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)     

The Effect of Mutations in the T-DNA Encoded Auxin Pathway on the Endogenous Phytohormone Content in Cloned Nicotiana tabacum Crown Gall Tissues

We analyzed the endogenous auxin and cytokinin levels of clonedNicotiana tabacum SR 1-lines induced either by the wild-typeAgrobacterium tumefaciens C58 strain or by mutants affectedin the T-DNA-encoded IAA biosynthesis pathway. The wild-typeSR1-C58 line contained up to 20 times more IAA than a nontransformedSRI-callus line. The mutant lines affected in gene 1 (iaaM^–)or gene 2 (iaaH^–) contained intermediate levels of IAA. Analysis of the endogenous levels of indole-3-acetamide (IAM)in the nontransformed SR 1 callus line, the wild-type SR1-C58and the two mutant lines confirmed the T-DNA-induced IAA biosynthesispathway in the transformed tumor cells. Supplementing auxinto the mutant lines resulted in complete suppression of theshoot-forming ability, but no changes in the endogenous IAAlevels. There was no marked difference in the cytokinin level betweenthe nontransformed callus line and the wild type tumor line.The two mutant lines, however, showed a 20- to 30-fold highercytokinin level which was not affected by the addition of NAA.The T-DNA encoded hormone biosynthetic pathways are discussedin relation to pathways of the host plant. (Received July 29, 1986; Accepted February 14, 1987)     

Myo-inositol Biosynthesis and Galactose Utilization by Wild Carrot (Daucus carota L. var. carota) Suspension Cultures

Wild carrot (Daucus carota var. carota) cell suspensions (63–120µm in diameter) were grown on a mineral salt medium containingdifferent carbon sources in the presence (10 mM) and absenceof myo-inositol. The data obtained after 14 and 21 days of growthshow that an external supply of myo-inositol is not essentialfor growth and development of wild carrot embryos. A linearrelationship was found between growth (d. wt) and embryo numberin the presence and absence of myo-inositol. Standard stock cell suspensions never exposed to exogenous myo-inositoland grown in the absence of 2, 4-D with glucose or galactoseas the carbon source synthesized radioactive myo-inositol whenexposed to D-[1–¹⁴C]glucose or D-[1–¹⁴C]galactose.Gas chromatographic analyses revealed the presence of myo-inositolin the bulk tissue grown in the presence of 2.25 µM 2,4-D with glucose, galactose, fructose or mannose as the solecarbohydrate. We could not detect any component indicating anisomer or a methylated derivative of an inositol in the tissueextracts. Stock cultures were maintained (with 2, 4-D) successfully forat least three successive sub-cultures on D-galactose as thesole carbohydrate. The growth achieved over this culture periodshowed that wild carrot cells used by us could quickly adaptto grow on D-galactose as rapidly as they grow on sucrose. Daucus carota L., wild carrot, suspension cultures, myo-inositol, galactose     

The Association of Ascorbate and Ascorbate Oxidase in the Apoplast with IAA-Enhanced Elongation of Epicotyls from Vigna angularis

The level of (ascorbic acid (AA) plus dehydroascorbic acid (DHA))and the ratio of the level of AA to that of AA plus DHA in intercellularwashing fluid (IWF) of epicotyl segments from Vigna angularisdecreased from 2.8±0.3 to 1.2±0.5nmol (g fr wt)^–1and from 0.23±0.03 to 0.13±0.01, respectively,after incubation of the segments without IAA for 20 h at 27°C.However, these values changed to 5.3±1.7 nmol (g fr wt)^–1and 0.07±0.05 after incubation with 0.1 mM IAA. The activityof cell wall-bound ascorbate oxidase increased by about 20%and 70% after incubation without IAA and with IAA, respectively.However, the activity of cell wall-bound peroxidase was notaffected by incubation with or without IAA. The activities ofascorbate oxidase and peroxidase in IWF decreased by about 85and 75% after incubation without IAA. IAA did not affect thesedecreases to any great extent. A lignin-like compound was formedduring the incubation of epicotyl segments in the absence ofIAA. Formation of this compound was inhibited by IAA. The resultssuggest that one of the causes of the enhancement of elongationgrowth by IAA is the inhibition of peroxidase-dependent lignificationas a result of increases in levels of AA and DHA and in ascorbateoxidase activity. (Received August 16, 1993; Accepted December 6, 1993)     

