichlorophenoxyacetic acids on three phases of rooting in Azukia cuttings

In adventitious root formation of disbudded epicotyl cuttingstaken from light-grown, 5-day-old Azukia angularis seedlings,indoleacetic acid (IAA), 1 x 10^–4 M, applied during thefirst day showed no effect, but enhanced the effect of IAA,1 x 10^–4 M, applied during the second day. Treatment duringthe second day promoted rooting by about 70%, and a combinationof treatments for the first and second days promoted rootingsome 200%. p-Chlorophenoxyisobutyric acid (PCIB), 3 x 10^–4M, and2,4,6-trichlorophenoxyacetic acid (2,4,6-T), 2 x 10^–44M, applied the first day also enhanced the effect of IAA, 2x 10^–4 M, applied the second day. When applied the second day, PCIB, 2 x 10^–4M, increasedthe number of root primordia or clusters of small cells, butnot die number of protruded roots. Formation of the cell clusterwas inhibited by 2,4,6-T, 3 x 10^–4M, applied the secondday. Rooting processes in Azukia cuttings seem to include at leastthree phases: the first phase is induced not only by IAA butalso by PCIB or 2,4,6-T, the second phase is induced by IAAor PCIB and the diird phase depends specifically on IAA. (Received October 28, 1970; )     

Effect of some Metabolic Inhibitors on Rooting Cuttings of Phaseolus mungo under Varying Light Conditions and its Relationship with Auxin and Nutrition

Water and 1 mg1^–1 each of IAA and IBA completely inhibitedon the cuttings of Phaseolus mungo obtained from the seedlingsraised under far-red light but rooting took place in the darkand under white and red lights. Sucrose, however, caused rootingunder far-red light and its effectiveness increased with theaddition of IAA and IBA to the sucrose medium IBA being moreeffective. Culturing in 1 and 5 mg 1^–1 each of FudR, actinomycin-D,cycloheximide and chloramphenicol after pre-treatment with water,IAA, IBA, sucrose and IAA/IBA + sucrose inhibited rooting. Theeffect increased with the concentration of each inhibitor. Incontrast to this, the culturing in water, IAA, IBA, sucroseand IAA/IBA + sucrose after pre-treatment with these metabolicinhibitors produced varying effects. While the inhibition persistedin water, IAA and IBA and even in 5 mg 1^–1 of each inhibitorthat in sucrose stimulated rooting and the effect increasedwith the addition of IAA and IBA to sucrose, the effect of IBA+ sucrose being more pronounced. This stimulation was irrespectiveof the inhibitor and light condition except that 1 mg 1^–1actinomycin-D inhibited rooting in the dark and under far-redlight.     

Combined Effect of Some Growth Regulators on Growth and Gametangial Formation in the Liverwort Riccia gangetica Ahmad

The present work deals with the effect of IAA-kinetin, IAA-GA³and GA³-ABA interactions on growth and gametangial formationin Riccia gangetica in vitro. Inhibitory effect of high concentrationof IAA on vegetative growth is overcome by the co-addition ofkinetin. The best response in terms of fresh and dry weightyields of thalli is obtained by a combination of 10^–5mol dm^–3 kinetin+ 10^–7 mol dm^–3 IAA. Interactionof IAA and kinetin has an additive effect on archegonial formation.Co-addition of IAA and GA³ decreases production of archegoniaand antheridia as compared to those produced in response toIAA and GA³ alone, respectively. Combination of GA³ and ABAreduces vegetative growth, as well as the number of anthendiaand archegonia. Key words: Riccia gangetica, growth, growth regulators, gametangial formation     

Effect of Indoleacetic Acid on Growth and Dinitrogen Fixation in Cyanobacteria

The effect of IAA on growth, dinitrogen fixation, and heterocystsfrequency of Anabaena PCC 7119 and Nodularia sp. have been investigated.Concentrations of IAA ranging from 10^–10 to 10^–4M did not change the growth of Anabaena PCC 7119. Concentrationshigher than 10^–4 M were inhibitory. Similar results werefound in Nodularia sp. although in this case the inhibitoryeffect appeared with 10^–5M of IAA. Neither the nitrogenaseactivity nor the heterocysts frequency were enhanced by IAAtreatment. (Received June 17, 1986; Accepted January 22, 1987)     

Tracheary Element Differentiation Induced in Isolated Cylinders of Lettuce Pith: a Bipolar Gradient Technique

To study the influence of morphogenetic gradients on vasculardifferentiation patterns, a new technique was developed whichallows different substances to be applied at opposite ends ofa tissue block. It yielded information on the mobility of particularmorphogens and on the dependence of callus formation and trachearyelement differentiation on their presence. Application of indol-3ylacetic acid (1AA) (10 mg l^–1), zeatin (0.1 mg l^–1)and sucrose (3 per cent, w/v) in various combinations to theends of cylindrical explants of lettuce pith (Lactuca sativaL.) showed that (a) callus formation was stimulated by IAA,whereas induction of tracheary elements required both IAA andzeatin; (b) callus was confined to a few millimetres at theends of the explants, and tracheary elements occurred mainlywithin the callus; (c) sucrose or its metabolic products diffusedthe 10 mm length of the explants, while IAA and zeatin wereeffective only close to the application site; and (d) some callusand tracheary elements formed when no sucrose was applied, butboth increased with sucrose application, though inhibition oftracheary elements formation occurred with high sucrose concentrations. differentiation, pith explant, tissue culture, xylogenesis, indol-3yl acetic acid, sucrose, zeatin, lettuce, Lactuca sativa     

