Changes in Cytokinin Activities and Mass Spectrometric Analysis of Cytokinins in Root Exudates of Rice Plant (Oryza sativa L.) : Comparison between Cultivars Nipponbare and Akenohoshi

Changes in exudation rate and cytokinin activities in the exudates were measured in two varieties of rice (Oryza sativa L.), cv Nipponbare (a Japanese normal cultivar) and cv Akenohoshi (a high-yielding cultivar). The exudation rates of Akenohoshi, the leaves of which remained green for a longer time, were higher than those of Nipponbare after the booting stage. Cytokinin activities in the exudates of Akenohoshi were higher than those of Nipponbare during the ripening period. Cytokinins in the exudates collected during the middle of the ripening stage were analyzed with mass spectrometry using deuterium-labeled standards. trans-Zeatin, trans-ribosylzeatin, and N⁶-isopentenyladenosine were detected as free cytokinins, and zeatin was detected in the hydrolysates of highly polar fractions (“conjugated zeatin”) in the exudates of both cultivars. Conjugated zeatin was the predominant cytokinin in both cultivars. Therefore, we suggest that conjugated zeatin is an important form of cytokinin during the ripening stage. The level of each of the cytokinins in Akenohoshi was higher than that in Nipponbare. Also, we discuss the correlation between the leaf senescence and cytokinin content in root exudates.     

Loss of the Photosynthetic Capacity and Proteins in Senescing Leaves at Top Positions of Two Cultivars of Rice in Relation to the Source Capacities of the Leaves for Carbon and Nitrogen

Senescing leaves are a source organ of both carbon and nitrogenbut, because degradation of chloroplast proteins and exportof their degradation products to sink organs give rise to lossof the photosynthetic capacity, the leaves serve as the sourceof nitrogen only at the cost of their source capacity for carbon.Changes in the photosynthetic capacity and levels of proteinsin leaves at the top four positions of two cultivars of rice,Nipponbare and Akenohoshi, during the ripening stage were investigatedtaking the trade-off relationship between the two source capacitiesinto consideration. Rate of light-saturated photosynthesis (Pmax)in leaves decreased more rapidly in Nipponbare than in Akenohoshiafter heading. Various proteins were also degraded during senescence,with Nipponbare leaves showing faster loss of proteins thanthe corresponding leaves of Akenohoshi. Decline in Pmax wascorrelated, similarly in the two cultivars, with losses of ribulose-l,5-bisphosphatecarboxylase/oxygenase, soluble proteins, chlorophyll a thatbound to the reaction center complexes of the two photosystems,the activity of whole-chain electron transport, and a majorpart of insoluble proteins during senescence. The results suggestthat degradation of proteins during senescence of rice leavesis coordinated so as to enable the leaves to perform photosynthesiswith a high use efficiency of protein and export nitrogen ata low or nearly minimum cost of the source capacity for carbon. ⁵ Present address: Section of Biology, University of California,Davis, California 95616, U.S.A. ⁶ Present address: Faculty of Agriculture, Utsunomiya University,350 Mine-machi, Utsunomiya, 321 Japan.     

Endogenous cytokinins in rose petals and the effect of exogenously applied cytokinins on flower senescence

In extracts from rose petals cytokinin activity was detected by Amaranthus bioassay in HPLC eluates corresponding to the standards: Z, ZR, 2iP and 2iPA; subsequently, the presence of two groups of endogenous cytokinins was confirmed by ELISA.Measurements of senesence indicators (cell sap osmolarity and conductivity) and observations of flower vase-life indicated that when the above cytokinins were applied as holding solutions they delayed flower senescence by 34–56% and prolonged rose longevity.Abbreviations B.H.T. 2.6-di-t-buytl-4-methyl phenol - ELISA Enzyme linked Immunosorbent Assay - HPLC High Performance Liquid Chromatography - 2iP isopentenyladenine - 2iPA isopentenyladenosine - Z trans-zeatin - ZR trans-zeatin riboside     

Induction of in vitro flowering in Dendrobium Madame Thong-In (Orchidaceae) seedlings is associated with increase in endogenous N(6)-(Delta (2)-isopentenyl)-adenine (iP) and N (6)-(Delta (2)-isopentenyl)-adenosine (iPA) levels