Effects of Abscisic Acid and Cytoplasmic pH on Potassium and Chloride Efflux in Arabidopsis thaliana Seedlings

The effects of ABA, isobutyric acid (IBA) and nicotine on K⁺and Cl⁺ efflux were studied in Arabidopsis thaliana seedlings,and the role of pH_cyt, and E_m in the regulation of the effluxof these ions was discussed. The data show that treatments withIBA and nicotine influenced in opposite directions the effluxof either K⁺ or Cl^–: K⁺ efflux was increased by nicotineand reduced in the presence of IBA, whereas Cl^– effluxwas stimulated by IBA and decreased by nicotine treatment. Underall the conditions tested ABA induced cytoplasmic acidificationand inhibition of K⁺ and Cl^– net efflux. Experiments aimedto estimate the individual contribution of pH_cyt and E_m in modulatingK^– efflux indicated that, within the range of acidic pH_cytvalues, a regulation of K⁺ efflux was imposed by pH_cyt on thecontrol exerted by E_m, the efflux being inhibited by lower pH_cytvalues. Conversely, in the alkaline side of pH_cyt K⁺ effluxseemed linked only to the E_m values. These results are consistentwith the hypothesis that the decrease in K⁺ efflux observedin non-stomatal tissues in the presence of ABA may be mediatedby the cytoplasmic acidification induced by the hormone. (Received August 6, 1996; Accepted January 19, 1997)     

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).     

Isolation of a Type I Topoisomerase from Carrot Cells

Carbonera, D., Cella, R., Montecucco, A. and Ciarrocchi, G.1988. Isolation of a type I topoisomerase from carrot cells.—J.exp. Bot. 39: 70–78. A DNA topoisomerase activity has been isolated from suspension-culturedcells of Daucus carota, the enzyme has been chromatographedon CM-cellulose, DNA-cellulose and Sephadex G100. Its Mr appearsto be about 100000 by gel filtration. Carrot DNA topoisomeraserelaxes both positively and negatively supercoiled DNA by changingthe linking number of the substrate in steps of one and is notable to unknot the knotted P4 DNA. It does not require ATP orMg²⁺ , has an optimal salt concentration between 40 and 120mol m^–3 KCl and is resistant to nalidixic acid and novobiocine.This carrot enzyme can be designated as a eukaryotic type IDNA topoisomerase. Key words: DNA topoisomerase, Daucus carota     

Auxin Transport in Secondary Tissues1

Auxin transport was investigated in excised stem segments ofNicotiana tabacum L. by the agar block technique using [1-¹⁴C]indol-3yl-acetic acid (IAA). The ability of the stems to transportauxin basipetally increased as secondary development proceeded;by contrast the ability of the pith to transport auxin declinedwith age. By separation of the stem tissues it was shown thatthe great majority of auxin transport took place in cells associatedwith the internal phloem and in cells close to the cambium;in both cases similar velocities of transport were found (c.5.0 mm h^–1 at 22°C). The effects of osmotic gradientson auxin transport through the internal phloem were investigated.IAA was found by chromatography to account for practically allthe radioactivity in receiver blocks and other extracts of stemsegments. The significance of these results is discussed.     

Inoculation with Azospirillum lipoferum Affects Growth and Gibberellin Status of Corn Seedling Roots

Maize (Zea mays L., hybrid Cargill 147) seedlings, grown inaseptic conditions, were inoculated with three strains of Azospirillumlipoferum (Al op 33, Al iaa 320, and ATCC 29708) or culturedin different concentrations of indol-3-acetic acid (IAA) orgibberellin A₃ (GA₃). After 48 h, root length, root surfacearea, root dry weight, and shoot dry weight were measured inall treatments. Gibberellin content was evaluated in selectedroots of control and Azospirillum inoculated seedlings by gaschromatography-mass spectrometry-selected ion monitoring withthe use of deuterated gibberellins as internal standards. Thethree strains of A. lipoferum, IAA (2 ng ml^–1), and GA₃(40 to 400 pg ml^–1) significantly enhanced root growth.Improvement of root hair growth and density was obtained mainlywith A. lipoferum strain Al op 33 and GA₃ 40 pg ml^–1.GA₃ was identified by gas chromatography-mass spectrometry (byboth, full scan and selected ion monitoring) in a free acidfraction from roots of the seedlings inoculated with A. lipoferum.In the roots of the non inoculated seedlings GA₃ was found afterhydrolysis of a fraction expected to contain glucosyl conjugates. (Received April 26, 1993; Accepted September 27, 1993)     