Callus, Organogenesis and Plantlet Formation in Tissue Cultures of Clitoria ternatea

Decoated seeds of Clitoria ternatea L. germinated on Murashigeand Skoog (Physiologia Plantarum 1962, 15, 473–97) basalmedium (BM) and differentiated callus and bipolar embryoids(two-step method) in low frequency. Calluses developed on lateralroots [BM+KN(0.1 mg 1^–1)], on roots and hypocotyls [BM+KN(0.5mg 1^–1)], and on roots [BM+KN+IAA (0.5 mg 1^–1 ofeach)]. On basal medium with KN (0.5 mg 1^–1) and withKN+IAA (0.5 mg1^–1 of each), multiple shoot buds and embryoids(one-step method) were differentiated directly on split hypocotylsand roots. In the former, shoot buds developed even on unsplithypocotyls. Rhizogenesis on isolated shoot buds occurred efficientlyin BM+indole butyric acid (IBA 0.1 mg 1^–1) and BM+IAA(0.1 mg 1^–1 and 0.5 mg 1^–1). Profuse direct embryoidsand shoot buds developing on root systems are interesting morphogeneticphenomena rarely reported. Clitoria ternatea L., callus, embryoids, multiple shoot buds, regeneration     

Shoot and Plantlet Formation in Organ and Callus Cultures of Albizzia lebbek Benth

Plantlets were produced in vitro from root and hypocotyl explantstaken from seedlings of the tree legume, Albizzia lebbek. Theseexplants formed shoots when cultured with 5.0 mg l^–1 kinetinand 1.0 mg l^–1 IAA in MS medium. Shoots were also inducedin large numbers from callus treated with benzylaminopurine.About 20 per cent of the shoots rooted and were grown into plants. Albizzia lebbek Benth, tree legume, hypocotyl, root, in vitro cultures, shoot-plantlet induction     

Hormonal Control of Xylogenesis in Pith Parenchyma Explants of Lactuca

The time course of xylem differentiation was determined in explantsof lettuce pith parenchyma (Lactuca sativa L. cv. Romana) culturedon Murashige and Skoog (1962) medium using different concentrationsof auxin (IAA) and one cytokinin (zeatin or kinetin). Increasinglevels of auxin from I mg 1^–1 to 15 mg 1^–1 in thepresence of a constant level of a cytokinin (zeatin or kinetin)yielded up to 10 mg 1^–1 IAA, an increase in the numberof tracheary element formations. Cytokinin concentrations aboveand below o.1 mg 1^–1 interacting with an optimal xylogenicamount of auxin inhibited xylogenesis. The IAA (10 mg 1^–1)-zeatin(0.1 mg ^1–1) treatment produced the greatest number oftracheids, while kinetin compared to zeatin did not producesuch an effect. The different effectiveness of zeatin and kinetinin inducing tracheary element formations was not due to a differentcapacity of the two cytokinins to stimulate cell division butit seems likely that zeatin, because of interaction with IAA,is more active than kinetin in the determination of the dividingcells in a specific type of cytodifferentiation. The IAA (10mg 1^–1)-zeatin (0.1 mg 1^–1) treatment produced about6.9 per cent tracheids with respect to cell division while IAA(10 mg 1^–1)-kinetin (0.1 mg 1^–1) produced 4.2 percent. These results are discussed with reference to the problemsof hormonal control of xylem differentiation.     

In vitro Organogenesis and Plantlet Formation in Cashew (Anacardium occidentale L.)

Rapid induction of multiple plantlets of Anacardium occidentalewas obtained from cotyledonary explants. Lin and Staba medium,containing 05 mg 1^–1 of both IAA and KN, promoted directorganogenesis and plantlet formation. Plantlets developed froman organized hemispherical mass of meristematic tissue arisingfrom single epidermal cells. Bipolar differentiation resultedin the formation of shoot and root primordia in a sequentialmanner Anacardium occidentale L., cashew, cotyledon explant, organogenesis, plantlet formation     

Epidermal Growth Factor Interacts with Indole-3-Acetic Acid and Promotes Coleoptile Growth