We analysed the endogenous cytokinin levels of Dendrobium Madame Thong-In seedlings grown in vitro during vegetative and flowering-inductive periods. HPLC was used to fractionate the extracts and radioimmunoassay (RIA) was used for assay of zeatin (Z), dihydrozeatin (DZ), N(6)-(Delta(2)-isopentenyl)-adenine (iP) and their derivatives. Coconut water used in experiments was found to contain high level (>136 pmol ml(-1)) of zeatin riboside (ZR). Protocorms and seedlings cultured in medium with coconut water were found to contain 0.5-3.9 pmol g(-1) FW of the cytokinins analysed. Seedlings (1.0-1.5 cm) cultured in flowering-inductive liquid medium containing 6-benzyladenine (BA, 4.4 muM) and coconut water (CW, 15%) contained up to 200 and 133 pmol g(-1) FW of iP and iPA, respectively. These levels were significantly higher than all other cytokinins analysed in seedlings of the same stage and were about 80- to 150-folds higher than seedlings cultured in non-inductive medium. During the transitional (vegetative to reproductive) stage, the endogenous levels of iP (178 pmol g(-1) FW) and iPA (63 pmol g(-1) FW) were also significantly higher than cytokinins in the zeatine (Z) and dihydrozeatin (DZ) families in the same seedlings. Seedlings that grew on inductive medium but remained vegetative contained lower levels of iPA. The importance of the profiles of iP and its derivatives in induction of in vitro flowering of D. Madame Thong-In is discussed.     

Identification and quantification of the major endogenous cytokinins in pistachio seedlings

The major endogenous cytokinins, Z, ZR, DHZ, DHZR, iP and iPR in pistachio seedlings (Pistacia vera L. cv. Ohadi) were purified by HPLC and their identities confirmed using GC-MS. The aerial parts of two-year old pistachio seedlings including mature leaves, young leaves, lateral buds, debarked stems and bark were subjected to analysis. All of the above mentioned cytokinins were identified in the aerial parts except DHZ which was only present in mature leaves. Z-type cytokinins contributed almost 43% of the total cytokinins. ZR and DHZR were identified as the major ribosides and iP as the main base. The greatest concentration of ZR was detected in the bark, amounting to about 48%. DHZR and ZR constituted the major portion of the total cytokinins detected in both young and mature leaves while Z was detected as a minor cytokinin in leaves. The sharp increase of iP concentration during leaf maturation indicates that mature leaves are probably capable of de novo biosynthesis of cytokinins. The absence of DHZ (except in mature leaves) and the presence of considerable concentrations of DHZR in pistachio stems suggest that these tissues are able to metabolize DHZ to DHZR. The large amount of ZR in pistachio leaves suggests that root-derived ZR is transported into the leaves after loading into the xylem. The presence of high amounts of iP in pistachio lateral buds indicates that iP has been accumulated in these parts. The occurrence of a totally different cytokinin distribution pattern in buds, as compared with the other aerial parts, possibly results from their different metabolism.     

Gas Chromatography-Flame Thermionic Detector Analysis of Cytokinins in Etiolated Squash Seedlings using 14C-BA as an Internal Standard

Cytokinin contents in cotyledon, hypocotyl and root of etiolatedsquash (Cucurbita maxima Duch.) seedlings were determined byinstrumental analysis using ¹⁴C-benzyladenine (¹⁴C-BA) as aninternal standard. Crude extracts were purified using insolublepolyvinylpyrrolidone, cellulose-phosphate column and SEP-PAKC18 cartridge, then applied to a Sephadex LH-20 column to separatezeatin riboside (ZR), isopentenyl adenosine, isopentenyl adenine,¹⁴C-BA and a mixture of zeatin (Z) and dihydrozeatin (DHZ).The recovery rate for the cytokinin fractions after LH-20 wascorrected by ¹⁴C-BA. Each cytokinin fraction was further purifiedby HPLC which also separated Z and DHZ in the LH-20 fraction.Before permethylation, ¹⁴C-BA was added to each of the cytokininfractions to correct the methylation rates. Each methylatedcytokinin fraction was again purified by HPLC, then subjectedto gas chromatography with a capillary column and flame thermionicdetector. The detection limit of cytokinins by this system was0.1 ng. cis-ZK was the most abundant cytokinin in all tissues of theetiolated squash seedlings. Active cytokinins such as trans-ZRand trans-Z were mostly found in cotyledons with lesser amountsin the roots. DHZ was most abundant in the cotyledon. All cytokininsisolated by this procedure were confirmed by gas chromatographyselectedion monitoring. (Received December 26, 1986; Accepted June 1, 1987)     