Partition of 14C Assimilate between Organs and Fractions of Contrasting Varieties of Carrot during Initiation of the Storage Root

This work examines the differences in partition and activityof ¹⁴C in two varieties of carrot (Daucus carota L.) contrastingin shoot to storage root ratio at maturity. Plants were grownin a controlled environment of 20 ?C and 500 µmol m^–2s^–1. During initiation of the storage root (10–25d from sowing) plants were exposed to ¹⁴CO₂ for 1 h and theradioactivity in ethanol-soluble and -insoluble fractions ofshoots, storage and fibrous roots estimated at various timesup to 48 h after exposure. Between 35% and 40% of radioactivityinitially present in the plants was respired during the first24 h and 25–35% of that remaining after 24 h was foundin the roots, depending on age. The proportion found in thestorage region remained fairly constant between 15 and 25 dand was smaller than at 10 d. In the variety with a larger proportionof storage root at maturity (cv. Super Sprite), there was agreater proportion of label in both ethanol-soluble and -insolublefractions of the storage region soon after storage root initiationhad begun than in the variety with a smaller proportion of storageroot at maturity (cv. Kingston). There was no varietal differencein specific activities of the storage roots, but fibrous rootsof cv. Super Sprite showed a greater specific activity thanin cv. Kingston. Differences in shoot to storage root ratiomay thus be associated with characteristics of the fibrous roots.Partition and specific activities are discussed in relationto the initiation and development of the storage organ. Key words: Daucus carota, carrot, assimilate, partition, ¹⁴C, storage root     

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)     

Somatic Embryogenesis from Clonal Leaf Tissues of Cassava

Leaf lobes were isolated from palmate leaves of clonal cassava(Manihot esculenta Crantz) material growing in vitro or in glasshouseconditions and subjected to a two-stage culture procedure involvingincubation on Murashige and Skoog (MS2) basal medium supplementedwith 2–12 mg l^–1 2,4-D for 20 d (Stage I) beforetransfer to MS2 basal medium supplemented with 0.01 mg l^–12,4-D and 0.1 mg l^–1 6-benzylamino purine (BAP) (StageII medium). Embryogenetic tissues, foliose structures and somatic embryosdeveloped from leaf lobes at all Stage I 2,4-D concentrations,except on those explants isolated from shoot-tip cultures incubatedon MS2 basal medium supplemented with 0.1 mg l^–1 NAA and1.0 mg l^–1 BAP. Leaf lobes isolated directly from glasshouse plants showed optimalembryogenetic competence when subjected to a Stage I cultureperiod of 17 d, although foliose structure initiation was optimalwith shorter Stage I durations. Leaf lobes of 2–4 mm lengthand those isolated from phyllotaxic leaf numbers 4 and 5 showedthe greatest embryogenetic competence. Manihot esculenta, cassava, somatic embryogenesis, tissue culture, morphogenetic competence     

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.     

Auxin Transport in Roots: V. EFFECTS OF TEMPERATURE ON THE MOVEMENT OF IAA IN ZEA ROOTS