We used coleoptile sections of Avena sativa, Sorghum bicolor,and Zea mays seedlings to examine interactions between epidermalgrowth factor (EGF) and indole-3-acetic acid (IAA) that mayaffect plant growth and development. Our 24-h bioassays employedthree controls ranging in dilution from 10^–4 to 10^–8g ml^–1: (1) 50 mM potassium-phosphate buffer solution(pH=6.0), (2) bovine serum albumin, a nonspecific protein; and(3) IAA; plus two treatments: (1) mouse epidermal growth factor(EGF) ranging from 10^–6 to 10^–10gml^–1, and(2) EGF + IAA. In all three species growth in IAA, EGF, andEGF + IAA treatments showed significant increases over controls;EGF+IAA showed significant increases in growth over IAA alone.As the concentrations of IAA decreased, the EGF and IAA interactionbecame more pronounced. At the highest IAA concentrations, EGF+ IAA increased growth rates ca. 2% to 39%, whereas at lowerIAA concentrations EGF + IAA promoted growth as much as 121%,thereby lowering the normal IAA physiological set point up tothree or four orders of magnitude. Our data suggest that aninteraction between EGF and IAA may allow plants to recognizeand respond to animal biochemical messengers, resulting in changesin plant cell elongation that ultimately may alter plant growthpatterns. (Received April 27, 1994; Accepted September 5, 1994)     

Growth and swainsonine production of Swainsona galegifolia (Andr.) R. Br. untransformed and transformed root cultures

Untransformed and transformed root cultures of Swainsona galegifollawere established for swainsonine production. Transformed rootsgrew faster and produced higher swainsonine levels (62.3 µgg^–1 DW) than untransformed roots (23.6 ,µg g^–1DW) or roots of intact plants (8.7 µg g^–1 DW). Transformationof a number of plant genotypes using A. rhizogenes strain LBA9402 showed that plant genotype Influences swainsonine levelin transformed roots but that a wide range of swainsonine levelscan be induced by separate transformation events in the samegenotype. Enhancement of swainsonine production was attemptedby treatment with sugars and induction of polyploid roots. Key words: Agrobacterium rhizogenes, root cultures, Swainsona galegifolia, swainsonine     

Ethylene and 1 -Aminocyclopropane-1-Carboxylic Acid Production in flacca, a Wilty Mutant of Tomato, Subjected to Water Deficiency and Pre-treatment with Abscisic Acid

Neill, S. J., McGaw, B. A. and Horgan, R. 1986. Ethylene and1-aminocyclopropane-l-carboxylic acid production in flacca,a wilty mutant of tomato, subjected to water deficiency andpretreatment with abscisic acid —J. exp. Bot. 37: 535–541. Plants of Lycoperstcon esculentum Mill. cv. Ailsa Craig wildtype and flacca (flc) were sprayed daily with H²O or 2?10^–2mol m^–3 abscisic acid (ABA). ABA treatment effected apartial phenotypic reversion of flc shoots; leaf areas wereincreased and transpiration rates decreased. Leaf expansionof wild type shoots was inhibited by ABA. Indoleacetic acid (IAA), ABA and l-aminocyclopropane-l-carboxylicacid (ACC) concentrations were determined by combined gas chromatography-massspectrometry using deuterium-labelled internal standards ABAtreatment for 30 d resulted in greatly elevated internal ABAlevels, increasing from 1?0 to 4?3 and from 0?45 to 4?9 nmolg^–1 fr. wt. in wild type and flc leaves respectively.Endogenous IAA and ACC concentrations were much lower than thoseof ABA. IAA content ranged from 0?05 to 0?1 nmol g^–1 andACC content from 0?07 to 0?24 nmol g^–1 Ethylene emanationrates were similar for wild type and flc shoots. Wilting of detached leaves induced a substantial increase inethylene and ACC accumulation in all plants, regardless of treatmentor type. Ethylene and ACC levels were no greater in flc leavescompared to the wild type. ABA pretreatment did not preventthe wilting-induced increase in ACC and ethylene synthesis. Key words: ABA, ACC, ethylene, wilting, wilty mutants     

Involvement of cellulose synthesis in actions of gibberellin and kinetin on cell expansion. Gibberellin-coumarin and kinetin-coumarin interactions on stem elongation

In azuki bean (Azukia angularis = Vignia angularis) epicotylsections, 5 ? 10^–4 M coumarin inhibited the incorporationof radioactivity from [U^–14C]glucose into the cellulosefraction by 35% in the absence of indole-3-acetic acid (IAA)and by 40% in the presence of 1 ? 10^–4 M IAA. There wasno inhibitory effect on the incorporation of radioactivity intothe other fractions. Coumarin at 5 ? 10^–4 M reversed thepromoting effect of 1 ? 10^–5 M gibberellin A₃ (GA) andthe inhibitory effect of 1 ? 10^–5 M kinetin on IAA-inducedelongation of sections with no significant effects on IAA-inducedelongation. Neither GA nor kinetin had any appreciable effectson cellulose synthesis. No inhibition of cellulose syntheiswas observed with 1 ? 10^–3 M colchichine, which has beenreported to have effects similar to those of coumarin on GA-or kinetin-affected stem elongation. Coumarin at 5 ? 10^–4M was ineffectual in breaking up wall microtubules, while adisrupting effect on wall microtubules was clearly demonstratedwith 3 ? 10^–4M colchicine. From these results, the possible involvement of cellulose synthesisin cell expansion controlled by GA or kinetin was suggested. (Received August 3, 1973; )