Quantification by ELISA of Cytokinins in Root-Pressure Exudates of Urtica dioica Plants Grown under Different Nitrogen Levels

ELISAs for [9R]iP, [9R]Z and (diH) [9R]Z have been worked out which, in combination with HPLC., allow the quantitative determination of cytokinins of the isopentenyladenin-, zeatin- and dihydrozeatin-type in samples of plant material. The three ELISAs are optimally applicable in the range from 0.5 to 20 pmoles of cytokinins per assay but reliable measurements can still be performed down to 0.05 pmoles. The assay has been applied to root pressure exudates obtained from Urtica dioica plants grown under controlled conditions and at different nitrogen (nitrate) levels. The predominant cytokinins of the xylem exudate were Z, Z-nucleotide, iP, [9R]iP and iP-nucleotide, while (diH)Z was present to a minor extent. The concentrations of these compounds were found to range between 0.2 and 3 pmoles per ml exudate. Interestingly, no effect of the different nitrogen nutrition on the pattern and concentration of cytokinins in the xylem exudate could be established.     

Identification of N6-(3-methyl-but-2-enyl) adenosine, zeatin, zeatin riboside, gibberellin A19 and abscisic acid in shoots of the hop plant

Young shoots of the hop plant (Humulus lupulus L.) have beenfound to contain at least three cytokinins, one gibberellin,and abscisic acid. Two of the cytokinins were identified aszeatin riboside and N⁶-(3-methyl-but-2-enyl)adenosine (2iPA)based on gas chromatography-mass spectrometry (GC-MS) afterseveral purification steps. Another cytokinin was assumed tobe zeatin from its chromatographic behavior. Gibberellin A₁₉was identified by GC-MS and mass fragmentography, and ABA characterizedby GC-MS. (Received March 30, 1978; )     

Isolation and identification of substances inducing formation of tracheary elements in cultured carrot-root slices

Data are presented on the cytokinin status of seeds and seed components, at different stages of development in Phaseolus coccineus L., as determined with the soybean callus growth bioassay: A change in cytokinin types according to developmental stage occurred: from biologically very active less polar types (zeatin=Z) at early stages to more polar types (zeatin glucoside=Z₉G and zeatin riboside=Zr), with relatively low biological activity, at intermediate and late stages of seed development: When cytokinins were analyzed separately in embryos (embryo proper) and suspensors at two embryonic stages: heart-shaped (A) and middle cotyledonary embryos (stage B) respectively, it was found that: i) at stage A, the suspensor showed cytokinin activity at the level of Z, 2iPA (2-isopentenyladenosine) and Zr, whereas more polar cytokinins (Z₉G, Zr) were present in the embryo; ii) at stage B, when the embryo seems to become autonomous for cytokinin supply, there was a relative abundance of active cytokinins (Z, 2iPA) in the embryo to which Z₉G activity in the suspensor corresponded. It is concluded that the suspensor plays an essential role in embryogenesis by acting as a hormone source to the early embryo.Abbreviations GA gibberellic acid - 2iPA 2-isopentenyladenosine - Stage A heart-shaped embryo - siage B middle cotyledonary embryo - Z zeatin - Z₉G zeatin glucoside - Zr Zeatin riboside     

Xylem fluxes of fixed N through nodules of the legume Acacia littorea and haustoria of an associated N-dependent root hemiparasite Olax phyllanthi