The effects of temperature on the polar movement of IAA through6-mm and 12-mm segments of Zea mays roots have been investigatedover the range from 1 to 50°C. At all temperatures an acropetal polar movement of IAA predominated,although at low temperatures and at 50°C the 6-mm segmentsshowed a transient basipetal polarity, before the persistentacropetal polarity developed. At 1°C the differences betweenacropetal and basipetal movement of IAA were less distinct thanat the other temperatures. There is, however, a marked metabolically-dependentacropetal movement of IAA through the tissues at 1°C, becausewhen the segments were deprived of oxygen the acropetal movementwas severely reduced while the basipetal movement was reducedto a smaller extent. At 1°C and at 5°C there was alwaysa persistent basipetal polarity of IAA movement through 6-mmand 12-mm segments under anaerobic conditions. The velocity of acropetal movement (mm h^–1) was the samethrough the 6-mm and the 12-mm segments and was markedly affectedby temperature. It increased from 1°C to a maximum valueof 8 mm h^–1 at 31°C and then decreased again at 40and 50°C. The velocity of basipetal movement could be assessedonly at 1 and 5°C at which temperatures it was greater thanthe velocity of acropetal movement, and virtually independentof segment length. The acropetal flux of IAA (cpm h^–1) was much less through12-mm segments than through 6-mm segments. For both lengthsof segment, however, the flux showed a complex relationshipwith ambient temperature, increasing from 1°C to a maximumat 10–15°C, declining to a minimum value at 31°Cand then rising again at 40 and 50°C. The basipetal fluxof IAA could be astimated only at 1 and 5°C at which itwas very much smaller than the acropetal flux. The amount of IAA in the receiver blocks increased linearlywith time at the lower temperatures. At temperatures withinthe range 15°C to about 31°C, however, the amount ofIAA in the receiver blocks began to decline if the transportperiods exceeded a certain length. The time at which this declinein the IAA in the receiver block began was related to the ambienttemperature. Chromatographic analysis indicated one radioactive substancein receiver blocks at the apical end of segments supplied withIAA-1-¹⁴C at the basal end after transport periods of 6 h at25°C, and 72 h at 5°C. The Rf of this substance wasclosely similar to that of the radioactive IAA supplied in thedonor blocks.     

Biosynthesis of Indole-3-Acetic Acid from L-Tryptophan in Coleoptile Tips of Maize (Zea mays L.)

Coleoptile tips (about 2.5 mm in length) were excised from 3-day-olddark-adapted maize (Zea mays L.) seedlings and incubated indarkness in potassium phosphate buffer that contained ¹⁴C-L-tryptophan(Trp). Subsequent analysis by gas chromatography-mass spectrometryindicated that a significant portion of endogenous indole-3-aceticacid (IAA) had been labeled with ¹⁴C. About 8% of the IAA thatdiffused from the tissue into the medium during incubation from0.5 to 2 h was labeled, and 12% of the IAA extracted from thetissue after a 2-h incubation was labeled. On the other hand,30% of the Trp extracted from the tissue after a 2-h incubationwas ¹⁴C-Trp, which was more than those determined for IAA. Sincethe experiments were carried out under the non-steady-stateconditions in which the tissue content of ¹⁴C-Trp increasedwith time, and since the extracted Trp included the ¹⁴C-Trpin the apoplastic space, it seemed that synthesis de novo fromTrp was the major means by which IAA was produced in the coleoptiletip. The conversion of Trp to IAA was not detected in sub-apicalsegments (5–7.5 mm from the top) that were incubated similarly,an indication that synthesis of IAA occurs specifically in thetip region. When intact seedlings were irradiated with a pulseof red light 2 h before excision of tips and the applicationof ¹⁴C-Trp, the amounts of extractable and diffusible IAA werereduced by 40–60% without a change in the rate of ¹⁴Cincorporation. This result indicated that the production ofIAA from Trp is controlled by a red-light signal. (Received May 15, 1995; Accepted September 1, 1995)     

Dynamics of Competition in Populations of Carrot (Daucus carota)

Populations of carrot (Daucus carota) were raised over a widerange of densities (79–5763 plants m^-2) to examine thedynamics of competition in terms of yield–density relationshipsand size variability, and to investigate the effects of nutrientsupply on competition. While the relationship between shootyield and density was asymptotic, the relationship between rootand total yield and density tended to be parabolic. For a giventime and density series the relationship between yield per unitarea and density could best be described by the model: y=w_mD_(1+aD)^b wherey is the yield per unit area,D is density,w_m, a andb arefitted parameters. The parametersw_m anda increased over timebut nutrient availability affected onlyw_m. An extension of thebasic yield-density model is proposed to describe the dynamicsof the yield–density relationship over time: y=kD_{[1+cexp(-rt)]{1+}     

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     

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; )