Nodulated 1-1.5-year-old plants of Acacia littorea grown inminus nitrogen culture were each partnered with a single seedlingof the root hemiparasite Olax phyllanthi. Partitioning of fixedN between plant organs of the host and parasite was studiedfor the period 4–8 months after introducing the parasite.N fluxes through nodules of Acacia and xylem-tapping haustoriaof Olax were compared using measured xylem flows of fixed Nand anatomical information for the two organs. N₂ fixation duringthe study interval (635 µg N g FW nodules^–1 d^–1)corresponded to a xylem loading flux of 0.20 µg N mm^–2d^–1 across the secretory membranes of the pencycle parenchymaof the nodule vascular strands. A much higher flux of N (4891µg mm^–2 d^–1) exited through xylem at the junctionof nodule and root. The corresponding flux of N from host xylemacross absorptive membranes of the endophyte parenchyma of Olaxhaustorium was 1.15 µg N mm^–1 d^–1, six timesthe loading flux in nodules. The exit flux from haustorium toparasite rootlet was 20.0 pg N mm^–1 d^–1, 200-foldless than that passing through xylem elements of the nodule.Fluxes of individual amino compounds in xylem of nodule andhaustorium were assessed on a molar and N basis. N flux valuesare related to data for transpiration and partitioning of Cand N of the association recorded in a companion paper. Key words: Olax phyllanthi, host-parasite relationships, N flux, Acacia, N₂ fixation     

Involvement of cytokinins in the germination of chick-pea seeds

Eight cytokinins detected in germinated chick-pea (Cicer arietinum L. var. Castellana) seeds were first present in the embryonic axes but appeared in the cotyledons after 12h of germination. The cytokinins detected in the cotyledons originate in the embryonic axes, but no passage of these substances from the cotyledons to the axes was detected, except when the seeds were treated with red light.It is concluded that the role played by the embryonic axis in mobilizating the main reserves of the cotyledons is mainly effected through these cytokinins. Both natural and synthetic cytokinins exert an important regulatory role in the hydrolysis of reserve proteins and calcium could be involved as an intermediate.Abbreviations BA benzyladenine - cot. cotyledon - (diH)Z dihydrozeatin - (diH)ZR dihydrozeatin riboside - GZR glycosyl zeatin riboside - 2iP 277-1 - iPA 277-2 riboside - Kin kinetin - Z zeatin - ZG zeatin glucoside - ZR zeatin riboside     

Effects of cotton rootstock on endogenous cytokinins and abscisic acid in xylem sap and leaves in relation to leaf senescence

Leaf senescence varies greatly among cotton cultivars, possiblydue to their root characteristics, particularly the root-sourcedcytokinins and abscisic acid (ABA). Early-senescence (K1) andlate-senescence (K2) lines, were reciprocally or self-graftedto examine the effects of rootstock on leaf senescence and endogenoushormones in both leaves and xylem sap. The results indicatethat the graft of K1 scion onto K2 rootstock (K1/K2) alleviatedleaf senescence with enhanced photosynthetic (Pn) rate, increasedlevels of chlorophyll (Chl) and total soluble protein (TSP),concurrently with reduced malondialdehyde (MDA) contents inthe fourth leaf on the main-stem. The graft of K2 scion ontoK1 rootstock enhanced leaf senescence with reduced Pn, Chl,and TSP, and increased MDA, compared with their respective self-graftedcontrol plants (K1/K1 and K2/K2). Reciprocally grafted plantsdiffered significantly from their self-grafted control plantsin levels of zeatin and its riboside (Z+ZR), isopentenyl andits adenine (iP+iPA), and ABA, but not in those of dihydrozeatinand its riboside (DHZ+DHZR) in leaves in late season, whichwas consistent with variations in leaf senescence between reciprocallyand self-grafted plants. The results suggest that leaf senescenceis closely associated with reduced accumulation of Z+ZR, andiP+iPA rather than DHZ+DHZR, or enhanced ABA in leaves of cotton.Genotypic variation in leaf senescence may result from the differencein root characteristics, particularly in Z+ZR, iP+iPA, and ABAwhich are regulated by the root system directly or indirectly. Key words: Abscisic acid, cotton, cytokinins, grafting, leaf senescence Received 23 October 2007; Revised 17 January 2008 Accepted 23 January 2008     

Cytokinins in Gladiolus (Gladiolus grandiflorus) Corms

Ten cytokinin-like substances, termed X₁, X₂, X₃, X_4a, X_4b,X_5a, X_5b, X₆, X₇, and X₈, active in the soya bean hypocotyltest, were detected in gladiolus corms. The factors X_4a, X_5aand X₄ were tentatively identified as zeatin (Z), isopentenyladenosine (iPA) and isopentenyl adenine (iP), respectively,based on their behaviour in Sephadex LH-20 column chromatography,paper chromatography and high pressure liquid chromatography.Factor X₃, which behaved like zeatin riboside (ZR) in the abovesystems could be ZR and/or dihydrozeatin riboside (DHZR). Thebehaviour in Sephadex and ion-exchange column chromatographysuggested that X₁ and X₂ may be cytokinin glucosides and X₅a cytokinin nucleotide or a cytokinin conjugate similar to lupinicacid. The total cytokinin content and the concentration of Z,ZR/DHZR and iPA were higher in non-dormant than in dormant corms.The concentrations of X₁ and X₂ were higher in dormant corms. Gladiolus grandiflorus, gladiolus, dormancy, cytokinins, Sephadex column chromatography, high pressure liquid chromatography     

Quantification of the Daily Cytokinin Transport from the Root to the Shoot of Urtica dioica L.*

Cytokinins are predominantly root-born phytohormones which are distributed in the shoot via the xylem stream. In the hormone message concept they are considered as root signals mediating the transport of the photosynthates to the various sinks of a plant. In this paper the cytokinin relations of Urtica dioica L., the stinging nettle, are described, based on the daily flux from the roots to the shoot. Trans-zeatin-type cytokinins predominate in the various tissues of Urtica (Wagner and Beck, 1993), and accordingly trans-zeatin riboside and trans-zeatin are the forms transported by the xylem sap. The daily time-course of cytokinin concentration in root pressure exudates and in xylem sap collected from a petiole after pressurizing the root bed showed high concentrations in the morning, followed by a substantial drop to a level of 15–30% of the initial concentration which was then maintained during the afternoon. This time-course is interpreted as resulting from continuous synthesis and exudation of cytokinins into the xylem fluid of the roots whose cytokinin concentration is then modified by the dynamics of the transpiration stream. Loading of cytokinins into the xylem sap could be enhanced several times by increasing the flux rate of the xylem stream to the maximal transpiration rate when a maximum export rate was reached. The total daily cytokinin gain by the shoot depended on the nitrogen status of the plant. Roots of Urtica plants grown on a sufficient nitrogen supply had a significantly higher cytokinin content and exuded more cytokinins into the shoot than those of plants raised under nitrogen shortage. A positive correlation was found between the steady rates of cytokinin export measured during the afternoon and the shoot to root-ratios of biomass which, in turn, corresponded to the nitrogen status of the plants.     

Effects of Zinc on Meristem Size and Proximity of Root Hairs and Xylem Elements to the Root Tip in a Zinc-tolerant and a Non-tolerant Cultivar of Festuca rubra L.

A 4 d exposure to zinc (0.1 and 02 µg Zn cm^–3) reducedthe length of the root apical meristem in a Zn-sensitive cultivar(S59) of Festuca rubra L. to a much greater extent than in aZn-tolerant cultivar (Merlin). In S59, Zn treatment also inducedroot hair and xylem formation much closer to the root cap boundarythan in control roots, whereas Merlin was only marginally affectedby Zn treatment. The data are discussed in relation to previouslyestablished effects of Zn on the cell cycle and other cellularcharacters of the two cultivars. zinc, meristem size, root hair, xylem, Festuca rubra     

Transfer RNA, a Possible Supplier of Free Cytokinins, Ribosyl-cis-zeatin and Ribosyl-2-methylthiozeatin: Quantitative Comparison between Free and Transfer Cytokinins in Various Tissues of the Hop Plant

Cytokinins in both free pool and tRNAs have been identifiedand quantified in the unfertilized cone, fertilized cone, seed,cotyledon, epicotyl and root of the hop plant (Humulus lupulusL. cv. Shinshu-wase) on the basis of combined gas chromatography-massspectrometry and combined gas chromatography-selected ion currentmonitoring analysis. Based on the quantitative comparison betweenfree pool cytokinins and tRNA cytokinins, it is suggested thatribosyl-cis-zeatin and ribosyl-2-methylthiozeatin might be derivedfrom tRNA catabolism and that the level of ribosyl-trans-zeatinmight be controled independently of the tRNA catabolism. ¹ Present address: Taisho Pharmaceutical Co. Ltd., Yoshini-cho,Omiya-shi, Saitama 330, Japan. (Received December 1, 1981; Accepted February 9, 1982)     

Spatial and temporal changes in endogenous cytokinins in developing pea roots

Germination and seedling establishment follows a distinct pattern which is partly controlled by hormones. Roots have high levels of cytokinins. By quantifying the fluctuations in endogenous cytokinins over time, further insight may be gained into the role of cytokinins during germination and seedling establishment. Radicles were excised from sterile Pisum sativum L. seeds after 30 min and 5 h imbibition. Seedlings germinated on agar were harvested after 1, 3, 6 and 9 days. The roots were divided into the root tip, root free zone, secondary root zone and from day 6, the secondary roots. Samples were purified by various chromatographic methods and endogenous cytokinins detected by LC(+)ES-MS. Benzyladenine levels doubled after 5 h imbibition and then gradually decreased over time. Low concentrations of cis-Zeatin (cZ) type cytokinins were detected in the radicle after 30 min imbibition. After 5 h imbibition, cis-zeatin riboside-5′-monophosphate had greatly increased. The total cytokinin content of the roots increased over time with the ribotides being the predominant conjugates. From day 3 onwards, there was a gradual increase in the free bases, O-glucosides and their ribosylated forms. Mainly N ⁶ -(2-isopentenyl)adenine (iP)-type cytokinins were detected in the root tip, whereas trans-zeatin- (tZ), dihyrozeatin- (DHZ) and iP-type cytokinins were found in the secondary roots and root zone. Cytokinin biosynthesis was only detected after day 6. Biosynthesis of iP and tZ derivatives was quite rapid, whereas biosynthesis of cZ derivatives remained at a low basal level. These fluctuations in cytokinin types and concentrations suggest the cytokinins may be synthesized from various pathways in pea roots.     

Identification of Cytokinins in Root Exudate of the Rice Plant

Cytokinins, cis-zeatin and cis- and (trans-ribosylzeatin, wereidentified in the root exudate of the rice plant (Oryza sativa,indica cultivar IR-24) after several chromatographic separationsand combined gas-liquid chromatography-selected ion monitoring(GC-SIM) analysis. The presence of trans-zeatin ribotide wassuggested by enzyme hydrolysis, subsequent chromatographic separationand GC-SIM. The comparatively high content of the ribotide inthe root exudate suggests the form of cytokinins to be transportedfrom roots to other parts in the rice plant. (Received July 22, 1982; Accepted November 25, 1982)     

The effect of aluminum on cytokinins in the mycelia of Amanita muscaria

High performance liquid chromatography analysis of immunoaffinity-purified extracts of mycelia of Amanita muscaria, and the Amaranthus bioassay of the eluted fractions, revealed the following seven cytokinins: zeatin, zeatin riboside, zeatin N-9-glucoside, dihydrozeatin, dihydrozeatin riboside, isopentenyl adenine, and isopentenyl adenosine. The decreased growth of aluminum-treated mycelia correlated with a 35% decrease in the total amount of the cytokinins. Among individual cytokinins, zeatin was the most affected, exhibiting a reduction of about 90%. The results are compared with previous investigations of aluminum effects on cytokinins in the mycelia of Lactarius piperatus, whose growth is stimulated by aluminum.Abbreviations ZR zeatin riboside - iPA isopentenyl adenosine - Z zeatin - DHZ dihydrozeatin - iP isopentenyl adenine - DHZR dihydrozeatin riboside - Z-9G zeatin N-9-glucoside - iP-9G isopentenyl N-9-glucoside - HPLC high performance liquid chromatography - DHZRMP dihydrozeatin riboside monophosphate - ZRMP zeatin riboside monophosphate     

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)